Systems Biology: Symbiosis between Analytical Sciences, Biostatistics and Biology
Jan van der Greef
TNO Systems Biology
In recent decades various scientific domains ranging from physics, biology to cosmology have been focusing towards a systems-based view. The key topic being the interconnectivity of systems and the study of the organizing principles, realizing that new properties emerge at different levels of complexity. In Life Sciences this focus is initiated in Systems Biology research. In Pharmaceutical industry the challenges in drug discovery are huge as highlighted by the fact that target-centered drug discovery, practiced by pharmaceutical companies for the past 30 years and recently amplified by the availability of genomic data, seems to become unproductive to the point where the economic future of the industry could even be questionable. The steady decline of new drug approvals in the USA since 1996 is in sharp contrast with the almost doubling of expenditures on pharmaceutical R & D during the same period. A major step forward is not only comprised of a technological one, but encompasses a conceptual shift towards systems thinking. Since the development of comprehensive bodyfluid/tissue profiling combined with pattern recognition (chemometrics) in the beginning of the eighties, tremendous amounts of data at different biological levels (transcripts, proteins and metabolites) have been generated in recent years to demonstrate differences in different biological states or the impact of pharmaceuticals or food on biological systems. Translating this data into biological information and knowledge to improve R&D processes has become the major challenge. The concept of systems-based strategies in medicine is emerging, with systems pathology guiding an understanding of the multidimensional aspects of disease system fingerprints and systems pharmacology providing insight into dynamic system responses upon perturbations. Knowledge of the changes of system characteristics during disease progression creates a framework for the design of novel combinatorial treatment strategies. Such a systems-based, combinatorial-therapies approach re-addresses the value of the synergistic actions of components of treatments based on natural products and highlights new methodology to study multidimensional intervention via reversed-pharmacology. It bridges nutrition and pharma related research. Examples will be used to discuss important questions such as: “How can a systems-based approach alter our view on drug discovery and development or, more generally, of human healthcare? and “What would be the impact of practising a systems-strategy in medicine?” .
Post-genomic Systems Integration as an Opportunity to build Better Drugs
Biosystems Informatcs Institute, Newcastle upon Tyne, U.K.
Our best estimates of distinct protein-protein binding interactions possible within the human body runs to 900 trillion. Proteins represent about 98% of all known drug targets, yet we have yet to make serious in-roads into dissecting this potential drug-binding diversity or the biochemical cascades induced by drug action. Adverse drug effects are annually responsible for > 100,000 deaths; > 2.1 million hospitalizations; and > 5 million illnesses in the USA alone, while the investment community was rocked by Adverse Drug Effects to Vioxx TM in September 2004. Since almost two years and for the first time ever, the scientific community and the pharmaceutical industry has access to an accurate parts list of all known potential drug targets within the human body. Efforts to translate this information into better functional annotations; protein characterization; protein-protein interaction prediction; dynamic pathway modeling; and data integration in a systems context with be presented with respects to extant activities at the Biosystems Informatics Institute. In other scientific disciplines, high-end complexity has been dealt with effectively since many decades. Biologists must now turn to mathematics as the first step in discovery and in efforts to build better drugs.
Session 2, Developments in MicroScale Bio-Analysis
New Nanoflow LC/MS Approaches to Comprehensive Trace Characterization of Post-Translationally Modified Proteins in Biological Systems
Barry L. Karger
The need to characterize proteins comprehensively in biological systems, e.g. cell lines, tissues, etc. is great. By comprehensive we mean high sequence coverage and detailed post-translational structure analysis, e.g. phosphorylation, glycosylation, etc. In this talk, we will examine the role of sample preparation, chromatography, mass spectrometry and bioinformatics in achieving this goal. Special emphasis will be placed on LC and MS, where the appropriate integration of the coupled technologies can lead to low level detection. Specifically, use of ultranarrow monolithic LC columns with mobile phase flow rates of 20 nL/min in combination with an LTQ-FT mass spectrometer can result in comprehensive characterization at the low fmole to high attomole level for proteins as large as 180,000 Da. After a review of the principles and practices involved with this approach, we will present several applications of the technology in biological systems. One of the examples will explore that stimulation of the phosphotyrosine receptor, EGFR, in a cancer cell line, followed by a detailed temporal analysis of the phosphorylation of multiple sites on the receptor. We will end the lecture with a discussion of future directions of trace detection of proteins in biological systems by LC/MS.
Oxidative Stress in Desease and Aging:New Analytical Strategies for Looking at Old Biological Problems.
Fred Regnier, Hamid Mirzaei
Department Chemistry, Purude University, West Lafayette, USA
Free radical initiated oxidation of proteins stemming from either ones diet, the environment, or a disease is a serious health risk, particularly when the capacity of cellular control and repair systems is exceeded. One of the most devastating outcomes is the introduction of aldehydes and ketones into proteins that either alters their biological activity, causes them to cross-link with other biological macromolecules, or triggers their degradation by proteosomes. Because this oxidative process is non-enzymatically driven it should theoretically occur randomly in proteins throughout cells. This paper will focus on the development of separation methods that enable oxidized proteins to be selected from complex biological extracts and identified along with sites of oxidation. Use of these methods in the study of four types of biological problems will be reported; environmental oxidative stress in yeast, protein oxidation in Parkinson's disease, oxidation of proteins in diabetes, and aging. In all four cases it will be shown that oxidation is relatively specific and multiple amino acids are altered within a small number of specific structural domains. The presentation will end with a discussion of new separation problems suggested by the results of this work.
Session 3A, FUNDAMENTALS OF MICROSCALE SEPARATIONS
Optimization of Capillary Electrophoresis Experiments
Stellan Hjertén, Sheila Mohabbati, Douglas Westerlund, Ŕkos Végvári
Department of Biochemistry, Uppsala University, Biomedical Center, P.O. Box 576, SE-75123, Uppsala, Sweden Analytical Pharmaceutical Chemistry, Uppsala University, Biomedical Center, P.O. Box 574, SE-75123, Uppsala, sweden
The first part of our presentation will be devoted to the difference between true and apparent separation parameters (plate number, the width of a peak, the distance between two peaks, resolution, etc.) since only true separation parameters should be used in optimization studies. Recording of the separation pattern by a scanning detector is preferable to recording by a stationary detector (which, unfortunately, is standard in all commercial CE equipment) since the former detector gives true parameter values, whereas the latter gives apparent values, because the width of a peak is affected by the speed at which the zone passes the detector: the higher the speed, the narrower the peak. The distortion of the peaks can be still more pronounced for charged analytes in EOF-assisted capillary electrophoresis and in pressure-assisted capillary electrochromatography. The second part of the lecture will deal with how to minimize the length of the starting zone and the running time in order to reduce zone broadening caused by diffusion while retaining a satisfactory resolution between two adjacent zones (the variance for the zone broadening caused by diffusion is about half the total variance for zone broadening in polyacrylamide-coated capillaries, which illustrates the importance of short running times). High field strengths decrease the migration time and thus the diffusional zone broadening, but increase the risk for zone broadening caused by interactions of analyte with the capillary wall and buffer constituents. We will show how to distinguish between these two types of interactions. If the latter interaction dominates, which is often the case in polyacrylamide-coated capillaries, other types of buffers should be tested. We will devote some time to discussion of the transformation of apparent separation parameters to true parameters in different types of chromatography and electrophoresis, using very simple equations.
Electrokinetic Transport in Sphere Packings and Monoliths: From High-resolution Numerical Simulations and Quantitative Imaging to Macro-scale Phenomena
Otto-von-Guericke-Universität, Magdeburg, Germany
Electrical field-induced transport of charged analytes is particularly complex in hierarchically structured porous media like sphere packings and monoliths which contain discrete ion-permselective regions (mesopore space) in addition to quasi-electroneutral macropore space. Besides perfusive electroosmotic flow (EOF)  concentration polarization (CP) is induced by the externally applied electrical fields due to coupled mass and charge transport normal to the charge-selective interfaces and it results in spatially extended convective diffusion boundary layers [2,3]. The intensity of these zones and their effect on analyte migration, retention, and dispersion depends on applied field stength, surface charge density, characteristic pore sizes, and ionic strength . An electrical field-induced, nonequilibrium electrical double layer and instability of the induced-charge EOF are extreme scenarios of CP which may be employed in miniaturized devices for nonlinear pumping of fluid and efficient pore-scale mixing at low Reynolds numbers [2,3]. In separation science, the realisation and control of lateral velocities is a unique mechanism for reducing axial dispersion by faster lateral exchange of analytes beween mobile phase velocity extremes [2,4]. Relevant transport phenomena including pore-scale EOF profiles, electrophoresis, and CP are analyzed locally in monoliths and particulate beds with respect to macroscopic transport behaviour [4-6]. Effects related to the actual (electrical field-dependent) intensity of CP are a key to understanding retention, migration, and dispersion behaviour in electrochromatography.  Tallarek, U., Paces, M., Rapp, E., Electrophoresis 2003, 24, 4241-4253.  Tallarek, U., Leinweber, F. C., Nischang, I., Electrophoresis 2005, 26, 391-404.  Leinweber, F. C., Tallarek, U., Langmuir 2004, 20, 11637-11648.  Nischang, I., Tallarek, U., Electrophoresis 2004, 25, 2935-2945.  Leinweber, F. C., Pfafferodt, M., Seidel-Morgenstern, A., Tallarek, U., Anal. Chem. 2005, 77, 5839-5850.  Hlushkou, D., Seidel-Morgenstern, A., Tallarek, U., Langmuir 2005, 21, 6097-6112.
Recent Developments in pH-Biased Isoelectric Trapping Separations of Ampholytic Compounds
Gy. Vigh, E. Shave, R. Estrada, H. C. Fleisher, B. Sinajon, S. Lalwani, P. Lim, R. North, E. Tutu
Texas A&M University
Recently, we developed pH-biased isoelectric trapping for the pI-based analytical and preparative-scale separation of ampholytic components. pH-biased isoelectric trapping is carried out in multicompartmental electrolyzers in which the adjacent compartments are joined through buffering membranes whose pH values bracket the pI of the ampholytic component to be trapped in the compartment. Since the solubility of ampholytic components is often low in their isoelectric state, a pH biaser, an isoelectric buffer is also trapped in every compartment, along with the ampholytic compound of interest. The pH biaser is selected to satisfy two requirements: (i) its pI value is between the pH values of the buffering membranes delimiting the respective compartment, and (ii) its pI value is sufficiently different from the pI values of the target component to insure that the target components are kept in charged state throughout the entire separation process. We have synthesized a family of new isoelectric buffers with good buffering capacities and adequate conductivities in the 3 < pI < 10 range for use in pH-biased isoelectric trapping. To create hydrolytically stable electrode membranes, we have grafted appropriate buffering groups to poly(vinyl alcohol)-based hydrogels. The pH values of these buffering membranes can be as low as about 1.8 and as high as about 13. The new membranes can withstand current densities as high as 80 mAcm-2. The new buffers and membranes were used to rapidly desalt ampholytic samples, prefractionate complex samples for further analysis and isolate ampholytic components from complex matrices. Transfer rates as high as 0.4 mg min-1cm-2 were achieved at an electrophoretic energy consumption of 0.22 Wh mg-1 ampholytic component.
What for is Useful Theory of Electrolyte Solutions: From Oscillating BGEs through Resonance Phenomena to Designing well Behaving BGEs for Electrophoresis
B. Gas, V. Hruska, M. Jaros, M. Stedry
Faculty of Science, Charles University, Prague, Czech Republic
The theories of movement of charged species in solutions stem from fundamental physico-chemical laws, which form an inherently nonlinear mathematical model. Its direct numerical solution (simulation) gives a complete picture about behavior of the electrophoretic systems in the electric field. Another approach is formulation of the approximate linear model. The linear model reveals that any solution of electrolytes possesses a set of certain characteristics - eigenmobilities, which play a substantial role when the electrolyte solution is used as the background electrolyte (BGE) in electrophoresis. The eigenmobilities are responsible for occurrence of system peaks, and, moreover, when the mobility of the separated analyte coincides with the BGE eigenmobility, it leads to the resonance causing a heavy deterioration of the analyte zone. Recently, we discovered a new class of electrolyte solutions, which have even oscillating behavior. When such an electrolyte is used as the BGE, it leads to its complete instability and oscillation of the electrolyte concentration in the separation channel (Fig. 1). This is an analogy to chemical oscillations, where the driving force is not the chemical potential but rather a gradient of the electric potential. Both the nonlinear and linear model of electromigration are implemented in two computer programs we developed, Simul and PeakMaster, respectively. Simul helps to understand what takes place during the electrophoretic run. Specifically, it can be used for (i) optimizing analytes’ stacking to obtain initial preconcentration, (ii) inspecting unusual peak broadening, and (iii) simulation of isotachophoresis. PeakMaster serves rather for computer design of background electrolytes with optimized properties to reach (i) more sensitive detection, (ii) higher efficiency of separation, and (iii) better selectivity of separation. Many practical examples and video sequences are given to demonstrate the ability of the programs, which can be downloaded from http://www.natur.cuni.cz/gas
Pores, Pressure and Temperature: Comparing Kinetic Advantages
G. Desmet, D. Clicq, D. Cabooter, P. Gzil
CHIS-TW, Vrije Universiteit Brussel, Brussel, Belgium
In the past years, several new approaches have been proposed to increase the performance of chromatographic systems. A first approach consists of altering the basic geometrical structure of the stationary phase. The best known examples of this approach are the silica and polymer monoliths. A second approach plays on the physico-chemical conditions of the mobile phase. In High-Temperature-HPLC the viscosity of the mobile phase is lowered by operating at higher temperatures. In UPLC the pressure limitation is alleviated using specially developed equipment, withstanding pressures of several thousands of bars, and opening the road to 1µm particles packed beds. When it comes to comparing the merits and potential advantages of these highly differing approaches, the traditionally used van Deemter curve representation (in either absolute or reduced terms) no longer shows the complete picture, for it does not incorporate the differences in permeability, viscosity, pressure gradient, retention capacity,…. that inevitably exist between all these different systems. In this context we have recently proposed (1,2) to recombine the variables u0 and H that make up a traditional van Deemter curve into two very simple expressions, respectively yielding the analysis time and the plate number. With this so-called kinetic plot approach the entire kinetic performance potential of a given support type or operating condition is visualized in a single curve. The "currencies" used in these plots are so universal that they yield a completely unbiased and system-independent comparison. The approach of a time versus plate number plot has already been used since the very beginning of HPLC by leading scientists like Giddings, Knox and Poppe. Having demonstrated that such plots can be established without the need for any iterative computer algorithm (1,2) it is hoped that now the entire field will start reporting their plate height measurements in a kinetic plot format. Instead of mentioning the Hmin and uopt-values of different systems it is for example much more informative and universal to compare the time they need to yield 10,000 and 50,000 plates (respectively denoted as t10,000 and t50,000). Using the kinetic plot method we were already able to show (1) that the current silica monoliths can only outperform the traditional packed bed column in the case of difficult separations (N>50,000). In the present study we have further developed the kinetic plot method, now also including the possibility to incorporate real world constraints (maximal velocity, maximal column length, minimal required peak volume, etc…), and have applied it to a panoply of experimental and simulated data (using CFD), obtained under varying pressures (including UPLC) and temperatures (High-Temperature-HPLC) and for several different compounds, mobile phases and column types. (1) Desmet, G. et al., Analytical Chemistry 2005,77,4058-4070. (2) Desmet, G. et al., LCGC Europe 2005,18(7),403-409.
Session 3B, PROTEOMICS
Toward Positive Identification of Structural Oligosaccharide Isomers in Functional Glycomics: a Combined use of Electromigration Data and Mass-spectral Information
M.V. Novotny, Y. Mechref, Z. Kyselova, P. Kang, M. Madera, I. Kouckova, J. Starkey
Department of Chemistry, Indiana University, Bloomington, IN, USA
The inherent complexity of mammalian glycomes and proteomics dictates the need to use different methodologies that provide complementary information, being dependent on different resolution principles. Although mass spectrometers (MS) using single mass analyzers can now readily provide compositional data on glycans, determination of structural isomers (including branching, linkage position and monomer anomericity) requires a combination of chromatographic/electrophoretic and sophisticated MS tools in a tandem mode. To analyze effectively complex glycan mixtures derived from glycoproteins, we have previously demonstrated the utility of CEC and CZE in coupling with different MS analyzers. This work has now been followed with advances in on-line permethylation, nanoscale LC or CEC and, ultimately, the use of ESI- or MALDI/MS-MS to yield highly informative data through cross-ring fragmentation. As “testing cases” for the new glycomic platforms, we present examples of human IgG, transferrin and rat liver glycoproteins.
A Novel Approach to Plasma Glycoproteomics using Multi-lectin Affinity Chromatography (M-LAC) Combined with other Depletion Methods
William Hancock, Marina Hincapie, Tatiana Plavina
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA
Serum and plasma are potentially the most valuable specimens for biomarker development studies. However, due to their complexity and extremely wide dynamic range, it is a challenge to analyze them and obtain valuable data. Clearly, there is a need in proteomic community for methods that would allow in-depth analysis of large plasma or serum sample sets in a relatively high-throughput manner. Here we report development of a robust and reproducible method for proteomic analysis of human plasma or serum that combines depletion of most abundant proteins with the enrichment of glycoproteins using variety of lectins, followed by LC-MS/MS analysis of digested proteins. When applied to analysis of human plasma, these methods lead to identification of proteins that are present in plasma at concentrations 10-100 ng/mL. Numerous tissue leakage proteins as well members of protein families that may be of particular interest when studying variety of disease conditions have been identified. A key advantage of the method is that the depletion can be optimized to certain classes of proteins or types of protein glycosylation patterns by changing the depletion method or type of lectins used for glycoprotein enrichment. The developed method may be successfully utilized for a relatively high throughput in-depth analysis of plasma or serum in search for potential biomarkers. We have achieved a dynamic range in excess of 106 in terms of identifying low level proteins relative to the most abundant protein, albumin.
