DOCSLIB.ORG

UC San Diego UC San Diego Electronic Theses and Dissertations

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
UC San Diego UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title A whole blood protease assay Permalink https://escholarship.org/uc/item/1r6885gb Author Lefkowitz, Roy Brian Publication Date 2010 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO A Whole Blood Protease Assay A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Roy Brian Lefkowitz Committee in charge: Professor Michael J. Heller, Chair Professor Sadik C. Esener Professor David Gough Professor Geert W. Schmid-Schönbein Professor Mark A. Talamini 2010 Copyright Roy Brian Lefkowitz, 2010 All rights reserved 2 The Dissertation of Roy Brian Lefkowitz is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2010 iii DEDICATION To my mother, my late father, my brother, and my wife, Thank you very much for all of your love and support. I owe my success to all of you. iv EPIGRAPH “I've failed over and over and over again in my life and that is why I succeed.” Michael Jordan v TABLE OF CONTENTS Signature Page ................................................................................................................... iii Dedication.......................................................................................................................... iv Epigraph...............................................................................................................................v Table of Contents...............................................................................................................vi List of Figures......................................................................................................................x List of Tables .................................................................................................................... xii List of Abbreviations ....................................................................................................... xiii Acknowledgements............................................................................................................xv Vita ....................................................................................................................................xx Abstract of the Dissertation ........................................................................................... xxiii Chapter 1: Introduction to Protease Detection.....................................................................1 1.1 Motivation for Protease Detection.............................................................................1 1.2 Proteases in Physiological Shock ..............................................................................4 1.3 Proteases in Other Diseases.......................................................................................6 1.4 Previous Detection and Concentration Strategies ...................................................10 1.4.1 Detection of Activity ........................................................................................10 1.4.2 Detection of Concentration Levels ...................................................................14 1.4.3 Sample Concentration Strategies......................................................................17 1.5 The Issue with Sample Preparation .........................................................................21 1.6 A Whole Blood Protease Assay...............................................................................23 Chapter 2: Charge-Changing Substrates for Whole Blood Protease Detection ................31 2.1 Introduction .............................................................................................................31 2.2 Materials and Methods ............................................................................................32 2.2.1 Materials ...........................................................................................................32 2.2.2 Detection Limit in 1X PBS and Plasma............................................................33 vi 2.2.3 Detection Limit in Whole Blood ......................................................................34 2.2.4 Specificity .........................................................................................................35 2.2.5 Kinetics .............................................................................................................36 2.2.6 Trypsin Detection in Multiple Gel Formats......................................................36 2.3 Results and Discussion ............................................................................................37 2.3.1 Detection Limit in 1X PBS...............................................................................38 2.3.2 Detection Limit in Human Plasma....................................................................39 2.3.3 Detection Limit in Whole Blood ......................................................................40 2.3.4 Substrate Specificity and Kinetics ....................................................................41 2.3.5 Trypsin Detection in Multiple Gel Formats......................................................42 2.4 Concluding Remarks ...............................................................................................44 2.5 Acknowledgements .................................................................................................44 Chapter 3: Polyanionic Focusing Gel Electrophoresis ......................................................53 3.1 Introduction .............................................................................................................53 3.2 Materials and Methods ............................................................................................55 3.2.1 Materials ...........................................................................................................55 3.2.2 Detection in 1X PBS using Various Classical Gel Formats .............................56 3.2.3 Detection in 1X PBS using Focusing Gels .......................................................57 3.2.4 Time Course of Signal Enhancement in Focusing Gel.....................................58 3.2.5 Detection in Whole Blood and Plasma with Focusing Gels.............................59 3.2.6 Detection of Spiked Trypsin in Whole Blood ..................................................60 3.3 Results and Discussion ............................................................................................61 3.3.1 Detection in 1X PBS using Various Classical Gel Formats .............................62 3.3.2 Detection in 1X PBS using Focusing Gels .......................................................64 3.3.3 Time Course of Signal Enhancement in a Focusing Gel..................................66 3.3.4 Detection in Whole Blood and Plasma with Focusing Gels.............................67 vii 3.3.5 Detection of Spiked Trypsin in Whole Blood ..................................................69 3.4 Concluding Remarks ...............................................................................................71 3.5 Acknowledgements .................................................................................................72 Chapter 4: Substrates for Elastase, Chymotrypsin, MMP-2, and MMP-9 ........................84 4.1 Introduction .............................................................................................................84 4.2 Materials and Methods ............................................................................................86 4.2.1 Materials ...........................................................................................................86 4.2.2 Substrate Specificity .........................................................................................87 4.2.3 Detection in 1X PBS using 25% Polyacrylamide Gels ....................................88 4.2.4 Detection in 1X PBS using Focusing Gels .......................................................89 4.2.5 Detection of Spiked Protease in Whole Blood .................................................90 4.3 Results and Discussion ............................................................................................91 4.3.1 Substrate Specificity .........................................................................................92 4.3.2 Detection in 1X PBS using 25% Polyacrylamide Gels ....................................96 4.3.3 Detection in 1X PBS using Focusing Gels .......................................................97 4.3.4 Detection of Spiked Protease in Whole Blood ...............................................100 4.4 Concluding Remarks .............................................................................................103 4.5 Acknowledgements ...............................................................................................104 Chapter 5: Measurement of Protease Activity in Disease ...............................................113 5.1 Introduction ...........................................................................................................113 5.2 Materials and Methods ..........................................................................................118 5.2.1 Materials .........................................................................................................118
Load more

Recommended publications
  • Control of Molecular Motor Motility in Electrical Devices
    Control of Molecular Motor Motility in Electrical Devices Thesis submitted in accordance with the requirements of The University of Liverpool for the degree of Doctor in Philosophy By Laurence Charles Ramsey Department of Electrical Engineering & Electronics April 2014 i Abstract In the last decade there has been increased interest in the study of molecular motors. Motor proteins in particular have gained a large following due to their high efficiency of force generation and the ability to incorporate the motors into linear device designs. Much of the recent research centres on using these protein systems to transport cargo around the surface of a device. The studies carried out in this thesis aim to investigate the use of molecular motors in lab- on-a-chip devices. Two distinct motor protein systems are used to show the viability of utilising these nanoscale machines as a highly specific and controllable method of transporting molecules around the surface of a lab-on-a-chip device. Improved reaction kinetics and increased detection sensitivity are just two advantages that could be achieved if a motor protein system could be incorporated and appropriately controlled within a device such as an immunoassay or microarray technologies. The first study focuses on the motor protein system Kinesin. This highly processive motor is able to propel microtubules across a surface and has shown promise as an in vitro nanoscale transport system. A novel device design is presented where the motility of microtubules is controlled using the combination of a structured surface and a thermoresponsive polymer. Both topographic confinement of the motility and the creation of localised ‘gates’ are used to show a method for the control and guidance of microtubules.