Bio-specific Interaction (Affinity) Nano-Liquid Chromatography and Capillary Electrochromatography for Proteomics/Glycomics
Ziad El Rassi, Yazen Jmeian, Fred Okanda
Department of Chemistry, Oklahoma State University
Biospecific interaction (i.e., affinity) chromatography/electrochromatography (BIC) is well suited for the isolation of biomolecules pertinent to proteomics and glycomics. Furthermore, BIC in miniaturized formats, e.g., capillary electrochromatography (CEC) and nano-liquid chromatography (nano-LC) offers the convenience for the isolation of biomolecules present at low level in small sample size. This talk will introduce novel nano-scale affinity methods based on monolithic capillary columns having immobilized lectins or oligosaccharides to perform BIC based on carbohydrate-protein interactions and in turn achieve the separation of sugar binding proteins and glycoconjugates. In addition, affinity monoliths with immobilized metal chelate ligands will be presented in the aim of describing chromatographic systems for depleting high abundance proteins and concentrating low abundance proteins in real proteomic samples. To facilitate the separation of multi-component, two-dimensional separations involving affinity in one dimension and reversed phase in another dimension will be described.
Biomolecular Sreening Applications of Protein-DNA Interactions in Capillary Electrophoresis
A.P. Drabovich, M. Berezovski, S.N. Krylov
York University, Toronto, Canada
Non-covalent protein-DNA interactions could be classified as binding of natural DNA-binding proteins to oligonucleotides or as interaction of in-vitro selected DNA aptamers with their target proteins. Both types of interactions could be used in developing methods for high-throughput screening of affinity ligands and drug candidates. We will demonstrate that gel-free capillary electrophoresis can serve as an indispensable tool to apply protein-DNA interactions in biomolecular screening assays. First, we will introduce CE-based schemes for in-vitro evolution of DNA aptamers for protein targets. Second, we will show kinetic approaches in CE to select “smart aptamers” - DNA ligands with predefined binding parameters . Finally, we will demonstrate DNA-mediated screenings of small molecule ligands to protein targets. We anticipate that protein-DNA interactions will be widely used in comprehensive CE methods for screenings of biomolecules.  Drabovich, A.; Berezovski, M.; Krylov, S.N. JACS, 2005, 127, 11224-11225.
On-line Coupling of Enzymatic Reactor, Capillary Electrophoresis and Mass Spectrometry for Protein Analysis
J. Krenková, F. Foret
Institute of Analytical Chemistry, Czech Academy of Sciences, Brno, Czech Republic
The complexity of biological materials, especially when dealing with protein analysis, mostly requires the use of multidimensional analysis for identification and quantification of individual components. Enzymatic digestion of the analyzed proteins, separation of the tryptic peptides and coupling with mass spectrometry are the most common steps in most analytical protocols. Many of the practical problems of enzymatic cleavage in a homogenous solution can be eliminated by immobilization of the enzyme on a solid support such as monolithic column, beads or capillary wall. Methods used for coupling of proteins on solid supports, most frequently rely on covalent bonds for minimization of the immobilized enzyme leakage. In this work a multidimensional approach for protein analysis by peptide mapping will be described. The preparation and characterization of the system integrating capillary enzymatic reactors, on-line protein and peptides separation, and electrospray ionization time-of-flight mass spectrometry analysis will be presented.
Poster Session 1
Session 4A, Stationary Phases and Column Technology
Preparation of RPLC Stationary Phases by Polymerization Reactions on Monolithic Silica in Capillary
Nobuo Tanaka, Tohru Ikegami, Wataru Kajiwara, Kanta Horie, Jun Ichimaru
Department of Polymer Science and Engineering, Kyoto Institute of Technology
Several types of stationary phases were prepared on monolithic silica in a capillary for reversed-phase HPLC. Methacrylate functionality was first bonded onto silica surface followed by radical polymerization reactions of methacrylate or styrene monomers. Alkyl, aryl, cyanoalkyl, and fluoroalkyl groups were attached and the chromatographic properties were evaluated. The stationary phases prepared from octadecyl methacrylate showed an increase in retention with the increase in monomer concentration in feed. They also showed comparable hydrophobic selectivity with, and the greater retention as well as much greater steric selectivity between planar and nonplanar compounds than octadecylsilylated stationary phase prepared on monolithic silica using a monomeric or polymeric octadecylsilane. The increased retention provided by polymerized alkyl methacrylates somewhat compensates the small phase ratio of a monolithic silica capillary column having high porosities. The stationary phases generally showed preferential retention for polar compounds over nonpolar compounds when compared to a C18 phase. The columns showed permeability twice as high as a column packed with 5 um particles and plate heights of about 10 um at an optimum linear velocity. The retention property was controlled by the type of monomers used for preparation. Other stationary phases including cyanoalkyl-, styrene-, and fluoroalkyl-bonded phases showed retention properties expected from their functionality. The possibility of 2D-HPLC using these columns as a second-dimension column will be discussed.
Monolithic Materials in Microfluidic Devices for Applications in Proteomics
Monolithic materials in microfluidic devices for applications in proteomics
Monolithic supports prepared in situ enable both vastly improved mass transport and rapid separation. Recently, we have developed a simple "molding" process based on photoinitiated free radical polymerization of liquid precursors achieved at room temperature by UV irradiation through a mask. Using this procedure similar to photolithography, the monolith can easily be located at a specific location within the device. Simultaneously, we also introduced photografting of pore surface of our monoliths with chains of functional polymers, a process that is a very powerful tool affording precise nanoscale control of surface chemistry within the pores. This procedure affords monoliths which pore surface can be covered with multiple layers of spatially segregated chemistries, co-contiguous chemistries, or with a longitudinal gradient of functionalities. Applications of these monoliths will be exemplified on rapid nano-HPLC separations, the preparation of immobilized enzyme reactors, as well as on design of complex fluidic devices.
Monoclonal Behaviour of Molecularly Imprinted Polymer Nanoparticles in CEC
Staffan Nilsson3, Feliciano Priego-Capote13, Lei Ye3, Sadia Shakil3, Shahab A. Shamsi2
1 Department of Analytical Chemistry, Córdoba University, Annex C-3 Building, Campus of Rabanales, 14071 Córdoba, Spain 2 Department of Chemistry, Center of Biotechnology and Drug Design, Georgia State University, GA 30303 Atlanta, USA 3 Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
A new approach for synthesis of very small imprinted nanoparticles (30-150 nm) based on miniemulsion polymerization is proposed. This approach consists of the use of a new monomer for its inclusion in the MIP nanoparticles structure. The MIP performance was tested as pseudostationary phase in CEC analysis of rac-propanolol (using (S)-propanolol as template) by partial filling technique mode. In contrast to previous methods based on the use of MIP in CEC, no tailing was obtained for any of the enantiomer peak and baseline separations (25.000-60.000 plate number) were achieved. Also, the size of MIP nanoparticles makes it possible to think in the direction of fabricating MIP-receptors by reducing the number of imprinting sites, near to the ideal situation with only one imprinting site.
Chiral Ion-exchange Type Monoliths for Enantioselective Capillary Electrochromatography
M. Lämmerhofer, B. Preinerstorfer, D. Lubda, W. Lindner
(a) Christian Doppler Laboratory for Molecular Recognition Materials, Institute of Analytical & Food Chemistry, University of Vienna, Waehringer Strasse 38, A-1090 Vienna, Austria (b) Merck KGaA, Frankfurter Strasse 250, Darmstadt, Germany
In a recent study, the high potential of enantioselective CEC with slurry packed particulate capillary columns (3.5 µm silica particles) containing an ion-exchange type chiral stationary phase based on a penicillamine-sulfonic acid derivative could be demonstrated by its application for quality control (QC) of enantiomeric ephedrine samples after assay validation (1). Benefits of CEC over CE and HPLC in QC of enantiomeric compounds with high enantiomeric excess could be clearly proven. However, limited column longevity appears to be the Achilles' heel of packed column CEC. In our efforts to overcome this shortcoming and obtain a more robust CEC method, we have drawn our attention to the monolithic column technologies. First, effective ion-exchange type selectors have been developed that fulfill both requirements of enantioselective CEC: 1) They provide electroosmotic flow due to the charged ion-exchange site, and 2) they are the stereorecognition site for distinction between the enantiomers. Along this line, cinchona alkaloid derivatives are used for enantiomer separations of acidic chiral solutes, while aminosulfonic acid or aminophosphonic acid derivatives turned out to be effective chiral selectors for basic chiral analytes. The most significant characteristics of these selectors are their suitable EOF beaviour owing to the co-electrophoretic flow and the ion-pair supported adsorption that needs to be properly balanced. These selector chemistries have been transferred to organic polymer and inorganic sol-gel silica monolith technologies: Thereby, the chiral selectors have been either directly incorporated into the polymer matrix by in-situ copolymerization (organic polymer technology) or have been immobilized subsequently to the preparation of the monolithic capillary column in a post-modification step (organic polymer and silica monolith technologies). Our experience with the obtained enantioselective monolithic columns in CEC will be shared with the participants and the advantages and drawbacks of each column technology will be discussed. (1) W. Bicker et al., Electrophoresis, 2003, 24, 2532-2542.
Fast Liquid Chromatographic Separations on Capillary Monolithic Columns
Pavel Jandera, Jirí Urban, Dana Moravcová
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Nám. Čs. Legií 565, 532 10 Pardubice, Czech Republic
The performance of several types of monolithic columns was investigated and compared. The separation media studied included commercial silica gel based conventional and capillary Chromolith columns, organic polymer CIM separation discs and polymethacrylate monolithic capillary columns prepared in the laboratory by in-situ polymerization in fused silica capillaries. The factors affecting pore formation, namely the concentrations of the monomer components and the porogen solvents in the polymerization mixture were investigated and an approach was suggested for preparing continuous separation media with desired porosity and low flow resistance. The parameters of the columns tested included the efficiency (van Deemter plots), the “equivalent dispersion particle size”, the “equivalent dispersion particle size”, total, inner-pore and mesopore size distribution (investigated by inverse size-exclusion chromatography) and chromatographic tests for hydrophobic and polar group selectivity, using homologous series, ionic samples and proteins. In addition to totally monolithic capillary columns, we prepared a new type of “hybrid interparticle monolithic” capillary columns, by polymerization in capillaries previously packed with spherical beads, 37 - 50 mm, covered by a superficially porous silica gel and bonded C18 layer. The “hybrid” columns showed excellent stability and improved hydrodynamic flow properties with respect to the “totally” monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space. The new materials are suitable for fast gradient separations of proteins. The authors acknowledge the financial support of this work by the Ministry of Education, Youth and Sports of the Czech Republic under research project No. MSM 0021627502 and by the Grant Agency of Czech Republic under project No. GA 203/02/0023.
Pressure Driven Separations in Channels Structured with Micropillars
M. De Pra1, W. De Malsche2, W. Th. Kok1, G. Desmet3, P. J. Schoenmakers1
1)Universiteit van Amsterdam, van’t Hoff Institute for Molecular Sciences, Amsterdam, NL, 2)MESA Research Institute, University of Twente, Enschede, NL, 3)Vrije Universiteit Brussel, Department of Chemical Engineering, Brussel, BE
In liquid chromatography (LC) with packed columns a lower limit for the reduced plate height (h) is found in practice. This limit is related to the low degree of order in a packed bed. Band dispersion is caused by variations in flow path length and local flow velocity. The effect is commonly known as Eddy diffusion. A possible way to further improve the efficiency of liquid-phase separations is by using separation channels with perfectly ordered structures. Band dispersion has been measured in micromachined channels structured with orderly disposed cylindrical micropillars. It was found that with an optimal channel design the band broadening could be lower by a factor of 3 than in packed columns with a comparable particle size. The positioning of the row of pillars closest to the side wall was a decisive factor in influencing band broadening. Different pillar shapes and different porosities were also tested. Besides LC, pillars structured microchannels can be an ideal platform for Hydrodynamic Chromatography separations.
Session 4B, Metabolomics
Capillary Electrophoresis for High Sensitive and Selective Metabolome Analysis
S. Terabe, L. Jia, P. Britz-McKibbin, M.J. Marukuszewski, B.-F. Liu, J.-B. Kim, H. Chen, T. Nishioka
University of Hyogo, Graduate School of Material Science, Kamigori, Hyogo, Japan
Most metabolites in bacterial or animal cells are ionic or polar small molecules and therefore, capillary electrophoresis (CE) is the most appropriate analytical technique for the metabolome analysis. However, the concentration sensitivity of CE is not very high because of limited sample volumes injected and short light path-length for absorbance detector. To improve concentration sensitivity several techniques have been developed, among which on-line sample preconcentration is a simple and efficient technique to perform because no instrumental modification is required. In on-line sample preconcentration larger amounts of analytes are injected than those in conventional CE procedures, that is, a large volume of the sample solution is injected into the capillary or only analytes are injected from the dilute sample solution. The sample zone injected must be focused prior to the start of separation not to deteriorate high resolution in CE. Several on-line sample preconcentration techniques have been developed with different focusing mechanisms. In any mechanism the migration velocity of the analyte must be changed drastically at a boundary between two different zones for the analyte to be focused. Major factors to change the migration velocity are pH, conductivity (concentration of the buffer), composition of the buffer, and additive. Dynamic pH junction, field enhanced sample stacking (injection), transient isotachophoresis, and sweeping are the techniques available. Dynamic pH junction is useful for the concentration of weak acids or bases, sweeping is usually useful for hydrophobic compounds, stacking and isotachophoresis are useful for ionic or charged analytes. One technique cannot concentrate all analytes simultaneously and a combination of techniques is often useful or separate runs are necessary for metabolome analysis. In this paper, several on-line sample preconcentration techniques are to be discussed with applications to metabolite analysis.
Comprehensive Analysis of Metabolites and Peptides
Thomas Hankemeier1, Harrie Storms1, Kjeld Janssen1, Fernando Benavente1, Ubbo Tjaden1, Rob van der Heijden1, Jan van der Greef1,2
1 Division Analytical Biosciences, LACDR, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands; 2 TNO Systems Biology, Department Analytical Sciences, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Systems Biology has recently emerged as the integrated approach to study biological systems-intracellular networks, cells, organs and any biological entity-by measuring and integrating genetic, proteomic, peptide and metabolic data. In this context, metabolomics involves the comparative, non-targeted, holistic (simply measure ‘everything’) analysis of the complete set of metabolites in a biological systems. The key issue in metabolomics is the translation of the differences in the metabolomes into differences in the biological functioning of a cell, organism or complete systems. Similarly, peptidomics is the analysis of the complete set of peptides. With other words, methods have to be developed which allow the quantitative analysis of a wide range of metabolites or peptides. Several LC-based separation strategies have been set-up for the comprehensive analysis of metabolites. Although CE has some clear advantages such as its potential high separation efficiency to be used for metabolomics or peptidomics, the limited loadability of the capillary is a serious drawback. To improve detectability, sampling, focussing and separations have to be combined in such a manner that the overall sensitivity of the analytical method is satisfactory. Interesting options for this are capillary isoelectric focussing (CIEF), isotachophoresis and transient methods. In this presentation some examples of efficient electrodriven separation methods for the comprehensive analysis of metabolites and peptides are discussed. One example discussed is the coupling of CIEF with mass spectrometry (MS) for the analysis of peptides. Usually carrier ampholytes are used in CIEF, which, on the other hand, strongly affect detection by MS. The goal was to balance these two effects. The use of low concentrations of carrier ampholytes as mere spacers, or no carrier ampholytes at all, were investigated. Finally, a good compromise could be found, and the developed CIEF-MS method was evaluated by the analysis of protein mixtures. Aspects of the validation of these methods discussed. The importance of data-preprocessing and multi-variate analysis will be explained, and some applications discussed. Finally, current analytical challenges and the future perspective will be discussed.
Construction and Analytical Application of an On-column Photoreactor for Improved Detection of Iron-species as Plant Metabolites in Capillary Flow Injection and Capillary Electrophoresis
Y. Xuan, G. Weber, A. Manz
ISAS - Institute for Analytical Sciences, Dortmund, Germany
Post-column photoreactors are used in HPLC as a means of improving detection selectivity or sensitivity. Only few applications of photoreactors have been reported for CE, but the inadequate sensitivity of absorbance detection makes photochemically enhanced detection very attractive for CE. We present an on-column photoreactor for CE, which is constructed from UV-transparent capillaries and a small UV lamp. The photoreactor is placed directly in front of the on-column absorbance detector, illuminating only some cm of the capillary. In spite of the small sample volume (short exposure time), relatively large photolysis effects are observed. The photoreactor is used for CE and also for capillary flow injection analysis. In the latter case, pressure is applied to the capillary instead of HV. The main difference of the two approaches is the presence of an electric field (and EOF) in CE. Interestingly, the buffer molecules forming the electric double layer are influenced also by the photoreactor, and thus the photoreactor affects not only the detection, but also the separation mechanism. The proposed photoreactor is applied to the analysis of small, non-covalent iron-species, which play an important role in plant metabolism. Some of the respective iron-species have already been identified, e.g. phytosiderophores in grasses, but details of the metabolic changes of iron-species in planta are still unknown. CE is well suited for the separation of such compounds, but the detection is hampered by their low UV absorptivity and low concentration. The proposed photoreactor takes advantage of the well-known photochemical activity of iron-complexes. Photoinduced sensitivity changes for model iron-species and for plant extracts are investigated, using absorbance detection and contactless conductivity detection. The detection sensitivity for some iron-species is enhanced considerably, depending on pH and on the type of bio-ligand. Advantages of using photoinduced detection for metabolic profiling of iron-species in plants are emphasised.
Analyses of Plant Hormones using Capillary Electrophoresis-tandem Mass Spectrometry
Liya Ge, Jean Wan Hong Yong, Swee Ngin Tan
Natural Sciences and Science Education Academic Group Nanyang Technological University
Capillary electrophoresis (CE) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is reported for the first time as an alternative and powerful analytical method for the separation and determination of plant hormones, including indole-3-acetic acid, indole-3-butyric acid, abscisic acid, gibberellic acid, zeatin, N6-benzyladenine, a-naphthaleneacetic acid and 2, 4-dichlorophenoxyacetic acid. The success of using the CE-MS/MS approach was attributed to the electroosmotic flow reversal using a cationic polymer-coated capillary. The various parameters for CE-MS/MS optimization, such as buffer pH, concentration of buffer and organic modifier, applied voltage and sheath liquid were evaluated systematically. Under optimum conditions, a baseline separation of eight plant hormones was accomplished within half an hour. The accessibility of MS or MS/MS (in negative ion mode) results together with the characterization of migration properties obtained by CE qualify CE-MS as a powerful method for the analysis of endogenous plant hormones. Compared with the other analytical techniques, the current developed CE-MS/MS method has several advantages: (i) various types of plant hormones, which are present as anionic forms could be separated and analyzed without derivatization in a short analytical time; (ii) relatively high sensitivity were achieved; and (iii) plant hormones could be selectively determined without serious matrix interference. Furthermore, the present methodology provides high resolving power, excellent reproducibility and low sample consumption. The utility of the CE-MS/MS method was demonstrated by the comprehensive analysis of plant hormones in coconut (Cocos nucifera L.) water after preconcentration and purification through solid-phase extraction cartridges. It is also expected that the method could be applied to the analysis of plant hormones in a wide range of biological samples.
A Capillary Electrophoresis Device for High-Throughput Multi-Dimensional Microanalysis of Biomarkers Using an On-Line Solid-Phase Extraction Device Having a Staggered Configuration for Maximum Enrichment Purposes
Norberto A. Guzman
Bioanalytical Drug Metabolism, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202, Raritan, New Jersey 08869, U.S.A.
In the post-genomic era, the need to study gene products, and the importance in understanding the function of proteins/peptides, and metabolites as active or toxic entities, is becoming the next priority in order to answer questions that genome alone cannot. Biomedical research is moving towards an era aimed at understanding biology as a highly complexed cellular system of interdependent pathways. A number of low- and high-throughput technologies are currently available for the characterization of a wide range of substances. However, all fall short of providing the throughput, specificity and dynamic range required to provide today’s demands for bioassay technology. Accordingly, alternative tools that provide a global view of the proteome and metabolome are a priority. In the last five years, there have been a number of advances in the designs of miniaturized reaction and separation systems. Most of these miniaturized devices are designed to perform integrated analysis of one or more simple or complex chemical or biochemical assays. However, the challenge of building an integrated analytical system that performs multiple reactions in series and/or parallel still remains in the early stages of development.
This presentation describes the technological advances that have brought the application of capillary electrophoresis (CE) to the forefront of protein research, including the identification of molecular and structural changes of proteins, various degradation pathways, and the monitoring of co- and posttranslational modifications. The goal of CE is to overcome and improve the technological limitations of other methodologies that are still employed in many laboratories engaged in protein studies. The design and operation of a CE instrument that provides both multistep separation and assay of proteins and peptides are reported. This high-throughput multi-dimensional electrophoresis device provides significantly improved performance to other systems available today. The main feature of the instrument that is of significant value is its ability to isolate and enrich those analyses found at low abundance in complex mixtures, in particular peptide biomarkers. The instrument contains a series of solid-phase, microextraction devices fabricated in a staggered configuration for use in on-line, affinity capillary electrophoresis. The staggered configuration permits a maximum enrichment capability.
Guzman NA, Phillips TM. Immunoaffinity CE for Proteomics Studies. Analytical Chemistry 77(3): 60A-67A (2005).
Rapid Classification of Lung Diseases by Ion Mobility Spectrometry
J.I. Baumbach1, S. Bader12, W. Urfer2, V. Ruzsanyi1, M. Westhoff3, P. Litterst3, L. Freitag3
1 ISAS - Institute for Analytical Sciences, Department of Metabolomics, Bunsen-Kirchhoff-Str.11, 44139 Dortmund, Germany 2 Department of Statistics, University of Dortmund, Vogelpothsweg 87, 44221 Dortmund, Germany 3 Lung Hospital Hemer, Theo-Funccius-Str. 1, 58675 Hemer, Germany
Volatile Metabolites occurring in human exhaled air are correlated directly to different kinds of diseases. Ion mobility spectrometer (IMS) coupled to multi-capillary columns (MCC) are used to characterize and quantify volatile metabolites occurring in human exhaled breath down to the ng/L- and pg/L-range of analytes within less than 500 s and without any pre-concentration. The IMS investigations are based on different drift times of swarms of ions of metabolites formed directly in air at ambient pressure. The outer dimensions of the drift tube of the IMS are less than 10 cm x 1 cm x 1 cm. The whole system matches on a palm of the hand. Since an interrelation between lung diseases and metabolites in human breath seems to be manifest, the instrument of IMS was object of research into a new screening method for pneumological aspects. The spectra obtained from patients suffering on bronchial carcinomas are discussed in detail. During a pilot study, data obtained on 36 cases suffering on lung cancer and 54 control persons were investigated. A reduction from more than one million data points per IMS measurement to 25 meaningful variables enables full differentiation of the groups and correct classification of the whole test set. In addition, a distinction of different infections is aspired in a promising pilot study using the IMS Chromatograms achieved and different statistical methods applied. The final ambition is the identification of biomarkers characterized in statistical analyses by metabolic profiling, valuable for
Session 5 Microfluidics
Nanofabricated Fluidic Structures for Elucidation of Molecular Information
J. Michael Ramsey
University of North Carolina
Tremendous interest in microfabricated fluidic channel structures (microchips) has grown over the past decade due to the large number of powerful demonstrations that have appeared in the literature. These devices have low cost and small footprints while consuming miniscule quantities of reagents and producing rapid results. Moreover, the manufacturing strategy used to make these devices, i.e., photolithography, allows highly parallel systems to be fabricated at low incremental cost. All of these features suggest the possibility to perform chemical experimentation at a massive scale at low cost on a bench top. More recently we have been investigating the prospects of shrinking channel lateral dimensions by a factor of ? 1000, i.e., to molecular length scales. A number of interesting capabilities are possible with nanoscale channels and pores including the structural characterization of single molecules. We have fabricated one-dimensional nanochannels (nanoscale in only one lateral dimension) below ten nanometers and have studied fluid and charge transport through such structures. These devices are fabricated by using standard photolithography and wet chemical etching. The etching process is slowed and performed for short durations to obtain nanoscale channel depths while the channel widths are at the micron scale as determined by contact photolithography. We have also fabricated two-dimensional nanochannels using focused charged particles to mill substrate materials. Focused ion beams have been utilized to form features as small as 50 nm x 50 nm. Electron beam milling has been used to form nanoscale holes that are less than 2 nm in lateral dimension. Nanochannel fabrication, some fundamental aspects of transport in nanoconfined spaces, and experimental results will be presented.
Microfluidic Monitoring of Cellular Responses to Stimuli
Klavs F. Jensen1, Jacob Albrecht1, Jamil El-Ali1, Susanne Gaudet2
1 Department of Chemical Engineering 2 Department of Biology Massachusetts Institute of Technology
We present microfluidic devices for monitoring cellular response to stimuli as a step to towards obtaining the large sets of data for protein activities, concentrations, and states of modification needed to understand cell signaling pathways. Soft lithography techniques are used to realize devices for cell growth, stimulus, and lysis, as well as separation of organelles and proteins. Simulations support device design and provide quantitative interpretation of experimental observations. Multiphase rapid mixing, cell stimulus and lysis components integrated with antibody arrays are used to explore cell signaling pathway kinetics. Separation of organelles and proteins is achieved by a new free flow isoelectric focusing device capable of achieving rapid separation. The device uses chemically modified hydrogel electrode regions as a simple, reliable means of applying high electric fields to micro free flow electrophoresis and avoiding bubble generation.
Session 6A, MICROFLUIDICS
New Strategies for Improved Sensitivity in Mass Spectrometry
Johan Roeraade, Johan Sjödahl, Patrik Ek, Mĺrten Stjernström
Royal Institute of Technology, Dept of Analytical Chemistry, Stockholm, Sweden
Mass spectrometry is one of the most useful methods for analyzing biomolecules. In particular, MALDI TOF MS and electrospray ionisation provide excellent sensitivity and important molecular information. However, in many cases the amount of available material is extremely limited such as in analysis of the content of single cells or in proteomics, when dealing with weakly expressed proteins. Therefore, improved sensitivity remains an issue of great importance in mass spectrometry. In this paper, we present a number of new strategies. We show, for MALDI TOF MS, that optimized sample handling and miniaturized, chip-based sample confinement down to spots with a diameter of 30 µm or less and can result in detection levels at the low zeptomole level. Factors affecting the LOD level have been studied and will be discussed. In another part of the paper, different concepts for electrospray MS are discussed, and a route towards more versatile systems and improved performance is presented. Examples of such systems, fabricated by means of micromachining as well as conventional technologies will be shown.
Ultra-sensitive Chemical and Physical Detection and Sensing for Micro Space
Department of Applied Chemistry, School of Engineering, The University of Tokyo
Detection and determination of chemical species and sensing physical parameters in micro space are one of the most important and difficult factors for microchip technologies. Absolute amounts of the target molecules are extremely small and physical quantities such as heat capacity are also very small and the disturbance of the system by probing is serious. Therefore, ultrasensitive, in-situ and non-disturbing detection methods are indispensable. In addition, these detection and sensing instruments should be device sized matching to the micro chemical chips. We have developed a thermal lens microscope (TLM), fluorescent probe, Mach-Zehnder interferometer and other detection devices on microchips. These devices have enabled at ymol (10-24 moll) levels determination of non-fluorescent and fluorescent molecules, chiral detection, flow velocity sensing, refractive index measurement, and others. They are also applicable to capillaries. Detection mechanisms, device structures and their applications will be introduced in the lecture.
A Novel Embedded Liquid Chromatography System with Submicron-Sized Filter Structure and Capacitively-Coupled Contactless Conductivity Detector
Chi-Yuan Shih, Wei Li, Yu-Chong Tai
California Institute of Technology
We present a novel technology to fabricate the first embedded LC system that composes high-pressure-capacity parylene separation column, submicron-sized filter for stationary-phase bead-packing, capacitively-coupled contactless conductivity detector (C4D) for ionic/neutral analyte detection, laser-induced fluorescence (LIF) detection cell for ultra sensitive optical sensing and resistive heater for on-chip chromatography temperature control. The proposed channel structure stood inner pressure higher than 1000 psi without fracture. Photoresist sacrificial layer is not required here for channel cross-section definition and therefore column contamination is minimized. Modern trend of LC instrumentation focuses on miniaturization of system components. For example, using smaller stationary-phase beads to pack separation column result in much higher separation efficiency. However, packing smaller beads(submicron) into LC column requires using finer filter structures(submicron) and higher packing pressure(thousands of psi) while both remain to be challenging issues for chip-based LC technologies. To resolve these issues, we developed a novel embedded-channel technology to build separation column and filters. Not only the embedded-channel structure is mechanically more robust than the above-substrate channel structure, the planarized profile(height variation over the 1cm2 chip area is smaller than 2µm) of embedded system is perfect for direct-top-clamping procedure which significantly enhances system pressure capacity to thousands of psi. Moreover, we introduce a submicron-filter-formation mechanism which generates in-channel, submicron-sized filter that can be used for packing state-of-the-art submicron stationary-phase beads. While C4D technology has been demonstrated in capillary electrophoresis to be a reliable conductivity sensor for a wide range of analytes, C4D is demonstrated here for the first time to be used in the chip-based LC system. Fabrication started by growing 1µm-thick thermal oxide. Cr/Au was then deposited. Backside oxide was patterned and DRIE was used to etch backside silicon until a 50µm-thick membrane was left. Metal and front side oxide were patterned to define the interdigited C4D and the meandering resistive heater. DRIE was used to create 6µm-wide, 80µm-deep trenches to define fluidic channel boundaries. First parylene layer(5µm) was deposited and etched back to fill up trenches. Second parylene layer(2µm) was deposited and patterned to define XeF2 etching window. XeF2 was then used to etch silicon and create square-like channel cross-section and open liquid access holes. Third parylene layer(10µm) was deposited to complete channels and filters. Parylene was then patterned for contact electrodes. Finally, to evaluate LC performances of the proposed system, a 16.7 nL sample containing 8.3 pmole of daunorubicin and 16.7 pmole of doxorubicin mixture was used for separation. Reproducible chromatograms obtained using the fabricated column(2.2 cm) and LIF detection were successfully demonstrated.
Ceramic Microfluidic Microsystems for High Temperature and High Pressure Applications
Kamlesh D. Patel, Kenneth A. Peterson, Kyle W. Hukari
Sandia National Laboratories
As an alternative material to glass, silicon, and plastics, Low Temperature Cofired Ceramics (LTCC) substrate technology is becoming increasingly important for enabling microfluidic microsystems. LTCC simple fabrication method and unique properties to withstand high temperatures and high pressures make it well-suited for applications not possible with traditional materials. We will present the latest results on the development of two microdevices that take advantage of the inherent properties of LTCC: (1) a high temperature, flow-through thermal lyser for solubilizing bacterial spores, and (2) a high-pressure microfluidic device for microHPLC. LTCC is well known in the microelectronics circuit packaging industry and is widely used in radio frequency applications. LTCC can be commercially purchased as thin, flexible tapes consisting of glass/alumina nanoparticles held together with an organic binder. Four simple fabrication steps are required to create LTCC microdevices. The green flexible LTCC tape can be punched, stamped, or laser cut to form the desired microstructure. Multiple layers are stacked and laminated together to form an enclosed three-dimensional structure. Sintering at 850°C causes the organic binder to burn-out and the ceramic particles to fuse together. The result is a solid ceramic device. As part of Sandia’s initiative to develop an automated sample prep system for the µChemlab™ bioagent detector, an integrated microfluidic lyser using LTCC technology has been fabricated, which enables the use of aqueous buffers at high temperatures without boiling by using a pressurized system. Thermal lysing of bacterial spores in a flow-through microfluidic device at high temperatures (~185°C) and pressures (500 psi or 35 bar) establishes a new method for solubilizing spore proteins for identification and analysis, eliminating the reliance on harsh and time-consuming chemical reducing agents for lysing. We will report on the performance and implementation of this LTCC lyser configuration for solubilizing proteins of biological agents compared with standard benchtop protocols. In addition, we will also report on the development of a LTCC microdevice capable of achieving very high pressures (>5,000 psi or 345 bar) for enabling microHPLC. Ultimate pressure limits, flowrate sensor measurements, and chromatography results will be presented for a microHPLC LTCC manifold using an integrated flow sensor coupled with electrokinetic (EK) micropumps for direct gradient solvent delivery at high pressure.
A Novel Capillary Electrophoresis Microchip with an Integrated Fluorescence Detection System.
J. Vieillard1, R. Mazurczyk1, A. Bouchard2, B. Hannes1, S. Krawczyk1
1 Laboratoire d''électronique optoélectonique et Microsystčme (LEOM), Ecole centrale de LYON, Ecully, France. 2 Institut de microelectronique, electromagnétisme et photonique (IMEP), Grenoble, France
The lab-on-a-chip (LOC) concept of integrated analytical microsystems, has developed rapidly in recent years, particularly in the domain of biomedical applications . One of the major trends in the design and fabrication of the LOC devices is the integration of microfluidic separation part of the device with its detection optics. A variety of concepts has been reported in this field of research . In our paper, we present a novel approach to this problem. Both microfluidic and detection optics components of our device were fabricated in the common glass substrate. As a result, we obtained a truly integrated, monolithic LOC microsystem. Potentially, our devices may be developed to the high level of complexity (complicated microfluidic structure, various optical microcomponents), as only the standard methods of microfabrication were utilised in our technology and no hybrid solutions - like introducing optical fibres into the microfluidic system - were necessary. In the paper we describe the fabrication technology of our devices, their performance and preliminary results of the separation of a model mixture of dye and protein in the lab-on-a-chip microsystems. In addition, we demonstrate also the system sensitivity and the dependence of the fluorescence intensity on model dye concentration in different optical configurations. In order to test the feasibility of our concept in the microfluidic systems, the experiments on electrophoretic separations were carried out. The sample manipulation was based on electrokinetic phenomena. The appropriate sequences of electric potentials were applied in the injection step by electrokinetic focusing . In this way, we defined the size of the sample plug. After the electric potential arrangement had been switched the sample is injected into the separation channel, separated in its components, and all of them were detected on line at the detection point. To limit leakage during injection of the sample we used modified injection procedure. The distance from the intersection of the channels to the detection point and electric field strength amounted for 5 cm and 230 V/cm respectively. In the figure, we present the separation of the mixture of 10 µM Cy3 and 1 µM of Cy3-streptavidine. We compared efficiency of separation on 10 injection/separation cycles between different chips, but also from day to day. The development of the demonstrated concept, along with the experiments on electrophoretic separations of several proteins mixture, is the subject of our current studies. References:  K.Huikko, R.Kostiainen, T.Kotiaho, Eur.J.Pharm.Sci., 20 (2003) 149-171  K.B.Mogensen, H.Klank, J.P.Kutter, Electrophoresis, 25 (2004) 3498-3512.  C.H.Lin, R.J.Yang, C.H.Tai, C.Y.Lee, L.M.Fu, J.Micromech.Microeng., 14 (2004) 639-646.