    [Show full text]
  • Molecular Biologist's Guide to Proteomics
    Molecular Biologist's Guide to Proteomics Paul R. Graves and Timothy A. J. Haystead Microbiol. Mol. Biol. Rev. 2002, 66(1):39. DOI: 10.1128/MMBR.66.1.39-63.2002. Downloaded from Updated information and services can be found at: http://mmbr.asm.org/content/66/1/39 These include: REFERENCES This article cites 172 articles, 34 of which can be accessed free http://mmbr.asm.org/ at: http://mmbr.asm.org/content/66/1/39#ref-list-1 CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on November 20, 2014 by UNIV OF KENTUCKY Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2002, p. 39–63 Vol. 66, No. 1 1092-2172/02/$04.00ϩ0 DOI: 10.1128/MMBR.66.1.39–63.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Molecular Biologist’s Guide to Proteomics Paul R. Graves1 and Timothy A. J. Haystead1,2* Department of Pharmacology and Cancer Biology, Duke University,1 and Serenex Inc.,2 Durham, North Carolina 27710 INTRODUCTION .........................................................................................................................................................40 Definitions..................................................................................................................................................................40 Downloaded from Proteomics Origins ...................................................................................................................................................40
    [Show full text]
  • Protein Facility
    Supporting core service facilities for biotechnology research by faculty, student, government, and industry scientists. More Office of Biotechnology at www.biotech.iastate.edu/service_facilities. Protein Facility Iowa State University’s Protein purification of proteins and of protein samples from 1D or 2D Facility provides expertise for the peptides can be accomplished. gels. Gel spots can be digested analysis, characterization, and with a variety of enzymes, and synthesis of proteins and peptides. Mass Spectrometry the resulting peptides can be After training, users can operate A matrix-assisted laser desorption/ analyzed to identify the protein. many instruments themselves. ionization time-of-flight (MALDI- TOF) mass spectrometer can be used SDS-PAGE / Electroblotting for determining the molecular weight The facility conducts sodium of proteins, peptides, glycoproteins, dodecyl sulfate polyacrylamide oligosaccharides, oligonucleotides, gel electrophoresis (SDS-PAGE) of and polymers. A quadrapole- proteins for purity and molecular TOF tandem mass spectrometer weight estimation. Gels can be is also available for obtaining electroblotted to nitrocellulose peptide sequence information. or to PVDF for immuno- detection and protein/peptide Peptide Synthesis sequencing, respectively. The facility can do both large- and small-scale peptide synthesis, 2-D Gel Electrophoresis including phosphopeptides, peptides The facility does two-dimensional with unusual amino acids, and electrophoresis by separating proteins multiple antigenic peptides (MAP). in the first dimension according to charge (isoelectric focusing), Circular Dichroism Protein and Peptide Sequencing followed by separating the focused Researchers who want to detect The Protein Facility provides proteins in the second dimension and quantitate the chirality of N-terminal protein/peptide sequence according to their molecular weight.
    [Show full text]
  • Impact Factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192 REVIEW ON: ELECTROPHORESIS: METHOD for PROTEIN SEPARATION Shindedipa
    Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192 Pharma Science Monitor 7(2),Apr-Jun 2016 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com REVIEW ON: ELECTROPHORESIS: METHOD FOR PROTEIN SEPARATION ShindeDipa V.*, JasminaSurati Department of Quality Assurance, Shree NaranjibhaiLalbhai Patel College of Pharmacy,Umrakh -394 345,Bardoli, Gujarat, India. ABSTRACT Electrophoresis is one of the widely used techniques in molecular biochemistry, microbiology, biomedical research. It is a type of protein separation method .It is one of the highly efficient techniques of analysis and sole method for separation of proteins for western blot, RNA studies etc. It is a both qualitative and quantitative analysis technique. Separation depend upon electrophoretic mobility.Electrophoresis technique are of various type like Moving boundary electrophoresis ,Zone electrophoresis ,Affinity electrophoresis ,Pulsed field electrophoresis ,Dielectrophoresis.this technique mainly used in antibiotic analysis,vaccine analysis DNA analysis and protein analysis as well as fingerprint analysis. KEYWORDS:Electrophoresis, Electrophoretic mobility,Zone Electrophoresis, Moving boundary Electrophoresis, Dielectricphoresis. INTRODUCTION Electrophoresis is a physical method of analysis based on the migration of electrically charged proteins, colloids, molecules or other particles dissolved or dispersed in an electrolyte solution in the direction of the electrode bearing the opposite polarity when an electric current is passed through it. Separations may be conducted in systems without support phases (such as free solution separation in capillary electrophoresis) or in stabilising media such as thin-later plates, filins or gels. The electrophoretic mobility is the rate of movement in metres per second of the charged particles under the action of an electric field of I volt per metre and is expressed in square metres per volt second.