Session 6B, PHARMACEUTICAL ANALYSIS
Development of Robust and reliable CE Methods for Pharmaceutical Quality Control and Stability Evaluations
M. Ilias Jimidar, Willy Van Ael, Patrick Van Nyen, Maurits De Smet
Johnson & Johnson Pharmaceutical Research & Development, A division of Janssen Pharmaceutica n.v., Global Analytical Development, Beerse - Belgium
Ever since the introduction of Capillary Electrophoresis (CE) in the late eighties of the last century, the technique has been applied widely in different application areas. The separation power and efficiency of the methods do not need to be emphasised here. Indeed, many scientific papers, reviews and text books are available nowadays in the literature. The technique is supposed to be easy, versatile, high performance, low cost, allows rapid analysis and fast method development. These are all features that should be embraced by the industry, especially by the pharmaceutical establishment where a large number of samples has to analyzed, rapidly, at low costs and with consistency in performance. At Johnson & Johnson Pharmaceutical Research and Development (J&JPRD), a division of Janssen Pharmaceutica n.v., Capillary Electrophoresis (CE) is applied as the first choice analysis technique for enantiomeric separations. By adding a chiral selector into the buffer electrolyte enatiomeric separations may easily be achieved. The chiral selector will generate a complex with both enantiomers. If one enantiomer-chiral selector-complex is more stable than the other, a different mobility is achieved, resulting in separation of the enantiomers. Finding the appropriate chiral selector is usually done by a “trial and error” process. It is impossible to predict the selectivity of a selector to a certain enantiomer. Therefore, the affinity of all selectors has to be examined one by one. In order to speed up this process a strategy is proposed. The approach includes first a screening in function of the pH to determine the optimal migration conditions followed by a selection of the right chiral selector by means of experimental designs. Potentially successful selectors are being examined in a minimum of experiments. In the approach several variables such as the type of cyclodextrin, concentration of cyclodextrin, concentration of buffer electrolyte, and percentage of organic modifier are varied simultaneously to find initial separation conditions rapidly. The resulting initial separation conditions can be optimized in further steps to be more reproducible. Many methods have been developed and successfully applied routinely in both the R&D- and Operational environments for QC- and SM activities. Moreover, CE methods are transferred successfully worldwide and have been subjected to FDA auditing during pre-approval inspections (REMINYL®). The technique is considered to be the first choice for enantiomeric methods. In this presentation the crucial factors for success in making CE a routine QC technique are discussed. Strategies how to achieve robustness will be reviewed.
Why not using CE in Pharmaceutical Analysis?
Ulrike Holzgrabe, Frank Wienen, Christine Weber
Institute of Pharmacy, University of Würzburg, Würzburg, Germany
Capillary electrophoresis (CE) and related techniques are well established methods in many analytic fields, especially in the case of the separation of biomolecules, e.g. of DNA. Even though CE can be a rather good alternative to high performance liquid chromatography (HPLC) for the evaluation of drug quality it is rarely applied. This is due to the reservation of national licensing authorities and pharmacopoeia commissions for some reason such as lack of reproducibility and sensitivity. However, the aforementioned drawbacks are often no longer true. Reproducibility can be enhanced by applying efficient rinsing procedures between each run. The problem of the lack of sensitivity can be often overcome by pre-concentration techniques such as isotachophoresis and different stacking procedures. Beside the chiral analysis, e.g. the determination of the enantiomeric excess, CE has been often proved to be superior to HPLC. This can be demonstrated by the impurity evaluation of some representative examples, e.g. glutathione, gentamicin and bacitracin. Due to much better selectivity of the CE methods the quantification of the impurities is more reliable. In addition, the CE methods are more robust. Thus, CE should be more often considered in drug quality control in both industries and the pharmacopoeias.
Efficient Characterization of Biopharmaceuticals under Non-denaturing Conditions using Robust CE Methodologies
G.W. Somsen, J.R. Catai, J. Sastre Torańo, G.J. de Jong
Utrecht University, Utrecht, The Netherlands
The production and impact of novel pharmaceutical proteins targeting life-threatening diseases has expanded enormously. The characterization of these biopharmaceuticals is mandatory, but also very challenging as protein pharmaceuticals and their formulations represent samples of high complexity. Biopharmaceuticals often are heterogeneous comprising different iso/glycoforms. Proteins also are relatively unstable species and closely-related degradation products may be formed during manufacturing and storage. Furthermore, both the production and formulation process can give rise to various impurities and/or interfering compounds. Liquid chromatographic (LC) techniques often lack high performance for intact proteins and, therefore, may be less suited for the characterization of biopharmaceuticals. Capillary electrophoresis (CE) is an attractive tool for purity and stability analysis of proteins offering efficient and fast separations and requiring only small sample amounts. Changes in protein charge and shape - e.g. as a result of chemical degradation or unfolding - are reflected in the electrophoretic mobility and can thus be monitored. Moreover, in contrast to most LC methods, CE can be conducted under mild, ‘biocompatible’ conditions allowing analysis of proteins in their native, non-denatured state. However, without taking proper precautions, the CE performance may be compromised by adsorption of proteins to the inner capillary wall causing band broadening, unstable electroosmotic flow (EOF) and poor migration-time reproducibilities. This presentation outlines a robust CE methodology for the characterization of pharmaceutical proteins in non-denaturing buffers. It will be shown that physical adsorption of a bilayer of charged polymers provides a fast and straightforward means to produce effective capillary coatings. The bilayer coating ascertains a strong and particularly constant EOF, and minimizes protein-wall interactions yielding high separation efficiencies. The resulting CE system is very stable and not affected by alkaline rinses or protein samples containing large amounts of serum albumin. The capillary coating also shows full compatibility with MS detection. Overall, the system exhibits good potential for the reproducible and quantitative profiling of complex protein mixtures. The applicability of the developed CE and CE-MS methods will be demonstrated by the stability monitoring of biopharmaceuticals such as human growth hormone (hGH), interferon-alfa and insulin. The usefulness of the CE system will be further illustrated by the analysis of several expired hGH products of commercial sources revealing large numbers of degradation products and showing superior performance over standard LC and CE methodologies for hGH.
"Chiral Separations by CE in the Pharmaceutical Industry - Lessons Learned"
F. Stapf, S. Kiessig, F. Kálmán
University of Basel, Basel, Switzerland
The presentation will summarize our experience in the development of a robust chiral separation system for the analysis of several chiral model substances. These substances are obtained in the homogeneous chiral hydrogenation of achiral starting materials. These hydrogenations are used as a quality and performance test of specific sterically and electronically tuned ligand/catalysts. The enantiomeric excess (ee) of the chiral model substances has to be determined after hydrogenation of the respective achiral starting material. From the analytical point of view, this characterization task is quite challenging, since for an efficient ligand-catalyst development the characterization of many different racemates/products (showing a large variety in their basic chemical structure as well as in the attached functional groups) has to done in a short period of time. Analysis should be performed directly from the reaction mixture (slurry) without tedious sample preparation, meaning varying sample matrices as well as sample concentrations. High precision and sensitivity (0.5% LOQ of one enantiomer in the presence of the other) are required. During method development several chiral selectors and buffers were tested. Based on their electrophoretic mobility differences, the simultaneous analysis of mixtures of up to 4 racemates in one CE-run was developed using highly sulfated cyclodextrins. Depending on the sample solvent as methanol, ethanol, acetonitrile or dimetylsulfoxide the migration of the well separated racemates in double peaks was observed. The dependence of this phenomenon the on experimental settings as temperature, sample concentration, solvent, coating and injection plug length was investigated.
Enantiomeric Separation of Acidic Compounds in Nonaqueous Capillary Electrophoresis using Single-isomer Amino Cyclodextrin Derivatives as Chiral Selectors
J. Crommen, A.C. Servais, I. Fradi, P. Chiap, M. Fillet
University of Liege, Liege, Belgium
The usefulness of nonaqueous capillary electrophoresis (NACE) for the enantiomeric resolution of chiral pharmaceutical compounds has been demonstrated. Like in aqueous media, cyclodextrins (CDs) and their derivatives have been the most widely used chiral selectors in NACE. Charged CD derivatives were found to be particularly interesting for the enantioseparation of ionizable compounds in methanolic background electrolytes (BGEs). Some single-isomer sulfated beta-CDs were successfully applied as chiral additives to the enantioresolution of various kinds of basic drugs in methanolic BGEs acidified with formic acid. Recently acidic compounds in anionic form could also be enantioseparated in such NACE systems, but only in the presence of a cationic CD derivative. This clearly confirms that electrostatic interactions between the drug enantiomers and the CD are particularly important for chiral recognition in these systems. Single-isomer amino beta-CD derivatives were used as chiral additives in a methanolic BGE containing ammonium acetate for the enantioresolution of a series of acidic drugs, most of them belonging to the class of profens. A D-optimal design with 20 experimental points was applied to the optimization of the BGE composition. Both the nature and concentration of the cationic CD were found to have a significant influence on enantiomeric resolution for all acidic compounds studied. An interaction effect was also observed for most compounds between the ammonium acetate and the CD concentrations. Generic NACE conditions could be deduced in order to obtain high resolution values in relatively short analysis times for the enantiomers of these acidic compounds, depending on their relative affinity to the cationic CD.
Poster Session 2
Session 7A, NOVEL INSTRUMENTAL TECHNIQUES
Instrumentation for Detecting Single Copies of Human Papilloma Virus for Screening Cervical Cancer
Edward S. Yeung, Ji-Young Lee, Hung-Wing Li, Becky Li
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011
The electrophoretic mobility or the fluorescence spectrum of individual DNA molecules were determined at the rate of 2500 every 25 ms by using fluorescence imaging. The signal-to-noise ratio (S/N) did not decrease in the presence of up to 8% plasma or 8% raw blood. Single-molecule detection was still possible in the presence of 50% raw blood. Single-molecule CE of two differently labeled molecules was carried out in the presence of a transmission grating. Even when the mobility difference is not sufficient because of low S/N, identification using different fluorescence wavelengths can be performed at > 99% accuracy. So, when small differences in DNA sequence due to disease or mutation can lead to hybridization to labels with different dyes, the screening of the mutated DNA will be facilitated by on-line spectroscopy in addition to the electrophoretic information from CE. Examples of detection of single copies of mRNA in blood and of human papilloma virus in pap smear will be presented.
Improvement of Detectability in Capillary and Microchip Electrophoresis using Micro/Nano Particles and Thermal Lens Microscopy
K. Otsuka, Y. Okamoto, T. Tsuneka, Y. Akimoto, K. Sueyoshi, F. Kitagawa
Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan
To improve the detectability or to make highly sensitive detection scheme possible in capillary electrophoresis (CE) and microchip electrophoresis (MCE), the use of gold nanoparticles (GNPs) with thermal lens microscope (TLM) detection was investigated. TLM has been introduced mainly into MCE as an ultra high-sensitive detection scheme, where even nonfluorescent molecules can be detected due to the heat distribution resulting from the absorption of the excitation light. The possibility of the enhancement of the detectability in MCE by TLM was evaluated under a micellar electrokinetic chromatography (MEKC) mode using a cycloolefin polymer microchip. By applying sweeping as an on-line sample concentration method, the detection limit (LOD) with S/N=3 for a dye compound was found to be 4 nM (injected amount of 8 amol) in microchip MEKC. The application of TLM to CE was also investigated by using a GNP as a specific supporting material providing a label-free detection of several samples. The surface plasmon resonance (SPR) absorption of GNPs exhibits a sensitive response toward environmental changes, and thus the sensitive detection of non-absorbing species is expected. By using a background solution containing GNPs in CE-TLM, a successful label-free detection of several amino acids, which exhibit no absorption in visible region, was achieved. The plot of the peak area of glutamic acid against its concentration gave a good linear relationship. (This work has been supported in part by the Kyoto City Collaboration of Regional Entities for the Advancement of Technological Excellence “Development of Core Technology Forming Nano-medicine COE”, Japan Science and Technology Agency.)
Electro Membrane Isolation - a New Concept for Separation of Chemical and Biochemical Substances from Biological Samples
Stig Pedersen-Bjergaard, Knut Einar Rasmussen
School of Pharmacy University of Oslo Norway
In this paper, we show for the first time electrokinetic migration in a three-phase system where chemical and biochemical substances migrates from an aqueous sample, across a thin artificial liquid membrane (≈ 200 µm) immobilized in the pores of a porous hollow fiber, and into an aqueous acceptor solution inside the lumen of the hollow fiber. Also, we demonstrate that this new concept, which is called electro membrane isolation (EMI), may be a very fast and selective approach to isolation and pre-concentration of charged analytes from complicated biological samples like blood and urine. With several basic drug substances as model compounds, transport across a 2-nitrophenyl octyl ether membrane by the application of 10-300 V DC will be shown. The samples were 300 µl blood or urine with pH adjusted to 2.0, whereas the acceptor solution was 30 µl of 10 mM HCl. The transport is forced by the electrical potential difference sustained over the liquid membrane, resulting in electrokinetic migration in the 3-phase system. Within 5 minutes of operation at 300 V, the basic drugs were isolated with recoveries in the range 71-86 %. EMI has the potential to be an important tool for future isolation within chemical and biochemical analysis.
Microfluidic Tools for Autoimmune Target Discovery and Analysis
Anna Tüdős1, L.H.H. Silvertand2, W.P. van Bennekom2, G.J. de Jong2, K.K. Unger2, D. Kohlheyer1, G.A.J. Besselink1, S. Schlautmann1, R.B.M. Schasfoort1
1 MESA+ Institute for Nanotechnology, Biochip Group, University of Twente,P.O. Box 217, 7500 AE Enschede, The Netherlands 2 Pharmaceutical Analysis, University of Utrecht, P.O. Box 80082, 3568 TB Utrecht, The Netherlands
The development of integrated microfluidic tools will enable high throughput, sensitive and selective target discovery and analysis. The primary goal of this project is to develop and test an integrated “proteomics on a chip” device combining on-chip separations and surface plasmon resonance imaging (i-SPR) detection. Low sample capacity is a common problem in miniaturized separation systems such as microchip-based capillary electrophoresis (CE), yielding discrete plugs of the separated compounds on the nanoliter range. The small volumes, together with the often low concentrations of relevant bioactive compounds put high demands on the detection system. Free flow electrophoresis (FFE) is a semi-continuous separation method, providing bands and thus virtually a continuous supply of the separated components. In this presentation the results of isotachophoresis (ITP) and isoelectric focusing sample clean-up as well as free flow electrophoresis (FFE) separation will be shown. Experiments with Chemchip (Merck, Darmstadt) were carried out to optimize sample preparation on-a-chip. Since the ITP Chemchip is a powerful desalting and albumin depletion device, it offers unique sample clean-up possibilities when coupled to a lab-on-a-chip separation system. Preliminary results demonstrating the potential for desalting and protein separation will be shown. In this presentation an improved µ-FFE device is introduced consisting of two thermally bonded glass plates with a microfabricated separation chamber and integrated photopatterned ion-permeable salt bridges to separate the electrode compartments. The salt-bridges act as physical barriers towards the pressure driven flow but allow ions to pass to ensure electrical connection. In the µ-FFE device the electric field is perpendicular to the flow direction in order to obtain continuous lines of separated proteins. The device is used for highly efficient free-flow zone electrophoresis and isoelectric focusing separations and is also equipped with channels for hydrodynamic focusing, allowing control over position and width of the sample stream sandwiched between two buffer sheath flow streams. Using hydrodynamic focusing, bands of separated components can be guided to the desired outlet by adjusting the volumetric flow ratio of the sheath flow streams. The separated proteins at the outlet of the chip enter a so called “address flow” affinity area where biomolecular interactions are studied using a microarray. The bottom plate of the chamber contains the microarray equipped with entities (e.g. antibodies) for specific biomolecular interactions for imaging with surface plasmon resonance (iSPR). Separated proteins directed over the desired sample lane bind at their specific binding area. In this presentation new results will be shown on the FFE separation and the connection with imaging SPR.
Selective Quantitative Determination of Proteins in Biological Fluids by On-line Immunoaffinity Chromatography - Protein Digestion - LC-MS
Johannes S. Hoos, Wilfried M. A. Niessen, Henk Lingeman, Hubertus Irth
Vrije Universiteit Amsterdam
Quantitative bioanalysis of protein drugs in biological matrixes is frequently performed using enzyme immunoassays in combination with simple spectrometric detection methods. However, these methods do not provide additional information on the target measured, and as a result strongly rely on the selectivity of the immunoassay. In this work, a quantitative method for the determination of proteins in complex biological matrixes has been developed based on the selectivity of antibodies for sample purification, followed by proteolytic digestion and on-line desalination and separation of the resulting peptide mix coupled to mass spectrometry. An immunosorbent of polyclonal anti-Bovine Serum Albumin (BSA) antibodies, immobilized on CNBR agarose, is used on-line for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture is performed using a desalination procedure and a separation step coupled to an ion-trap mass spectrometer. This approach enhances substantially the selectivity compared to common analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and liquid chromatography-mass spectrometry is performed and an on-line quantification of the model protein BSA in different biological matrixes like human urine, human plasma and bovine urine was established. A detection limit of 170 nM and a quantification limit of 280 nM is obtained using 50 µL of sample. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures
Session 7B, CLINICAL DIAGNOSTICS
Capillary Electrophoresis Analysis of Carbohydrate-deficient Transferrin in Human Serum, a Marker for Chronic Alcohol Abuse
W. Thormann, C. Lanz
University of Bern, Bern, Switzerland
During the past decade, the use of capillary electrophoresis in clinical and forensic analysis has been actively explored and successfully introduced to the routine arena. Area of applications include i) analysis of drug seizures, ii) monitoring of drugs in body fluids, iii) screening for serum proteins, iv) analysis of specific blood proteins, such as transferrins and hemoglobins, and iv) DNA fingerprinting and mutation analysis. In this lecture, capillary electrophoresis with a dynamic double coating formed by charged polymeric reagents is shown to represent a very effective tool for the separation of iron-saturated Tf isoforms and thus the determination of carbohydrate-deficient transferrin (CDT), a marker for chronic alcohol abuse, in human serum [1-4]. CDT encompasses isoforms of the glycoprotein transferrin (Tf) with zero up to two sialic acid residues in the carbohydrate side chains of the molecule and is determined in relation to total Tf. The high-resolution CE assay used in our laboratory is demonstrated to provide reliable data for patient screening, long-term monitoring of individuals and confirmation analysis. It features simpler sample preparation, faster analysis time and higher isoform resolution compared to the most recent HPLC approach and is thus regarded as a candidate of a reference method for CDT.  Lanz, C., Kuhn, M., Bortolotti, F., Tagliaro, F., Thormann, W., J. Chromatogr. A 2002, 979, 43-57.  Legros, F.J., Nuyens, V., Minet, E., Emonts, P., Zouaoui Boudjeltia, K., Courbe, A., Ruelle, J.-L., Colicis, J., de L’Escaille, F., Henry, J.-P., Clin. Chem. 2002, 48, 2177-2186.  Legros, F.J., Nuyens, V., Baudoux, M., Zouaoui Boudjeltia, K., Ruelle, J.-L., Colicis, J., Cantraine, F., Henry, J.-P., Clin. Chem. 2003, 49, 440-449.  Lanz, C., Kuhn, M., Deiss, V., Thormann, W., Electrophoresis 2004, 25, 2309-2318.
Absolute Quantitation of Biomarker Proteins in Serum Using HPLC x HPLC-MS and Bioinformatic Tools
C. G. Huber1, N.Delmotte1, B. M. Mayr1, O.Kohlbacher2, K. Reinert3, C. Gröpl3, C. Klein
(1)Saarland University,Saarbrücken, Germany (2)University Tübingen, Germany (3)Free University of Berlin, Germany (4)European Commission, Ispra, Italy
In recent years, there has been growing interest in applying proteomics to clinical diagnostics and predictive medicine. Protein biomarkers can assist in the diagnosis of diseases in order to reduce the time and cost of clinical and medical treatment. The reliable absolute quantitation of proteins in serum or plasma as matrix still represents one of the most difficult analytical challenges. The difficulties arise from the presence of a few, but highly abundant proteins in serum, which have to be removed before quantification of medium to low-abundant proteins in serum can be performed, and from the non-availability of isotope-labeled proteins, which serve to calibrate the method and to account for losses during sample preparation. This study shows that first-dimension separation at the intact protein level, using either affinity chromatograph or ion-exchange chromatography, followed by digestion and second-dimension separation at the tryptic peptide level represents a suitable generic approach to reduce sample complexity before quantitation of selected proteins in serum by liquid chromatography-mass spectrometry. In order to measure myoglobin, a marker for myocardial infarction, directly in human serum, high-abundant serum proteins are first depleted by strong anion-exchange chromatography. The myoglobin fraction is digested and injected onto a 60 x 0.2 mm i.d. monolithic capillary column for quantitation of selected peptides upon mass spectrometric detection. The addition of known amounts of myoglobin to the serum sample is utilized for calibration and horse myoglobin is added as an internal standard to improve reproducibility. Calibration graphs are linear and facilitate the reproducible and accurate determination of the myoglobin amount present in serum. Manual data evaluation using integrated peak areas and an automated multi-stage algorithm fitting two-dimensional models of peptide elution profiles and isotope patterns to the mass spectrometric raw data are implemented and compared. Applying the automated method, a myoglobin concentration of 460 pg/µl serum was determined with a maximum relative deviation from the theoretical value of 10.1% and a maximum relative standard deviation of 13.4%.
Fast Mutation Detection by Microchip Electrophoresis Tandem SSCP/Heteroduplex Analysis (HA): 98% Sensitivity and Specificity in a Blinded Study of Over 100 Samples
C.N. Hestekin, D.Y. Kim, L. Senderowicz, A.E. Barron
Northwestern University, Evanston, IL
High-throughput genetic mutation detection technologies promise to revolutionize the diagnosis and treatment of cancer by enabling the correlation of prognosis with specific sequence alterations. We are developing a novel technological approach to rapidly and accurately screening for cancer-related mutations, based on microchip electrophoresis (ME). Mutations in the p53 gene, in particular, are known to be important in the pathogenesis of a variety of human cancers. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two excellent and complementary electrophoretic “mobility shift”-based methods for genetic mutation detection because of their simplicity, breadth of application, and low cost. To develop a clinically feasible mutation detection system, we have been working to optimize tandem-SSCP/HA for implementation on an ME platform by investigating the importance of variables such as polymer matrix and wall coating properties, applied electric field strength, and DNA sample concentration and purity. To pilot this technology on a relatively large and complex sample set, we performed a blinded study of over 100 genetic samples from exons 5-9 of the p53 gene. Results show that for this sample set, which derives from p53 exon 5-9, our novel microchip electrophoresis based-SSCP/HA screening technology provides an overall sensitivity and specificity of mutation detection of 98%, exceeding for example the performance of DNA hybridization chips and automated DNA sequencing. This high sensitivity and specificity as well as the relatively rapid analysis time (< 10 min) for the detection of single-base mutations in the p53 gene exons 5-9 demonstrates the powerful clinical potential of ME-SSCP/HA. This research is now being extended to a large set of node-negative breast cancer tumor samples, as we work to create a clinically applicable system for rapid and low-cost mutation screening.
Multi-Capillary Electrophoresis Analysis of Single Nucleotide Polymorphisms in the Deoxycytidine Kinase Gene
E. Szantai, Z. Ronai, M.Sasvari-Szekely, A. Guttman
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University
Investigation of the genetic background of complex traits is in the focus of recent interest, as several common diseases or the individual response to treatments of various illnesses have not yet been explored. These studies require the development and implementation of reliable and large scale genotyping methods. In this paper we introduce an efficient PCR-RFLP based technique for the analysis of the -360CG and -201CT single nucleotide polymorphisms (SNP) of the deoxycytidine kinase (dCK) gene. A high throughput multi-capillary gel electrophoresis instrument was used for the size determination of the DNA amplicons of interest and it was demonstrated that this 12-capillary-system can be applied for reliable genotyping at a 20 seconds / sample analysis rate. A healthy population of 100 individuals of Hungarian origin was investigated to determine the allele- and genotype frequencies for the two polymorphisms. Our technique can readily facilitate the analysis of these important SNP’s in other ethnic groups to clarify the role of these sequence variations in conjunction with Arabinosyl-Cytosin treatment in acute myeloid leukemia.
Rapid Screening of Drug-Protein Binding using a Single Run Measurement by High Performance Frontal Analysis - Capillary Electrophoresis and Mass Spectrometry
Hong Wan, Ĺsa Östlund, Stefan Jönsson, Walter Lindberg
DMPK and Bioanalytical Chemistry AstraZeneca R&D Mölndal SE-431 83 Mölndal SWEDEN
A novel method - single run measurement of drug-protein binding is developed. The method is based on high performance frontal analysis and capillary electrophoresis (HPFA/CE) but uses a single run measurement to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer (ESI-ion trap, Agilent technologies) is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to specific protein or drug-plasma protein binding. As the sample volumes of plasma required is typically one or two orders of magnitude lower than the conventional protein binding methods such as dialysis and ultrafiltration method, the proposed new method is particularly suitable for small volumes of plasma (e.g., mouse plasma). Typical applications of this new technique for drug-protein binding studies will be demonstrated.
Session 8 PROTEOMICS
The proteome that makes your heart beat
Albert J.R. Heck
Biomolecular Mass Spectrometry, Utrecht University, Utrecht, The Netherlands
Embryonic stem (ES) cells are pluripotent cells that have the capacity to form all somatic cells of the adult individual. Both mouse and human ES cells can be cultured in vitro, and their differentiation can be directed specifically towards heart muscle cells (cardiomyocytes). Since this process is poorly understood, and only a small proportion of the initial pool of ES cells reaches the cardiomyocyte stage, we set out to unravel the differentiation process of ES cells using a quantitative proteomics approach. To reach our ultimate goal of identifying cardiomyocyte-specific proteins in an early stage of differentiation, we have taken an approach comprising several steps: 1. Qualitative proteomic analysis of mouse and human ES cells by FT-MS. This has resulted in the identification of over 1800 unique proteins in each of these cell types, including well-known ES cell-specific transcription factors. In these datasets, the mass accuracy of the FT mass spectrometer ensures an extremely high confidence in protein identification with a false positive rate of <0.2%. 2. Qualitative comparison of ES cells with (randomly) differentiating cells to identify ES cell- and differentiation-specific proteins (both in mouse and human cells). 3. Quantitative analysis of ES cells differentiating towards cardiomyocytes. We have established a method to metabolically label ES cells with heavy isotope-containing amino acids, allowing us to follow protein expression levels. Our study so far makes up the largest proteomic dataset of both human and mouse ES cells known to date. Furthermore, the ability to isotopically label these cells provides a way to quantitatively determine protein expression during several stages of cell differentiation. Comparison of the results obtained from human and mouse ES cells will highlight commonalities and differences in cardiomyocyte development between these organisms. In addition, we develop and implement novel technologies to profile the phosphoproteome of the stem cells based on TiO2 affinity nanocolumns. This study was financed by the Netherlands Proteomics Centre (www.netherlandsproteomicscentre.nl).>
CE-SELEX: Isolating High Affinity Aptamers Using Capillary Electrophoresis
M.T. Bowser, S.D. Mendonsa, R.K. Mosing, E. Skowronek
University of Minnesota, Minneapolis, USA
SELEX is a process that selects DNA or RNA from a random library of sequences based on their affinity for a target molecule. These high affinity ligands (a.k.a. aptamers) have great potential for use as drugs or diagnostic agents. Traditionally, SELEX selection is performed using filter, panning or affinity chromatography separations. While relatively straightforward there are drawbacks to these approaches. None offer particularly high resolution separation of binding from non-binding sequences, making 8-12 selection rounds necessary before a significant fraction of the pool shows affinity for the target. Each of these separation techniques also exposes the library to relatively large stationary support surfaces, creating the possibility of selecting for non-specific binders with affinity for the stationary support. Recently we have developed an alternative SELEX procedure that uses capillary electrophoresis to perform selections (CE-SELEX). In this procedure a random sequence DNA library is incubated with the target in an injection vial. The mixture is injected onto the capillary and a separation voltage is applied. The size or charge of DNA with affinity for the target changes upon binding the target, inducing a mobility shift. This mobility shift allows binding and non-binding sequences to be collected into separate vials. Binding sequences are PCR amplified generating a new pool for further rounds of enrichment. We have identified highly selective aptamers with dissociation constants in the low nanomolar to high picomolar range for large protein targets such as IgE and HIV-RT. More recently we have identified aptamers with affinity for smaller targets such as neuropeptide Y. An important advantage of CE-SELEX results from the high efficiency, high selectivity separations characteristic of CE. This increased separation power increases the rate of enrichment between rounds. We have demonstrated nearly 100% enrichment of the library after as few as two rounds of selection, greatly shortening the SELEX process. Selection takes place in free solution, minimizing non-specific interactions, increasing the abundance of high affinity aptamers in the final DNA pool.
Session 9A, BIOPOLYMER ANALYSIS
A Comparison of Several New and Conventional Microchip Electrofocusing Techniques.
Cornelius F. Ivory
Washington State University
The segregation and analysis of low-abundance proteins from complex biological fluids will require the serial application of several separation techniques that can simultaneously fractionate and concentrate solutes. In general, these techniques will belong to the family of displacement methods, e.g., isotachophoresis, or gradient methods, e.g., gradient-elution HPLC. Isoelectric focusing is a member of the subset of the gradient methods refered to as equilibrium gradient methods (EGMs) and has the important property that, starting from an arbitrarily-distributed initial state, it evolves over time to a self-sharpening, stationary steady-state. Until the introduction of counteracting chromatographic electrophoresis by O’Farrell in 1985, isoelectric focusing was the only known electrokinetic technique with this property. Today the sub-family of electrokinetic equilibrium gradient methods has at least a half-dozen members and is slowly growing. This paper will describe some of the essential properties of the displacement methods, the gradient methods and the equilibrium-gradient methods showing how they can be applied in MEMS devices, how their performance can be predicted and how new members with orthogonal separation properties may be added to the EGM family.
Single MS, HPLC- and CE-MS Hyphenated Approaches towards Characterization of Glycopeptides
A. Rizzi, A. Plematl, S. Amon, T. Hrebicek, R. Ullmer, M. Lechner
Institute of Analytical Chemistry and Food Chemistry University of Vienna
Glycoproteins are known to play a very important role in a variety of biological processes and a majority of proteins occurring in serum and which can serve as disease markers are glycoproteins. Whereas “glycoproteomics” (performed in a high-throughput-mode) usually focus on the detection of glycosylation and identification of the corresponding proteins, “glyco-typing” addresses the more detailed analysis of glyco-structures attached to certain proteins particularly under consideration. Both questionings need different analytical strategies. As glyco-typing addresses the site-specific location of the glycans as well as a rough quantitative estimate of the abundances of certain glycan variations, this type of analysis is best done on the level of the glycopeptides obtained by enzymatic digestion. The paper deals with a comparison of various up-to date approaches for glycopeptide analysis and characterization, discussing strengths and weaknesses of the various procedures. It covers single mass spectrometric approaches, particularly multi-stage mass spectrometry, as well as the combinations of HPLC and CE techniques hyphenated to single- and multi-stage mass spectrometry in on-line or automated off-line mode. Affinity chromatography and capillary electrophoresis in the zone electrophoretic as well as in the isoelectric focusing mode will be used. Examples are given for which the final results obtained by different techniques are compared and the risks of generating analytical artifacts is discussed. In all instances the employment of HPLC or CE separation steps prior to mass spectrometry turned out to be essential for reaching a complete and reliable analysis of the various glycan structures.
The Potential of Capillary Electrophoresis in the Study of Amyloidoses
De Lorenzi Ersilia1, Chiara Carazzone1, Raffaella Colombo1, Stefania Sabella1, Milena Qualgia1, Vittorio Bellotti2
1 Department of Pharmaceutical Chemistry, University of Pavia, Pavia,ITALY 2 Biotechnology Laboratories,IRCCS S.Matteo Hospital; Department of Biochemistry,University of Pavia, Pavia,ITALY
Amyloidoses represent an heterogeneous category of diseases in which different proteins share the common property of misfolding and self aggregating to generate insoluble and toxic amyloid deposits, named fibrils, that are localised at the extracellular level in tissues and organs. Amyloid deposits are the basis of several conditions that have an enormous social and medical impact (Alzheimer's disease, prion associated diseases, immunoglobulin light chain amyloidosis), but also of rare diseases like those caused by genetically transmitted punctiform mutations in target genes. A detailed description of the folding process of an amyloidogenic protein, as well as of its fibrillogenic pathway, is essential for a full understanding of the underlying events leading to aggregation and for a full exploitation of our potential to design therapeutic strategies. We have focused our attention on beta2-microglobulin, a protein responsible for dialysis-related amyloidosis and on Abeta peptides, associated with the neurodegenerative changes of Alzheimer's disease. Capillary electrophoresis has proven to be an additional and complementary technique to the spectroscopic methods for the investigation of protein folding equilibria, providing a cross section of populated molecular states. It has also shown great potential in the monitoring of the nucleation steps leading to fibril deposition. The obtained separation and quantification of transient folding intermediates or of oligomers along the fibrillogenic pathway has been used as a starting point for new pharmaceutical approaches that consider such species as independent molecular targets. Off-line preincubation followed by CE analysis or affinity capillary electrophoresis have been exploited to search for drug-like molecules capable of binding the separated species and of perturbing the existing equilibrium. Suramin and nordihydroguaiaretic acid seem to disaggregate the toxic oligomeric intermediates of the Abeta 1-42 peptide. Affinity capillary electrophoresis turns out to be useful also for a medium-throughput screening and, a new ligand for beta2-microglobulin has been discovered.
Detection of Urinary and Recombinant Human Erythropoietin by Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry：Challenge and Solutions
Huwei LIU, Bing YU, Yiping LIAO, Feng LIU, Yuanzong LI
College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Erythropoietin (EPO) is produced primarily in the kidney, and plays a key role in regulating human erythropoiesis. In clinical application, various kinds of recombinant human erythropoietin (rhEPO) have been used for the treatment of some diseases. On the other hand, some athletes used rhEPO to improve their achievements in endurance competition sports, which have resulted in serious tragedies. Consequently, it is necessary and urgent to pursue an effective and prompt method for the doping control of rhEPO in sports games. In this article, the method for fast separation and detection of rhEPO and uEPO glycoforms by CE-ESI-MS was investigated, and the results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate (HAc-NH4Ac) running buffer was 4.80 and 5.50 respectively, and a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In CE-ESI-MS experiments, a baseline separation of three major rhEPO glycoforms was successfully and reproducibly achieved. Furthermore, the mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. It can be concluded that, contrasted with other indirect methods, the online CE-ESI-MS technique shows great potential for the separation and detection of rhEPO doping directly in competitive sports. However, Detection limit of MS for EPO is not low enough at the present for the direct detection of real sample, and potential solutions were discussed.
Physicochemical Characterization of Biopeptides by Capillary Electrophoresis in Background Electrolytes within a Broad pH Range (1.4-12.0)
V. Kasicka, D. Koval, V. Solinova, P. Sazelova
Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic
Capillary zone electrophoresis (CZE) has become a powerful tool not only for highly sensitive analysis of biologically active peptides but also for their physicochemical characterization . This will be demonstrated by CZE of two sets of structurally related biopeptides: insect oostatic peptides (IOPs) influencing the reproduction process of the flies, and phosphinic pseudopeptides (peptide isosteres with one peptide bond substituted by phosphinic acid moiety, -PO2H-CH2-) acting as inhibitors of Zn-metalloproteinases and aspartic proteinases. From the CZE analyses and separations of these biopeptides in the background electrolytes (BGEs) within a broad pH range, from strongly acidic, pH 1.4, via neutral, up to strongly alkaline, pH 12.0, their important physicochemical parameters, such as effective, actual and limiting mobilities, dissociation constants of ionogenic groups and Stokes radii, have been determined. In addition, the investigation of the structure-mobility relationship of homologous series of peptides allowed estimation of the shape of their molecules.
The diastereomers of a set of phosphinic pseudopeptides, derived from the structure N-Ac-L-Val-D,L-Alaψ(PO2H-CH2)-D,L-Leu-L-Xaa-NH2, where Xaa is one of twenty common proteinogenic amino acid residues, were separated by CZE in the BGEs within a broad pH range (1.4-12.0). From the measured pH dependence of their effective mobilities the dissociation constants (pKa) of their ionogenic groups (phosphinic acid, carboxyl, imidazolyl, amino) and the actual mobilities of some of their ionic forms were determined by non-linear regression analysis. Solution of problems resulting from application of the strongly acidic/alkaline BGEs in CZE (high ionic strength and high conductivity of BGE, high Joule heating and high temperature increase inside the capillary, very low electroosmotic flow (EOF)) will be shown. Experimentally determined effective mobilities were corrected to standard temperature 25°C and to constant ionic strength 25 mM, and the determination of EOF mobility was accelerated by pressure assisted measurement of the velocity of slow cationic EOF or by reversing and accelerating of EOF in anodic direction by internal coating of the fused silica capillary by the positively charged polybase (Polybrene).