    [Show full text]
  • Bioanalytical Chemistry 8. Gel Electrophoresis and Blotting
    91 Bioanalytical chemistry 8. Gel electrophoresis and blotting Suggested reading: Sections 9.1, 9.2.3, 9.2.4, 9.5.1, 10.1 to 10.7, 11.1 to 11.5, and 15.5 of Mikkelsen and Cortón, Bioanalytical Chemistry Primary Source Material • Chapter 4 and 6 of Biochemistry: Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert (NCBI bookshelf). • Chapter 3 and 7 of Molecular Cell Biology 4th ed. (Ch. 9, 5th ed.): Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. (NCBI bookshelf). • Chapter 12 of Introduction to Genetic Analysis Anthony: J.F. Griffiths, Jeffrey H. Miller, David T. Suzuki, Richard C. Lewontin, William M. Gelbart (NCBI bookshelf). • Some animations are from http://www.wiley-vch.de/books/info/3-527-30300-6/. • Cancer examples from Weinberg, Robert (2007). The Biology of Cancer. Garland Science. • http://www.piercenet.com/ Electrophoresis 92 The velocity of migration (v) of a molecule in an electric field depends on the electric field strength (E), the net charge on the protein (z), and the frictional coefficient (f). Ez v = f The frictional coefficient f depends on both the mass and shape of the migrating molecule and the viscosity (η) of the medium. For a sphere of radius r, f = 6πηr € The speed of migration is therefore proportional to the charge:mass ratio. z Will the charge to mass ratio differ v ∝ between proteins? Between r different DNA molecules? € • Electrophoresis is a technique for separating, or resolving, molecules in a mixture under the influence of an applied electric field.
    [Show full text]
  • Permease from Escherichia Coli
    Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli Mikhail Bogdanova,1, Philip Heacocka, Ziqiang Guanb, and William Dowhana,1 aDepartment of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, TX, 77030; and bDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710 Edited by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved July 15, 2010 (received for review May 9, 2010) Phosphatidylcholine (PC) has been widely used in place of naturally N-terminal two-TM helical hairpin of PheP and GabP are in- occurring phosphatidylethanolamine (PE) in reconstitution of verted with respect to their orientation in PE-containing cells bacterial membrane proteins. However, PC does not support native and the remaining TMs. These permeases do not carry out pro- structure or function for several reconstituted transport proteins. ton-coupled energy-dependent uphill transport of substrate in Lactose permease (LacY) of Escherichia coli, when reconstituted cells lacking PE, but still display energy-independent downhill in E. coli phospholipids, exhibits energy-dependent uphill and transport. LacY reconstituted into total E. coli phospholipids car- energy-independent downhill transport function and proper con- ries out uphill transport with domains C6 and P7 on opposite formation of periplasmic domain P7, which is tightly linked to sides of the membrane bilayer as observed in wild-type cells uphill transport function. LacY expressed in cells lacking PE and (9). Leaving out PE during reconstitution results in only downhill containing only anionic phospholipids exhibits only downhill trans- transport with domains C6 and P7 residing on the same side of port and lacks native P7 conformation.
    [Show full text]
  • Protein Blotting Guide
    Electrophoresis and Blotting Protein Blotting Guide BEGIN Protein Blotting Guide Theory and Products Part 1 Theory and Products 5 Chapter 5 Detection and Imaging 29 Total Protein Detection 31 Transfer Buffer Formulations 58 5 Chapter 1 Overview of Protein Blotting Anionic Dyes 31 Towbin Buffer 58 Towbin Buffer with SDS 58 Transfer 6 Fluorescent Protein Stains 31 Stain-Free Technology 32 Bjerrum Schafer-Nielsen Buffer 58 Detection 6 Colloidal Gold 32 Bjerrum Schafer-Nielsen Buffer with SDS 58 CAPS Buffer 58 General Considerations and Workflow 6 Immunodetection 32 Dunn Carbonate Buffer 58 Immunodetection Workflow 33 0.7% Acetic Acid 58 Chapter 2 Methods and Instrumentation 9 Blocking 33 Protein Blotting Methods 10 Antibody Incubations 33 Detection Buffer Formulations 58 Electrophoretic Transfer 10 Washes 33 General Detection Buffers 58 Tank Blotting 10 Antibody Selection and Dilution 34 Total Protein Staining Buffers and Solutions 59 Semi-Dry Blotting 11 Primary Antibodies 34 Substrate Buffers and Solutions 60 Microfiltration (Dot Blotting) Species-Specific Secondary Antibodies 34 Stripping Buffer 60 Antibody-Specific Ligands 34 Blotting Systems and Power Supplies 12 Detection Methods 35 Tank Blotting Cells 12 Colorimetric Detection 36 Part 3 Troubleshooting 63 Mini Trans-Blot® Cell and Criterion™ Blotter 12 Premixed and Individual Colorimetric Substrates 38 Transfer 64 Trans-Blot® Cell 12 Immun-Blot® Assay Kits 38 Electrophoretic Transfer 64 Trans-Blot® Plus Cell 13 Immun-Blot Amplified AP Kit 38 Microfiltration 65 Semi-Dry Blotting Cells