Series of homologous IOPs, tetrapeptide - decapeptide, with increasing number of proline residues at the C-terminus of the peptide chain, H-Tyr-Asp-Pro-Ala-Prox-OH, x = 0-6, were separated by CZE in strongly acidic (pH 2.25-2.40) and weakly alkaline (pH 8.1) BGEs, and their effective electrophoretic mobilities, mef, were determined. Several semiempirical models of the dependence of effective mobility of peptides on their charge, q, and relative molecular mass, Mr, (mef versus q/Mrk) were tested to describe the electromigration behavior of IOPs, particularly the shape of their molecules, which is related to the value of exponent k in the above dependence.
The work was supported by the GACR, grants no. 203/04/0098, 203/05/2539, and by the Research Project Z40550506 of the ASCR.
 V. Kasicka, Electrophoresis, 2003, 24, 4013-4046.
Session 9B, BIOMARKER DISCOVERY
Biomarker Discovery: Methodological Challenges and Progress
N. Govorukhina, T. Reijmers, P. Horvatovich, A. van der Zee, R. Bischoff
University of Groningen and University Medical Centre Groningen, The Netherlands.
Most diseases manifest themselves by more or less severe changes in human physiology. This forms the basis for clinical chemistry and its value in helping to diagnose disease correctly and in following therapeutic interventions. Presently, many biochemical and cellular parameters are routinely measured in blood, plasma, serum or urine in any major hospital and the results of these measurements support decision making by clinicians. Due to new methodological advances in separation science, mass spectrometry and bioinformatics, there is a growing interest to apply these methods to the discovery of novel biomarkers or biomarker patterns. Body fluids like plasma, serum or urine are commonly used, since they are routinely sampled in the hospital. However, the analysis of body fluids with modern analytical methods presents challenges of sample preparation, separation and finally data processing and analysis. Focused on our work on serum analysis of cervical cancer patients by LC-MS, an approach will be highlighted combining Medical Sciences, Analytical Chemistry and Bioinformatics/Statistics.
Biomarker Discovery by Combining Monoclonal Antibody Technology with Multidimensional Microscale Seperations and Mass Spectrometry
Andras Guttman1, William S.Hancock2, Barry L.Karger2, Laszlo Takacs3
1 Horváth Laboratory of Bioseparation Sciences, Leopold Franzens University, Innsbruck Austria 2 Barnett Institute, Northeastern University, Boston, USA 3 Biosystems International SAS, Evry, France
Discovery and utilization of new, disease specific protein biomarkers will forward and accelerate the complex drug discovery and validation process. It also facilitates identification of the optimal patient population for important clinical trials. The two popular approaches of biomarker discovery are protein profiling by mass spectrometry and systems biology based exploration of multiple biomarkers. While these methods can generate some disease relevant candidates, the markers are generally based on abundant proteins and in most instances lack true disease specificity. Therefore, such activities are rarely translated into a clinical diagnostic assay. The bottleneck is in the validation/qualification process of the relevant candidates with sensitive, reproducible and easily applicable clinical assays. Here we describe a novel biomarker discovery strategy that combines high throughput monoclonal antibody-based global disease specific analyte screening technology, with multidimensional microscale liquid chromatography and mass spectrometry-based identification of protein biomarkers. Thus, in a single step, differential epitope profiling of the potential biomarker is obtained in terms of the immunogenic space of a given complex sample, in conjunction with protein IDs. Large-scale monoclonal antibody profiling also offers the sensitivity, efficiency and automation of ELISA assays. The monoclonal antibodies derived by this novel platform can be readily incorporated into the biomarker development process by the pharmaceutical and biotechnology industry to obtain faster product development cycles.
Implementation of Microfluidic Devices for Mass Spectrometry-based Glycomics. Applications to Carbohydrate Biomarker Discovery
Jasna Peter-Katalinic, Alina D. Zamfir
Institute for Medical Physics and Biophysics, University of Muenster, Germany
Recently, microfluidic systems in combination with mass spectrometry (MS) emerged as high throughput molecular-profiling technologies able to provide efficient platforms in searching for biomarkers. Alterations in carbohydrate abundance, structure, or functions were shown to act as useful indicators of pathological abnormalities prior to development of clinical symptoms and as such are often useful diagnostic and prognostic biomarkers. Their expression in either body fluids or different normal or afflicted tissues carry crucial histological information whose complementary determination may also serve for general therapy orientation. In this study, for the MS analysis of quantity-limited complex glycoconjugate mixtures derived from biological matrices and carbohydrate biomarker discovery, automated nanoscale liquid delivery and chip-based electrospray (ESI) interface techniques were for the first time introduced. Fully automated silicon chip-based nanoESI-MS and planar polymer microchip ESI methodologies were developed, optimized and applied for high throughput screening, sequencing and biomarker identification and characterization in complex mixtures of chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan (GAG) oligosaccharides from human decorin, O-glycosylated sialylated amino acids and peptides from urine of patients suffering from hereditary N-acetylhexosamine deficiency known as Schindler’s disease and complex mixtures of gangliosides from different histopathologically defined regions of the human brain and aggressive cerebral tumors. The structural analysis of the GAG components was greatly enhanced by the high sensitivity and mild nanoESIchip ionization conditions under which, the readily in-source desulfation of the molecule could be hindered and the multiple charging of the ions became a favorable event. Both processes have leaded to the MS detection of long fully and oversulfated GAG chains structurally characterized further by automatic tandem MS (autoMS/MS) experiments. For the glycopeptide mixture purified from urine of patients suffering from Schindler’s disease, by nanoESIchip-MS a high signal/noise ratio was obtained in only one minute of data acquisition. The sensitivity achieved in these experiments was several times higher than by conventional capillary-based nanoESI. Singly, doubly and triply charged ions, derived from tri- to octasaccharide peptides were detected and identified by autoMS/MS in data-dependent analysis mode. The high ionization efficiency of the nano and microESIchip systems, the ability to generate a sustained ESI signal and the capability of these techniques to discover new glycoforms were beneficial for identification of topo-and developmental-specific ganglioside composition in human brain and their altered expression in glioma, meningioma and hemangioma tumors. Molecular ions observed for diagnostic-marker gangliosides in the negative ion nano- and microESIchip-MS were structurally characterized by tandem MS experiments optimized in either precursor manual selection mode or automated MS/MS by CID at low energy energies. In all cases investigated, the potential of the microfluidics coupled to MS to detect, sequence and identify previously undetectable carbohydrate species of biomarker value has been demonstrated.
Serum Profiling with a Novel ''Label Free'' LC/MS Approach: The Analysis of Therapeutic Proteins and Diagnostic Markers for Lysosomal Storage Disorders
Matt Kennedy1, John Rontree1, Therese McKenna2, Hans Vissers1, Hans Aerts3
1 Waters Corporation., Almere, The Netherlands 2 Waters Corporation, Manchester, UK 3 Academic Medical Centre, Amsterdam, The Netherlands
Relative quantification of protein expression changes is important in understanding disease mechanisms and detecting protein biomarkers. Several approaches utilize stable isotope labeling of samples that enable quantitatively comparison of protein levels between samples and across conditions however unnecessary cost and complexity are inherent in such approaches. We have recently introduced an isotope label-free exact mass LC-MS strategy where quantification is achieved via normalization of the LC-MS datasets and comparison of the observed tryptic peptide intensities across samples. In addition, the multiplexed peptide (data independent) detection technique enables improved protein sequence coverage for relative quantitation and identification over traditional 'data directed' methods. Lysosomal Storage diseases are genetic defects, which are inherited, and result in an abnormal enzyme deficiency. Fabry disease is caused by deficient activity of the lysosomal enzyme alpha-galactosidase A. In affected patients progressive accumulation of the glycolipid substrate for this enzyme, occurs within vulnerable cells and tissues. Administration of recombinant a-galactosidase A has been shown to alleviate symptoms of the disease. Despite this, no specific therapeutic biomarker(s) for Fabry disease exist, and as such the therpeutic dose; and efficacy of treatment for this disorder is difficult to determine. Here we detail an investigation into the potential of this novel label free LC/MS method for analysing samples of human serum from patients under going therapeutic treatment for Gaucher and Fabry discease. In this work we have analysed serum samples from patients with both Fabry and Gaucher disease and compared them to control serum. We have investigated the affect of treatment, on global protein expression changes, and will present data from measurements made across patient sample sets.
Comprehensive Serum Proteome Profiling of BreastC Cancer with the Application of Multi-Dimensional Protein Identification Technology
Qinhua Cindy Ru1, Luwang Andy Zhu1, Jordan Silberman1, Craig D Shriver2, Michael Liebman1
1 Windber Research Institute, Windber, PA 15963 2 Walter Reed Army Medical Center, Washington, DC 20307
Proteomics has undergone dramatic improvements recently, and it is anticipated to discover the biomarkers for the early cancer diagnosis. Multi-dimensional protein identification technology (MudPIT) has been developed such that it becomes an effective alternative to the two-dimensional gel (2-D gel) based proteomic technology. Over hundreds human serum samples of breast cancer patients have been analyzed via the optimized MudPIT method. Only 10µL of serum was needed. Every sample was run three times to ensure the reproducibility and to maximize the identification. ProteomeX Workstation and Bioworks Browser 3.1 SR1 (Thermo Finnigan, San Jose, CA) were used in data collecting and searching. A semi-quantitative peptide profiling method based on peptide’s normalized relative intensity has been developed. The cluster analysis was done via SpotFireTM 8.0, and the artificial neural net work modeling was done via Clementine 8.0 (SPSS, Inc. Chicago, IL). Five hundred twenty-six proteins were identified from one hundred serum samples including 78 breast cancer specimens (23 invasive, 14 atypical, and 41 benign) and 22 normal controls, and among which twenty-four proteins was detected only in breast cancer samples. Further investigation revealed that, a subgroup containing four proteins (plasma protease C1 inhibitor precursor, baculoviral IAP repeat-containing protein 6, nesprin 1, and transthyretin precursor) has been identified in 87% of invasive samples and only in 11% benign samples. The initial protein profiling also exposed some limitations existing in the current protein database strategies. Thereafter, a semi-quantitative peptide profiling method has been developed, and a group of sixteen peptides was found from another 89 samples including seventy breast cancer sera (13 atypical, 20 invasive, and 37 benign) and 19 normal controls. Cluster analysis and neural network modeling have been done on further 120 sera samples and the models were proved to be predictive in breast cancer diagnosis.
Poster Session 3
Session 10A, YOUNG SCIENTIST SESSION
High Speed and High Sensitivity Two Dimensional Capillary Electrophoresis with Laser Induced Fluorescence of Barrett's Esophageal Tissues
J. Kraly, M. Jones, B. Reid, N. J. Dovichi
University of Washington, Seattle, Washington, USA Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Protein expression fingerprints of Barrett’s Esophageal biopsies and cultured cells are generated using a novel two dimensional capillary electrophoresis system. Proteins from cellular lysate are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), which reacts with lysine residues to produce a highly fluorescent product. Proteins are detected by laser-induced fluorescence inside a sheath flow cuvette using a fiber-coupled single photon counting module. The CE system requires only nL of sample, and has limits of detection in the zeptomole range (10-21). Protein separations are performed by capillary sieving electrophoresis (CSE) and micellar electrokinetic chromatography (MEKC). Field strengths in excess of 1000 V/cm produce CSE and MECC separation profiles in less than 3 minutes. Coupling the separation modes in two-dimensional capillary electrophoresis (2D-CE) increases the peak capacity. Proteins are separated according to their size by CSE on the first capillary. Fractions are then repeatedly transferred to the second capillary and subject to MEKC. 2D-CE analysis time has been reduced to less than 40 minutes. 2D-CE experiments are highly reproducible. Relative standard deviation in the CSE and MECC dimensions are less than 1% for the 50 most intense protein components. 2D-CE analysis provides high sensitivity detection and rapid separation of complex protein mixtures. Biopsies of esophageal epithelium are collected during endoscopic procedures and subject to 2D-CE analysis. Three tissue types (squamous, fundus, Barrett’s) are compared from each of four patients. 2D-CE protein profiles show distinct differences between tissue types, as well as similarities between the same tissue type from different patients. In a second study, biopsy tissues are treated with acidic bile salts as a model for gastrointestinal reflux. Exposure to acid produces dramatic differences is 2D-CE protein profiles. Investigation of these differences in protein expression may help in prognosis of the pre-cancerous condition Barrett’s Esophagus.
SPE-CE-MS using an In-line Valve for Sensitive Analysis of Peptides In biological Samples and Protein Digests
F. W. A. Tempels, W. J. M. Underberg, G. W. Somsen, G. J. de Jong
Department of Biomedical Analysis, Faculty of Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, NL-3508 TB, Utrecht, The Netherlands
Capillary electrophoresis (CE) enables fast and highly efficient peptide separations. However, concentration sensitivities in CE are often fairly low due to nL-injection volumes and small optical pathways if UV detection is employed. A considerable gain in sensitivity can be obtained by sample preconcentration using solid phase extraction (SPE) and/or by the use of more sensitive detection methods like mass spectrometry (MS). Recently, we developed an efficient on-line SPE-CE system using an in-line injection valve as interface . Relatively large sample volumes (100-250 µL) can be concentrated using a micro trapping column, which is desorbed with a small plug of acetonitrile towards the in-line valve interface. This interface allows efficient introduction of a part of the elution plug into the CE system. In this presentation, the combination of the microcolumn SPE-CE system with ion-trap mass spectrometry is presented. Electrospray ionization was employed using a coaxial sheath-liquid sprayer for CE-MS coupling. Formic acid (pH 2.5) was used as background electrolyte. To ensure a high and stable electro-osmotic flow and efficient peptide separations, the CE capillary was coated with a bilayer of polybrene and poly(vinylsulfonate). The SPE-CE-MS system provides high resolutions for peptides in short analysis times with good migration-time repeatabilities. For 100-µL samples of enkephalin peptides, detection limits are in the picomolar range. The applicability of the system will be demonstrated by the analysis of peptides in cerebrospinal fluid and protein digests. This system shows high potential as a flexible tool for the analysis of complex biological samples by CE.  FWA Tempels, WJM Underberg, GW Somsen, GJ de Jong, Anal. Chem. 2004, 76(15), 4432-4436.
Accurate, Repeatable, and Replaceable Constraint of Capillary Arrays Using a Micro-Fabricated Device
C.R. Forest, B. Woodruff, I.W. Hunter
Massachusetts Institute of Technology
Capillary arrays for electrophoresis instrumentation are typically purchased as a pre-packaged assembly, with 16-96 capillaries bonded onto a support bracket. To replace an inoperable, relatively inexpensive (~$5) capillary after merely 300 runs or bad fortune, one must typically replace the entire assembly (~$1K-$5.5K). We report the design, manufacture, and testing of a device which constrains one hundred capillaries and can be scaled to thousands, while permitting individual replacement and alignment. The device fundamentally consists of a sandwich of steel, silicone, and steel and contains an array of thru-holes manufactured by microelectrode discharge machining (microEDM) and laser micro-machining. A plunger is first fabricated using wire EDM, and then the hole array is die-sunk with the plunger through 5 mm thick steel plates. The hole array in the silicone layer is pre-drilled with a 75 W CO_2 laser. Clamping the sandwich compresses the silicone. Lateral deformation of the silicone, defined by the Poisson ratio, locates and seals around capillaries inserted loosely through the holes with alignment tolerances of 250 µm axial and 25 µm radial, spaced 1 mm apart. An axial load constraint limit of 3 N is achieved. These tolerances are sufficient for optical detection alignment, separation matrix injection, and operation. The design offers replaceability by unclamping the sandwich as well as sealing against fluid flowing axially. This device could contribute to consumable cost and downtime reduction for capillary array electrophoresis instrumentation.
Key to Analyte Migration and Retention in Electrochromatography
I. Nischang, K. Spannmann, U. Tallarek
Otto-von-Guericke-Universität, Magdeburg, Germany
We have investigated the fundamental retention behaviour of charged analytes in capillary electrochromatography (CEC) with silica-based particulate beds in dependence of applied field and mobile phase ionic strengths. Fixed beds of porous particles have a hierarchical structure characterized by discrete intraparticle mesoporous and interparticle macroporous spatial domains. While the macroporous domains contain quasi-electroneutral electrolyte solution, the ion-permselectivity (i.e., charge-selectivity due to electrical double layer overlap) of the mesoporous domains determines the co-ion exclusion and counter-ion enrichment at electrochemical equilibrium without superimposed electrical field. With an externally applied electrical field concentration polarization (CP) is induced in the whole material. It originates in electrical field-induced coupled mass and charge transport normal to the charge selective interfaces at the external surface of the particles. CP is characterized by the development of extended convective diffusion boundary layers around the particles. For charged analytes an important consequence of CP is related to their effective migration and retention behaviour because the local intensity of CP zones critically depends on applied field and mobile phase ionic strengths. Thus, CP affects the residence time of charged with respect to electroneutral analytes in conventional electrochromatographic media, and the retention factor becomes a complicated function of parameters that determine the local intensity of CP which we analyze in this work.
Miniaturized Fluorescence Detection Cell Based on Liquid-core Waveguides for Capillary Separation Methods
V. Kostal, M. Zeisbergerova, Z. Hrotekova, V. Kahle, K. Slais
Dept. of Analytical Chemistry,Palacky University, Olomouc, Czech Republic Dept. of Biochemistry, Masaryk University, Brno, Czech Republic Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic
The capillary separation methods comprising microcolumn liquid chromatography (µ-HPLC), zone electrophoresis (CZE), isoelectric focusing (CIEF) and electrochromatography (CEC) belong to high efficiency separation techniques today used in many scientific branches. These techniques usually operate with small sample amounts often in trace concentrations so the high sensitive detection methods are required. The technique with lowest detection limits is laser-induced fluorescence (LIF). However, classical arrangements are rather expensive, large and hard to manipulate. That is why the new, simpler configurations are developing. One of promising approaches is based on utilization of liquid core waveguide capillaries (LCWs) [1,2]. The LCW capillary is a fused silica capillary coated with special fluoropolymer (Teflon AF) with index of refraction lower than that of silica and even water. This fact in connection with the low absorbance properties of the coating and UV-VIS transparency makes it an excellent optical cladding material. A number of applications take advantage of this by using Teflon AF-coated capillaries for both separation medium and light guiding. This contribution presents the miniaturized post-column LIF detection cell for capillary separation methods based on utilization LCW capillary. Presented configuration offers flexibility, simple arrangement and easy optical alignment while maintaining reasonable sensitivity. The device also enables a high degree of miniaturization. The cell function is demonstrated on capillary electrophoresis of amino acids labeled by fluorescein based tags.  Dallas T., Dasgupta PK, Trends Anal. Chem. 23 (2004) 385-392.  Kostal V., Zeisbergerova M., Slais K, Kahle V., J. Chromatogr. A 1081 (2005) 36-41.
Implementation of Preconcentration Methods (Transient Isotachophoresis (t-ITP) or Field Enhanced Sample Injection (FESI) in Carrier Ampholytes Based Capillary Electrophoresis (CABCE).
J.M. Busnel1, S. Descroix1, V. Kašicka1, S. Terabe2, M.C. Hennion1, G. Peltre1
LECA,ESPCI,75005 Paris,France 1 Academy of Sciences of the Czech Republic, Institute of Organic Chemistry and Biochemistry,Prague,Czech Republic 2 Graduate Scool of Material Science, Kamigori,Japan
The concept of low conductivity background electrolytes (BGE) for capillary zone electrophoresis (CZE) has been introduced in 1995 by Hjerten et al. They proposed the use of different compounds as low conductivity buffers in CZE, for example: amino-acids and their derivatives, narrow pH cuts of carrier ampholytes (CAs) usually used in isoelectric focusing (IEF), or other amphoteric compounds. So far, only the first quoted have been more deeply studied. Since two years, we are focusing on the use of narrow pH cuts of CAs as low conductivity buffers in CZE. First, the narrow pH cuts of CAs have been prepared by preparative IEF fractionation of a wide pH range mixture of CAs. Then, a physico-chemical study has shown that this kind of solution presents a conductivity and a buffering capacity suitable for their use as BGE in CZE. The separative potential of the carrier ampholytes based capillary electrophoresis (CABCE) was demonstrated. Indeed, a successful separation of a protein mixture was obtained in CABCE with a better resolution than with classical buffers. Moreover the CAs low conductivity allows the achievement of rapid separations under high electric field without inducing any significant Joule heating. In this study, we focused on the preconcentration techniques which can be implemented in CABCE in order to enhance the analysis sensitivity. We first evaluated the possibilities to induce a transient isotachophoresis (t-ITP) step in CABCE. The carrier ampholytes being the sole buffering species in each narrow pH cut of CAs, the fraction pH is close to their isoelectric point. Consequently, their electrophoretic mobility is very low. We used this property to implement t-ITP in CABCE. The influence of the nature and concentration of the leading ion on the sensitivity enhancement was investigated. We showed that both, an addition of salt in the sample and the presence of an electrolyte plug containing salt allow to inject a very large sample volume (till 40 % of the effective capillary volume) without peak broadening and resolution loss. This method was successfully applied to different protein test mixtures. Then, a more complex sample was analysed, the t-ITP step followed by the CABCE one provides a sufficient sensitivity while maintaining a good resolution and permits the separation and the identification of the major proteins contained in skimmed milk. We also investigated the feasibility and the capabilities of field enhanced sample injection (FESI) in CABCE. In spite of their low conductivity, it appears that this preconcentration method can be easily implemented in the CAs based CE. The analysis of small organic compounds mixture or of peptide mixture have shown that a FESI step allows to reach high sensitivity enhancement factors (from 300 to 1500 depending on the analyte).
Enhanced DNA Separations in Physically Crosslinked Polymer Solutions with a Novel Sieving Mechanism as Visualized with Single Molecule Videomicroscopy
Thomas N. Chiesl, Karl Putz, Meena Babu, Annelise E. Barron
Northwestern University Department of Chemical and Biological Engineering
We report the development of a novel class of DNA separation media, “physically crosslinked” polymer networks, which provide substantially better peak resolution than conventional entangled polymer networks. Linear poly(acrylamide-co-dihexylacrylamide) comprising as little as 0.13% mol dihexylacrylamide (LPA-co-DHA) yields remarkably improved electrophoretic DNA separations when compared side-by-side to conventional LPA of matched molar mass. Single-molecule DNA videomicroscopic imaging reveals a novel separation modality, resembling inch-worm movement, which we can best describe as “stationary entanglement coupling.” These physically crosslinked polymer networks exhibit three distinct concentration regimes, which have dramatic consequences on the electrophoretic separation of DNA. At low concentrations, concentrations slightly higher than C* in unmodified polymers, DNA separations are faster than in LPA and have equal resolution. At moderate concentrations near CT, the concentration at which the polymer chains begin to interpenetrate and create viscoelastic solutions, the DNA separation performance of the LPA-co-DHAs is remarkably improved compared to unmodified LPA matrices. As concentration is further increased past CT , the separation performance of the modified and unmodified matrices become similar; however, improved separation is seen for DNAs with size less than 120 base pairs. Using this new type of replaceable polymer network, several hundred base pairs of DNA have been sequenced in microfluidic devices in under 10 minutes, with high peak efficiencies and excellent results compared to matched-molar mass LPA. These physically crosslinked systems have advantages over both linear polymers because of separation performance (or speed) and covalently linked crosslinked gels; the physical crosslinks can be broken (reversibly) by applied shear and loaded into microchannels.
Session 10B, PERSPECTIVES ON AND REQUIREMENTS OF MICROSCALE BIOSEPARATION INSTRUMENTATION
Macro to Nano-Multidimensional HPLC Fractionation of Intact Proteins as a Frontend to Nanoscale Microfluidic LC-MS Analyses for Proteomic Biomarker discovery and Characterization
A. Apffel1, T. Sana1, M. Tom-Moy1, R. Kincaid1, B. Curry1, A. Adler1, R. Brennen1, H-F Yin1, K. Killeen1, J. Hollenhorst1, Y.Dragan2
1Agilent Laboratories, Palo Alto, CA, United States, 2National Center for Toxicological Research, Dept. Systems Toxicology, Jefferson, AR, United States.
One of the key challenges facing proteomic analysis is coping with the huge dynamic range of analytes present in complex biological samples. In plasma, for example, protein concentrations can vary by 12 orders of magnitude. Depletion approaches can be used to remove the most abundant proteins, but there still remaines a huge concentration range of proteins of interest. This presents a two opposing requirements for an analytical protocol. On the one hand, the requirement to detect very low level proteins in complex samples may require the most sensitive detection systems to be run under there most sensitive operating modes, for example mass spectrometry with nano-electrospray ionization and nanoscale separations. On the other hand, even given high sensitivity detection systems, adequate sample volumes must be initially introduced into the system to provide sufficient analyte for detection. This in return requires relatively high sample capacity inlet systems. We have chosen to approach this problem by using an approach to initially fractionate a large volume, high concentration range proteomic sample into many discrete, simpler fractions of smaller volumes and lower concentrations at the intact protein level. Specific fractions of interest are enzymatically digested and analyzed by nanoscale LC-MS/MS. In such a “capacity funnel”, spanning macro to nano scales, at each step of separation, the concentration capacity, analytical scale and sample complexity are reduced while protein specificity, peak capacity and peak concentration are increased. In the field of Systems Toxicology, we have applied an approach based Multidimensional High Performance Liquid Chromatography (MDLC) of Intact Proteins combined with feature extraction and statistical analysis of abundance variations to identify components which are differentially expressed as a function of toxin dosage given normal between individual variations. In this approach, serum samples are initially immunodepleted to remove the 3 most abundant proteins using an Agilent MARS MS3 Column (100x 4.6mm i.d.) . The immunodepleted serum is separated by Strong Anion Exchange (SAX) chromatography (100x4.6mm i.d) , collecting 96 fractions. Each of the 96 fractions is rechromatographed on a Macroporous Reversed Phase (mRP) column (75x2.1mm i.d.) collecting 4 fractions, resulting a total of 384 fractions per sample. The resulting 96 RP chromatograms and associated UV spectra are combined and reconstructed into a multidimensional data matrix. Once a complete set of samples and controls has been run, the extracted features are processed as a set to discover statistically significant differentially expressed biomarkers. The corresponding collected fractions are characterized and validated by Microfluidic ChipHPLC-MS/MS (50x0.050mm i.d.). As an example, we have evaluated the effect of long term exposure to TCDD on rats through analysis of plasma samples.
Innovations in Liquid Chromatography in the Era of Miniaturization
Steven A. Cohen
One of the significant trends in technology for liquid chromatography has been the development of systems designed for sub-millimeter ID columns operating at nanoliter per minute flow rates. These systems play an important role in proteomics research, and despite a number of challenges in producing optimized nanoscale separation systems, such as minimizing system band spread and controlling compositional integrity reproducibly in gradient analysis, recent developments in instrumentation have shown that performance levels for nanoscale systems can be similar to those obtained for conventional HPLC instruments. Systems designed to operate at pressures up to 15,000 PSI with columns packed with particles less than 2 microns in diameter are the latest innovation in liquid chromatography. These provide increased resolution, often with faster analysis times and more sensitive detection, than traditional HPLC systems operating at 2 - 5000 PSI. Originally introduced for columns with 1 - 2 mm ID, these “ultraperformance LC” systems are now being configured for operation with nanoscale columns. New improvements in system design have focused on managing solvent delivery at nanoliter per minute flow rates, creating leak-free fluid paths that operate at elevated pressures, and improving the overall ease of use. In parallel to these developments in higher pressure analysis, several groups have been exploring HPLC systems with packed columns in microfluidic chip-based formats, in which one of the major goals is to simplify operational considerations, such as making leak tight, low band spread fluid connections. Combining high pressure operation with microfluidic technology would appear to be the ultimate solution for nanoscale chromatography, but the mating of these two concepts has yet to be achieved. In this talk I will review the current state of the art in chromatographic research in these areas, and discuss the challenges that remain to realize the lofty goals of an ideal system.
Microscale Separations of Small and Large Molecules for Real World Pharmaceutical Analysis
Nathan A. Lacher1, Yining Zhao1, Rob Dufield2, Jeffrey Schneiderheinze2, Chuck Demarest2, Anabel Fandino3, Martin Vollmer3, Georges L. Gauthier3
1 Analytical R&D, Pfizer Global Biologics, St. Louis, MO 63017 2 Analytical R&D, Pfizer, Groton, CT 06340 3 Agilent Technologies, Waldbronn DE
In the past 15 years, many research groups have been involved with the development of microfluidic chips, primarily for CE-based applications 1-3. Recently, commercial products for microchip HPLC applications have begun to appear with manuscripts demonstrating their use for different applications 4. These commercial products that have been developed result in improved separation performance as it pertains to reproducibility, theoretical plates, resolution, peak symmetry, retention time, etc. This is possible because smaller dimensions are used by fabricating the microchips with machining processes that are more reproducible, lowering system-to-system variability. Specifically, chip-based devices are produced by using microfabrication techniques (lithography, etching, ablation), which is standard in the microelectronics industry. Also, by integrating HPLC components onto a chip, fittings are subsequently eliminated which will dramatically reduce the dead volume resulting in improved separation performance. This is of great interest to the pharmaceutical industry since the quality of data obtained at this point can at least match that of conventional systems. Data will be presented that shows the performance of commercially available HPLC microchip-MS technology for the separation of pharmaceutical analytes, both small and large molecule. Progress towards the development of an HPLC microchip system utilizing UV detection will also be shown. 1 McClain, M.A.; Culbertson, C.T.; Jacobson, S.C.; Allbritton, N.L.; Sims, C.E.; Ramsey, J.M. Anal. Chem. 2003, 75, 5646-5655 2 Cheng, S.B.; Skinner, C.; Taylor, J.; Attiya, S., Lee, W.E., Picelli, G.; Harrison, D.J. Anal. Chem. 2001, 73, 1472-1479 3 Huynh, B.H.; Fogarty, B.A.; Martin, R.S.; Lunte, S.M. Anal. Chem. 2004, 76, 6440-6447 4 Yin, H.; Killeen, K.; Brennen, R.; Sobek, D.; Werlich, M.; van de Goor, T. Anal. Chem. 2005, 77, 527-533
Ionic Liquids in Capillary Elctrophoresis:Physical and Chemical Characterization and Interest for Electrokinetic Separations
Y. François, K. Zhang, M. Urbanek, D. Villemin, A. Varenne, P. Gareil
Laboratory of Electrochemistry and Analytical Chemistry, UMR CNRS
A great interest is being drawn towards ionic liquids (ILs), as alternatives for conventional molecular solvents used in organic synthesis and catalytic reactions. They supplement the family of “green solvents” including water and supercritical fluids. Among these, room temperature Ils are defined as materials containing only ionic species and having a melting point lower than 298 K. Their low vapor pressure and the versatility of their physico-chemical properties make them really attractive. Their interest in separation methods has appeared for stationary phases in gas chromatography, mobile phase additives in liquid chromatography and is recently being studied as electrolyte additives in capillary electrophoresis (CE). In this context, this work promotes the interest of CE instrumentation and related techniques for the physical and chemical characterization of ILs and conversely the interest of ILs for electrokinetic separations. The knowledge of physico-chemical properties (especially viscosity, conductivity, and absorbance) and impurity levels of ILs, has appeared mandatory for better targeting their applications and improving their performances. A new in-line process of viscosity, conductivity and absorbance measurement of pure ILs and IL containing-mixtures was developed with CE intrumentation, taking benefit from combined pressure delivery system, power supply, diode array absorbance detector, thermoregulation device, automatization and miniaturization. Furthermore, CE appeared as a well-adapted technique for the quantification of IL impurities. Concerning trace anionic impurities, the key points in the method development were the sample dilution factor, the on-line electrokinetic stacking, the addition of an internal standard and the implementation of indirect absorbance detection mode. For trace cationic impurities, a new two-dimensional single-capillary counter-flow isotachophoresis-zone electrophoresis method was developed. In a second step, the understanding of some interaction phenomena between analytes and ILs during electrophoretic separations was studied. Specific selectivities can be achieved by exploiting unique ion-ion or ion-dipôle interactions and proper selection of the cation and anion nature. These phenomena were exemplified in the case of the enantioselective separation of 2-arylpropionic acids, using chiral ILs, phenyl- and ethyl-choline of bis(trifluoromethanesulfonyl)amide. Non-aqueous media were investigated to favor ion pairing, applying a multivariate approach with IL concentration, ionic strength, acetonitrile-alcohol mixture composition and alcohol nature as factors. Antagonistic interaction phenomena were observed, due to the presence of the IL both in solution and adsorbed to the capillary wall. As the chiral ILs tested did not show any direct enantioselectivity with respect to the analytes studied, neutral cyclodextrins were added to the separation electrolyte. A decrease in resolution was generally observed, but a synergistic effect appeared in two cases. Better understanding of the results obtained was provided by the determination of the inclusion constants between the IL cation and the cyclodextrin.
Kinetic Capillary Electrophoresis (KCE): a Conceptual Platform for Kinetic Homogeneous Affinity Methods
S.N. Krylov, V. Okhonin, A. Petrov, M. Berezovski
York University, Toronto, Canada
We propose kinetic capillary electrophoresis (KCE) as a conceptual platform for the development of kinetic homogeneous affinity methods . KCE is defined as CE separation of species, which interact during electrophoresis. Depending on how the interaction is arranged, different KCE methods can be designed. All KCE methods are described by the same mathematics: the same system of partial differential equations with only initial and boundary conditions being different. Every qualitatively unique set of initial and boundary conditions defines a unique KCE method. Here, we: (i) present the theoretical bases of KCE, (ii) define four new KCE methods, (iii) and propose a multi-method KCE toolbox as an integrated kinetic tool. Using the KCE toolbox, we were able to, for the first time, observe high-affinity (specific) and low-affinity (non-specific) interactions within the same protein-ligand pair. The concept of KCE allows for the creation of an expanding toolset of powerful kinetic homogeneous affinity methods, which will find their applications in studies of biomolecular interactions, quantitative analyses, and screening of complex mixtures for affinity probes and drug candidates [1-6]. 1. Petrov, A.; Okhonin, V.; Berezovski, M.; Krylov, S.N. Proc. Natl. Acad. Sci. USA 2005, submitted. 2. Drabovich, A.; Berezovski, M.; Krylov, S.N. J. Am. Chem. Soc. 2005, 127, 11224-11225. 3. Berezovski, M.; Drabovich, A.; Krylova, S.M.; Musheev, M.; Okhonin, V.; Petrov, A.; Krylov, S.N. J. Am. Chem. Soc. 2005, 127, 3165-3171. 4. Okhonin, V.; Berezovski, M.; Krylov, S.N. J. Am. Chem. Soc. 2004, 126, 7166-7167. 5. Berezovski, M.; Krylov, S.N. J. Am. Chem. Soc. 2003, 125, 13451-13454. 6. Berezovski, M.; Krylov, S.N. J. Am. Chem. Soc. 2002, 124, 13674-13675.
Novel Strategies for the Control of Electroomotic Flow in Capillary Electrophoresis
Hervé Cottet, Grégoire Danger, Jacques Taillades
Organisation Moléculaire, Evolution et Matériaux Fluorés, UMR CNRS 5073, Equipe « Dynamique des Systčmes Biomoléculaires Complexes », Université de Montpellier 2, Place Eugčne Bataillon, 34095 Montpellier cedex 5, France
The electroomotic flow (EOF) is an important parameter for the optimization of capillary electrokinetic separations. Indeed, the electroomotic mobility is directly related to the selectivity of the separation. Moreover, EOF is much easy to implement in miniaturized devices in comparison with hydrodynamic flow. Thus, the control of the direction and the amplitude of the EOF is a main issue. Different strategies for the modification of fused silica capillary wall, using (non-covalent) physical adsorption of neutral and/or charged polymers, will be presented. Three main approaches were investigated: (i) the control of the surface charge density by adsorption of polyelectrolytes having different chemical charge rates; (ii) the control of the surface charge density by competitive adsorption of neutral and charged polymers; and (iii) the non-homogeneous modification of the capillary surface using partial filling approaches. The results obtained with the three aforementioned strategies will be discussed and compared in terms of electroosmotic flow control and stability, and in terms of separation performances on a mixture of peptides.