    [Show full text]
  • Post-Polymerization Modifications of Polymeric Monolithic Columns: a Review
    Chromatography 2014, 1, 24-53; doi:10.3390/chromatography1010024 OPEN ACCESS chromatography ISSN 2227-9075 www.mdpi.com/journal/chromatography Review Post-Polymerization Modifications of Polymeric Monolithic Columns: A Review Sinéad Currivan and Pavel Jandera * Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice 532 10, Czech Republic; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +420-460-037-023. Received: 18 December 2013; in revised form: 16 January 2014 / Accepted: 17 January 2014 / Published: 10 February 2014 Abstract: The vast cache of methods used in polymeric monolithic column modification is presented herein, with specific attention to post-polymerization modification reactions. The modification of polymeric monolithic columns is defined and can include the modification of pre-existing surface groups, the addition of polymeric chains or indeed the addition of structures such as nano-particles and nano-structures. The use of these modifications can result in the specific patterning of monoliths, useful in microfluidic device design or in the investigation of modification optimization. Keywords: polymer monoliths; modifications; HPLC; CEC; grafting; click chemistry; hypercross-linking; nano-particles List of Abbreviations: 2-acrylamido-2-methyl-1-propanesulphonic acid AMPS, α, α’-azoisobutyronitrile AIBN, adenosine triphosphate ATP, atom transfer radical polymerization ATRP, benzoyl peroxide
    [Show full text]
  • Capillary Electrochromatography for Analysis of Proteins and Metalloproteinases
    San Jose State University SJSU ScholarWorks Master's Theses Master's Theses and Graduate Research 2008 Capillary electrochromatography for analysis of proteins and metalloproteinases Vasudha Salgotra San Jose State University Follow this and additional works at: https://scholarworks.sjsu.edu/etd_theses Recommended Citation Salgotra, Vasudha, "Capillary electrochromatography for analysis of proteins and metalloproteinases" (2008). Master's Theses. 3572. DOI: https://doi.org/10.31979/etd.m48v-cr76 https://scholarworks.sjsu.edu/etd_theses/3572 This Thesis is brought to you for free and open access by the Master's Theses and Graduate Research at SJSU ScholarWorks. It has been accepted for inclusion in Master's Theses by an authorized administrator of SJSU ScholarWorks. For more information, please contact [email protected]. CAPILLARY ELECTROCHROMATOGRAPHY FOR ANALYSIS OF PROTEINS AND METALLOPROTEINASES A Thesis Presented to The Faculty of the Department of Chemistry San Jose State University In Partial fulfillment of the Requirements for the Degree Master of Science by Vasudha Salgotra August 2008 UMI Number: 1459696 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 1459696 Copyright 2008 by ProQuest LLC. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
    [Show full text]
  • Wave Electroblotting, 2D, Complete Electrophoresis Systems And
    Instruction Manual WAVE Standard and Tetrad Electroblotting, 2-D and Complete Electrophoresis Systems Electroblotting VS20WAVECBS AND WAVETETRAD1CBS Includes VS20WAVESYS and WAVEBI SW20 & WAVEBI 2-D Electrophoresis WAVEC2DS Includes VS20WAVESYS and VS20WAVEDCI VS20WAVEDC Complete (Electroblotting & 2-D Electrophoresis) VS20WAVECES AND WAVETETRAD1CES Includes VS20WAVECBS and VS20WAVEDCI 1 Contents:- Page 1) Safety Instructions 3 2) Packing Lists 4 3) Care and Maintenance 6 4) Usage Guidance and restrictions: 7 5) Set Up 8 6) Gel Casting 13 7) Gel Preparation 20 8) Gel Selection 20 9) Gel Pouring 22 10) Sample Preparation and Loading 23 11) Buffer Volume 25 12) Gel Running 25 13) Solutions 26 14) Vertical Gel Electrophoresis References 28 15) Combs 29 16) Protein Blotting using the WAVE 31 17) Buffer Volumes 32 18) Passive and Active Cooling 32 19) Run Conditions 34 20) Blotting References 35 21) Buffers 36 22) Blotting Troubleshooting 37 23) 1st Dimension Electrophoresis using the WAVEDCI Tube Gel Module 41 24) Capillary Tube Gel Pouring 41 25) 1st Dimension (IEF) Phase Tube Gel Running 43 26) 2-D, Size Determination Phase 44 27) Warranty 45 2 SAFETY PRECAUTION WHEN USED CORRECTLY, THESE UNITS POSE NO HEALTH RISK. HOWEVER, THESE UNITS CAN DELIVER DANGEROUS LEVELS OF ELECTRICITY AND ARE TO BE OPERATED ONLY BY QUALIFIED PERSONNEL FOLLOWING THE GUIDELINES LAID OUT IN THIS INSTRUCTION MANUAL. ANYONE INTENDING TO USE THIS EQUIPMENT SHOULD READ THE COMPLETE MANUAL THOROUGHLY. THE UNIT MUST NEVER BE USED WITHOUT THE SAFETY LID CORRECTLY IN POSITION. THE UNIT SHOULD NOT BE USED IF THERE IS ANY SIGN OF DAMAGE TO THE EXTERNAL TANK OR LID.
    [Show full text]
  • Power and Limitations of Electrophoretic Separations in Proteomics Strategies
    Power and limitations of electrophoretic separations in proteomics strategies Thierry. Rabilloud 1,2, Ali R.Vaezzadeh 3 , Noelle Potier 4, Cécile Lelong1,5, Emmanuelle Leize-Wagner 4, Mireille Chevallet 1,2 1: CEA, IRTSV, LBBSI, 38054 GRENOBLE, France. 2: CNRS, UMR 5092, Biochimie et Biophysique des Systèmes Intégrés, Grenoble France 3: Biomedical Proteomics Research Group, Central Clinical Chemistry Laboratory, Geneva University Hospitals, Geneva, Switzerland 4: CNRS, UMR 7177. Institut de Chime de Strasbourg, Strasbourg, France 5: Université Joseph Fourier, Grenoble France Correspondence : Thierry Rabilloud, iRTSV/LBBSI, UMR CNRS 5092, CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9 Tel (33)-4-38-78-32-12 Fax (33)-4-38-78-44-99 e-mail: Thierry.Rabilloud@ cea.fr Abstract: Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry( i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed. Keywords: electrophoresis, two-dimensional electrophoresis, isoelectric focusing, immobilized pH gradients, peptides, proteins, proteomics. Table of contents I. Introduction II. The principles at play III. How to use electrophoresis in a proteomics strategy III.A.
    [Show full text]
  • High Performance Capillary Electrophoresis
    High Performance Capillary Electrophoresis A Primer A Primer High Performance Capillary Electrophoresis Second completely revised edition by Henk H. Lauer and Gerard P. Rozing Foreword Electrophoresis was born at the beginning of nineteenth century, even earlier than chromatography. It has taken a long development path, which in the early days was framed by names like Kohlrausch, Tiselius and later Everaerts. At the beginning of eighties in the last century, Jorgenson cause a paradigm shift by executing electrophoresis in a very thin capillary with inner diameter less then 100 μm leading to capillary electrophoresis. Since then, capillary electrophoresis has been constantly improved and has become a routine technique. Instrument manufacturers like Agilent Technologies have developed instrumentation for capillary electrophoresis serving many analytical laboratories reliably for many years. After a decline at the beginning of this decade, the capillary electrophoresis market is recovering strongly. Especially, hyphenation with mass spectrometers has been very fruitful and brought a remarkable ability to unravel complex structures of biomolecules with invincible speed and sensitivity. And there are several modes of capillary electrophoresis, which make it useful for separation of really wide class of compounds, such as small ions, peptides, proteins, DNA fragments both sequencing and restriction ones, and even complete cells or uncharged molecules. When compared with liquid chromatography capillary electrophoresis has one big advantage: the separation process takes place in a rather simple environment – in homogeneous solutions or in a polymeric sieving network and in a separation space with simple geometry, like a cylindrical channel. The equations describing completely movement of matter in space and time can be than easily formulated and solved by advanced mathematical methods.
    [Show full text]