Session 11A, SINGLE CELL ANALYSIS
Single Cell Proteomics
Norman J. Dovichi, James Kraly, Megan Jones, Ryan Bonn, Md. Abul Fazal, Melissa Harwood
Department of Chemistry, University of Washington
We have developed two dimensional capillary electrophoresis for analysis of complex protein homogenates, and we have applied this technology to characterize the protein content of single mammalian cells. This talk will focus on the use of this technology to characterize the protein changes associated with progression of neoplasia to cancer. Our hypothesis is that the cell-to-cell variation in protein expression increases as the disease progresses. Our model system is Barrett’s esophagus, which is the only known precursor to esophageal adenocarcinoma. Patients with Barrett’s esophagus undergo surveillance endoscopy at the Fred Hutchinson Cancer Research Center, and we routinely obtain biopsies from these patients. We have demonstrated that two-dimensional capillary electrophoresis is remarkably reproducible and extremely sensitive method for the analysis of protein homogenates prepared from biopsies and single cells.
Chirality in your Brain: Detecting D-amino Acids and D-amino Acid Containing Peptides in Single Neurons
Jonathan V. Sweedler, Cory Scanlan, Liping Wang, Michael Ewing, Jane Wang, Stanislav S. Rubakhin
Departments of Chemistry and Neuroscience, The Beckman Institute, University of Illinois, USA
The amino acids and the polymers made from them in higher organisms are normally assembled from L-amino acids. However, in the nervous system of animals ranging from mollusks to mammals, D-amino acids are present. For example, D-Asp and D-Glu may be a neurotransmitter and the biosynthesis of D-asp has been observed in mammalian cells. In order to understand the functioning of D-Asp and D-Glu, we use a combination of small volume sampling and capillary electrophoresis with radionuclide detection, laser induced fluorescence and on-column absorbance detection. Using the invertebrate neuronal model system of Aplysia californica, we find that in specific identified neurons, the D-Asp levels represent up to 80% of the total Asp. In order to study the function of this molecule, we use single and subcellular characterization of the amino acids in the cell soma and neuronal processes, as well monitor its synthesis, transport and degradation. Our data supports a role of D-Asp in cell-to-cell signaling. In addition to free amino acids, signaling peptides, whether neuropeptides, trophic factors, or hormones, represent an important and functional part of the cell peptidome and have been implicated in almost all aspects of organism function, including learning and memory. One cannot understand and test the function of such molecules until they can be chemically characterized. One of the most unusual and elusive posttranslational modifications (PTMs), the switch of a single amino acid from the L-isomer to the D-configuration in a peptide, has now been documented in a variety of animals, including mollusks such as Aplysia californica. Detecting, identifying, and separating the D-amino acid-containing neuropeptides in a neuron is a challenging analytical task revolving around the difficulty in determining the stereochemical configuration of a trace amount of peptide in a complex environment. This is especially true as this is a zero Dalton mass change, making mass spectrometric approaches difficult to use without secondary techniques. Aminopeptidase N degrades the neuropeptides in the L-configuration faster than those with D-amino acids. We report a novel method of digesting tissue with the enzyme aminopeptidase N, used in a before-and-after approach with capillary scale separations and mass spectrometry, to find peptides that contain D-amino acids. We demonstrate the application of this approach to the Aplysia CNS, confirming the method with known D-amino acid-containing peptides and using the method to find new D-amino acid-containing peptides. Figures of merit of the method will be discussed, as well as reasons that several peptides are detected that contain no D-amino acids.
Single Cell Protein Electrophoresis in Microfluidic Device Format
W. Hellmich, A. Sischka, D. Anselmetti, A. Ros
Experimental Biophysics and Applied Nanosciences, Bielefeld University, Germany
Single cell analytics for proteomic analysis is a key method in the framework of nanosystems biology which allows novel proteomics without being subjected to ensemble-averaging, cell-cycle or cell-population effects. We demonstrate first results of a single cell analytical method for proteins which combines a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation and (label-free) protein detection. Our method is based on two main issues. First, single biological target cells were selected, trapped, injected, steered and deposited by means of optical tweezers in a tailored poly(dimethylsiloxane) (PDMS) microfluidic device, and consecutively lysed chemically or electrically at a predefined position. Second, separation and detection of fluorescent dyes, amino acids and proteins was achieved with confocal laser induced fluorescence detection in the visible (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the visible and protein separation and label-free detection could be achieved in the UV spectral range. Whereas the fluorescein detection sensitivity in the visible spectral range corresponds to roughly 50-100 analyte molecules, which is well below the anticipated number of low abundant proteins in a cell, the sensitivity with label-free UV-detection is currently in the nM range. The combination of single cell manipulation with microfluidic protein separation and detection allowed for analytical experiments with single Sf9 (Spodoptera frugiperda) insect cells in this tailored microfluidic device. The resulting single cell electropherograms exhibit distinct single component peaks of the GFP-construct protein proving the validity of our concept, thus allowing for novel and fascinating single cell experiments for nanosystems biology and single cell protein fingerprinting in the future.
Capillary electrophoretic analysis of individual organelles producing reactive oxygen species
E.A. Arriaga, D. Li
University of Minnesota, Minneapolis, Minnesota, USA
Our research group has previously reported the analysis of individual organelles and particles by capillary electrophoresis with laser-induced fluorescence detection. In this presentation, we extend this method to measure the production of superoxide inside individual mitochondria. Two simultaneous fluorescent measurements are carried out: (i) detection of individual organelles containing oxidized hydroethidine, a fluorogenic and membrane permeable probe for superoxide; (ii) detection of Mito Tracker Green, which selectively accumulates and labels mitochondria. The method was validated by analyzing mitochondria isolated from cells treated with antimycin A and rotenone. As expected, these electron transport chain inhibitors increase superoxide production in individual mitochondria. Surprisingly, these inhibitors also alter the electrophoretic mobilities of individual mitochondria. The origin of the electrophoretic alterations will be discussed.
The method was used to investigate one aspect of the mitochondrial theory of aging. According to this theory, the presence of mitochondrial DNA mutations causes an increase in reactive oxygen species, including superoxide. We monitored the distributions of individual mitochondrial superoxide generation in the 143B cell line and a derived cybrid cell line (DeltaH2-1) that has a long deletion in 70% of its mitochondrial DNA. This 7522 base pair deletion, affects multiple genes essential for oxidative phosphorylation. The results clearly show that the cybrid cell line has increased levels of superoxide generation and provides a direct correlation between mutational load and reactive oxygen species production.
Lastly, examples of capillary electrophoretic measurements of superoxide production in individual mitochondria released from single cells will be presented. The implications and interpretation of these studies will be used to highlight the importance enabling power of capillary electrophoresis to carry out single cell studies.
Session 11B, HYPHENATION OF MICROSCALE SEPARATION METHODS WITH MS
Microscale Bioseparations with MALDI and Electrospray Mass Spectrometry
G. Hopfgartner, E. Varesio
University of Geneva, Geneva, Switzerland
Liquid chromatography coupled to atmospheric pressure ionisation tandem mass spectrometry (LC-MS/MS) is widely applied for the analysis of pharmaceutical compounds, peptides and proteins. While narrowbore-LC remains very popular for quantitative bioanalysis, microscale separations become mandatory to achieve good sensitivities for the analysis of peptides. Since electrospray (ESI) mass spectrometry behaves like a concentration sensitive detector there is basically no limit in the diameter of the LC column and even separations on 10 um i.d. columns have been reported. MALDI mass spectrometry is an off-line technique and LC-MALDI is achieved, either by spotting LC fractions onto the target plate or by continuous liquid deposition. This setup requiring microscale separation has many advantages over on-line couplings because the mass spectrometer does not suffer anymore from the time constrains of the LC. This becomes even more evident when using fast LC separations with peak width below a few seconds and the separations can be multiplexed more easily. The decoupling of LC-MS with ESI is also of interest in particular with qualitative analysis. The LC fractions can either be stored into 96-well plates or for nanoLC separations into pipette tips. I a second step the samples are infused using chip based nanoelectrospray for a variable time period. An important difference is that with MALDI the sample is embedded into the crystals, while with chip based nanoelectrospray infusion the LC solvent can be completely removed and the sample is reconstituted in a more appropriate solvent mixture to enhance ionisation. Several applications will demonstrate the two complementary approaches for bioanalysis.
Capillary Electrophoresis-Mass Spectrometry - Instrumentation and key applications
C. Neusuess, M. Pelzing
Bruker Daltonik GmbH
Mass Spectrometry as a detector for capillary electrophoresis promises sensitive, universal, selective, and structure elucidating detection of highly efficient separated species from low sample amounts. Nevertheless, CE-MS is still judged as a difficult technique. Therefore, this combination is still not widespread, in contrast to LC-MS, which has become a routine tool in analytical laboratories. In this presentation the state-of-the-art of interfacing will be presented. Key parameter such as composition of the electrolyte (including non-aqueous CZE) or MS-compatible coatings will be discussed as well as main parameters of the sheath-liquid interface. Important applications for CZE-MS include both small molecule and protein analysis. The latter will be discussed based on recent developments for the characterization of isoforms of intact glycoproteins. CZE-separation of the glycoforms (based on the content of sialic acids as well as N-acetyl-lactosamine repeats), and high resolution of the ESI-time-of-flight-MS enables a detailed description of complex proteins like erythropoietin or fetuin. Complementary information is provided by the analysis of enzymatically released glycans by CZE-MS. The selectivity of CZE-MS in peptide analysis is especially of interest for those compounds not easy accessible by RPLC-MS and where the introduction of charge bearing groups significantly changes the separation selectivity. Here, examples of phosphorylated and glycosylated peptides will be presented. The ability of non-aqueous CZE to analyze hydrophobic peptides is illustrated by the characterization of alamethicin. The screening of bodyfluids in order to find biomarker is another interesting application of CZE-MS. The profiling of peptides will be discussed as well as the analysis of amino acids from various matrices. Examples for the structure elucidation of small molecules by MSn in an ion trap mass analyzer will be given. The separation and identification of drugs of abuse in hair samples is a nice showcase for the selectivity of non-aqueous CZE and the identification of small molecules based on the accurate mass of ESI-time-of-flight-MS.
Miniaturized, Selective Analytical Methods for the Determination of Peptides and Proteins in Biological Matrices
H. Irth, W.M.A. Niessen, H. Lingeman, J. Hoos, H. Krabbe
Vrije Universiteit Amsterdam Department of Analytical Chemistry and Applied Spectroscopy
The present lecture will focus on novel, miniaturized analytical methodologies that allow the selective detection of peptides and proteins in complex biological matrices. Two different approaches will be described: (i) on-line immunoaffinity preconcentration with on-line tryptic digest and reversed-phase LC-MS determination of digest peptides and (ii) post-column ligand-exchange mass spectrometry. The on-line immunoaffinity method is used to selectively enrich proteins from the biological matrix. Proteins enriched on the immunoaffinity column are eluted at a low pH and, after buffer adjustment, subjected to an on-line tryptic digest. Finally, peptides are reconcentrated on a reversed-phase SPE column and subsequently analyzed by LC-MS. The compatibility of immunoaffinity preconcentration with on-line tryptic digest will be discussed. In the second part, a post-column ligand exchange system coupled to electrospray mass spectrometry is described. The method is used for the selective detection of phosphorylated peptides that exhibit a strong interaction with iron(III) complex. Reporter ligands released during the ligand exchange reaction are selectively detected by MS. Simultaneously, the molecular characteristics of the phosphorylated species can be detected. The application of both methodologies in trace analysis of proteins and peptides in biological matrices will be presented.
Novel Approaches for Studying Drug-protein Interaction with CE, CE-MS and Model Calculations
F. Kilár12, M. Rezeli2, Cs. Páger1, P. Kuti2, L. Gagyi3, Á. Gyéresi3
1 Institute of Bioanalysis, Fac. Medicine, University of Pécs 2 Dept. Analytical Chem., Fac. Science, University of Pécs, Pécs, Hungary 3 Dept. of Pharmaceutical Chemistry, University of Medicine and Pharmacy, Targu-Mures/Marosvásárhely, Romania
Drug molecules interact reversibly with macromolecules (albumin, transferrin) in the serum. Several techniques are available to study this interaction in vivo and in vitro. Here we will discuss the possibility of applying capillary electrophoresis in conventional mode and also coupled to mass spectrometry for the characterization of the drug-protein complexes. Human serum trannsferrin is the primary object for the transportation of Fe3+ in the body. It has already been shown with in vitro experiments (chromatography, electrophoresis) that transferrrin separates the optical isomers of several drugs and small molecules. This protein is recognizing both, positively and negatively charged enantiomers (optical isomers) and the interaction can be easily examined by capillary electrophoresis. The racemic mixtures of the drugs are electrophoresed through a transferrin zone in a coated capillary (MES buffer, pH 6). The experiments were performed in a BioFocus 3000 CE equipment and an Agilent 3D-CE equipment coupled to an ion-trap MS (Agilent, XCT Plus). The changes in the migration properties of the drug isomers indicate the selective interaction with the surface of the protein. Special examples for the study of the interaction were chosen from beta-blockers, anti-histamines and non-steroid anti-inflammatory drugs. The parameters (resolution and pH dependence) are the most sensitive indicators of the stereoselective recognition. One of the most important questions of the drug action is how the eutomers are acting with the help of complex formation with macromolecules in the physiological fluids (serum). Therefore, we determine the separation order of the drugs, and follow the complex formation of the drugs (in vitro) by sensitive CE-MS measurements. To evaluate the CE and CE-MS results model calculations of the docking of the small molecules on the surface of the protein were performed. The Sybyl software (Tripos) provided numerous alternative conformations characterized by lipophylicity, binding energy, etc., and thus we obtained a picture of the complexes. The proper configuration that is in accordance to the real placement of the ligand and receptor was chosen by a systematic comparison of theoretical data to the real CE experiments. With the molecular modelling we tried to characterize the binding areas at the iron-binding site of iron-free transferrin. We will present cases where model-calculations are in excellent agreement with the capillary electrophoresis results. We conclude that this approach is a suitable technique for mapping interaction sites on protein surfaces.
Strategies for Conventional and Chiral CE-ESI-MS Analysis in Plasma
J. Schappler, D. Guillarme, J.-L.Veuthey, S. Rudaz
Laboratoire de Chimie Analytique Pharmaceutique, 20 Bd D’Yvoy, CH-1211 Genčve 4 School of Pharmaceutical Sciences, EPGL, UniGE, CH-1211 Genčve 4
Capillary electrophoresis (CE) is now recognized as a powerful separation technique in conventional and chiral analysis. The use of MS detection in selected ion monitoring (SIM) mode expands CE potential due to its selectivity and sensitivity. Therefore, CE-MS has evolved as an efficient technique for the analysis of drugs and metabolites in biological matrices. However, biological fluids contain several endogenous compounds which can interfere with CE separation and particularly with MS detection. Therefore sample preparation is a mandatory step to avoid such interferences to occur. In order to achieve fast and simplified sample preparation, protein precipitation (PP) is usually selected whereas liquid-liquid extraction (LLE) appears to be the most efficient extraction procedure. Furthermore, in CE two main techniques for injecting sample into the capillary could be assessed: hydrodynamic and electrokinetic injection. Generally, the former mode is preferred according to its better reliability and simplicity while the latter could provides much more sensitive determinations. MS signal suppression or enhancement effects have been widely reported in the literature when complex matrices are analysed. This undesirable phenomenon, termed matrix effect, is generally not reproducible between various sample batches or even samples and, thus, could compromises obtained results with electrospray ionisation (ESI). Matrix effect on CE-MS responses was thus investigated with a commercially available coaxial sheath-liquid ESI interface used as post-capillary infusion system to observe the MS signal alterations. Examples concerning pharmaceutical compounds of interest and amphetamines derivatives are presented in this work. The use of PP and hydrodynamic injection appeared dedicated to high concentration samples (>1 ppm) while the combination of LLE and electrokinetic injection allowed to obtain detection limit at the ppb level in conventional as well as stereoselective determination.
Poster Session 4
Session 12, FUTURE OF MICROSCALE BIOSEPARATIONS
High-speed Capillary Electrophoresis
University of Michigan
Performing electrophoresis separations in capillaries allows much higher electric fields to be applied than in conventional gel electrophoresis, which in turn allows separations to be performed much more rapidly. High resolution separations in < 1 s can be performed with fields of 3,000 V/cm and appropriate instrumentation. Such fast separations allows the possibility of electrophoresis being used for chemical sensing, high-throughput analysis (as in drug screening), and for detection of short-lived species. The advent of microfabricated electrophoresis devices has allowed such fast separations to be incorporated into more complex systems. In this talk we will describe the use of fast electrophoresis to monitor hormone secretion from live cells, neurotransmitters in vivo, and the kinetics of biochemical reactions. For the live cell studies, a microfabricated device is developed that houses a single islet of Langerhans (insulin secreting cells of the pancreas). The islet is perfused with cell culture media and continuously sampled on-chip. The insulin released is measured using an electrophoresis-based immunoassay. The method should be generally applicable to other cell types. For neurotransmitter analysis, an in vivo sampling probe is coupled to on-line to a rapid CE measurement. The high temporal resolution allows rapid changes in behavior to be correlated with neurochemical changes enabling a new generation of psychological/neuroscience experimentation. For biochemical kinetics measurements, two reactions are studied: G protein hydrolysis of GTP and binding interaction of SH2 domains. The former example utilizes fluorescent nucleotides as substrate and continuous monitoring of the reaction mixture stream by serial injections onto a fast CE system allows changes in substrate, complex, and product to be determined. Michaelis-Menton kinetics are measured. The method allows activation of the proteins to be detected leading to the possibility of using the method as a drug screen.
Reactions and Separations in Microfluidic Structures
Andreas Manz, Petra S. Dittrich, Xin Yang, Dirk Janasek, Eduardo Greaves, Gareth Jenkins, Joachim Franzke
ISAS-Institute for Analytical Sciences, Bunsen-Kirchhoff-Str.11, 44139 Dortmund, Germany
Microchip technology still offers new opportunities to explore, for example the formation of vesicle microtubes from bilayer membranes, circular liquid chromatography, free-flow isotachophoresis or spectroscopic detectors using x-ray or plasma emission.