Proteomics Sample Preparation E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 891

Veröffentlichungsjahr: 2011

Beschreibung

This long-awaited first guide to sample preparation for proteomics studies overcomes a major bottleneck in this fast growing technique within the molecular life sciences. By addressing the topic from three different angles -- sample, method and aim of the study -- this practical reference has something for every proteomics researcher. Following an introduction to the field, the book looks at sample preparation for specific techniques and applications and finishes with a section on the preparation of sample types. For each method described, a summary of the pros and cons is given, as well as step-by-step protocols adaptable to any specific proteome analysis task.

Leseprobe

Contents

Preface

List of Contributors

List of Abbreviations

Part I Perspectives in Proteomics Sample Preparation

1 IntroductionN. Leigh Anderson

2 General Aspects of Sample Preparation for Comprehensive Proteome AnalysisSven Andrecht and Jörg von Hagen

2.1 The Need for Standards in Proteomics Sample Preparation

2.2 Introduction: The Challenge of Crude Proteome Sample Analysis

2.3 General Aspects: Parameters which Influence the Sample Preparation Procedure

2.4 Summary and Perspectives

References

3 Proteomics: A Philosophical PerspectiveErich Hamberger

3.1 Introduction: “In the Beginning was the Word”

3.2 The Experiment as a Scientific Method and a Tool of Cognition

3.3 The Experiment as a Method (Tool) of Cognition Within the Scope of Biology: The So-Called “Life Sciences”

3.4 Proteomics as a Cognition-Theoretical Challenge

3.5 Conclusion

References

Part II Methods

4 Mass Spectrometry

4.1 A Practical Guideline to Electrospray Ionization Mass Spectrometry for Proteomics ApplicationJon Barbour, Sebastian Wiese, Helmut E. Meyer, and Bettina Warscheid

4.2 Sample Preparation for the Application of MALDI Mass Spectrometry in Proteome AnalysisAndreas Tholey, Matthias Glückmann, Kerstin Seemann, and Michael Karas

4.3 Sample Preparation for Label-Free Proteomic Analyses of Body Fluids by Fourier Transform Ion Cyclotron Mass SpectrometryCloud P. Paweletz, Nathan A. Yates, and Ronald C. Hendrickson

4.4 Sample Preparation for Differential Proteome Analysis: Labeling Technologies for Mass SpectrometryJosef Kellermann

4.5 Determining Membrane Protein Localization Within Subcellular Compartments Using Stable Isotope TaggingKathryn S. Lilley, Tom Dunkley, and Pawel Sadowski

References

5 Electrophoresis

5.1 Sample Preparation for Two-Dimensional Gel ElectrophoresisWalter Weiss and Angelika Görg

5.2 Sample Preparation for Native ElectrophoresisIlka Wittig and Hermann Schägger

5.3 Sample Preparation for LC-MS/MS Using Free-Flow ElectrophoresisMikkel Nissum, Afsaneh Abdolzade-Bavil, Sabine Kuhfuss, Robert Wildgruber, Gerhard Weber, and Christoph Eckerskorn

5.4 Sample Preparation for Capillary ElectrophoresisRoss Burn and David Perrett

References

6 Optical Methods

6.1 High-Throughput Proteomics: Spinning Disc Interferometry (SDI)Patricio Espinoza Vallejos, Greg Lawrence, David Nolte, Fred Regnier, and Joerg Schreiber

6.2 Optical Proteomics on Cell ArraysAndreas Girod and Philippe Bastiaens

6.3 Sample Preparation by Laser Microdissection and Catapulting for Proteome AnalysisKarin Schütze, Andrea Buchstaller, Yilmaz Niyaz, Christian Melle, Günther Ernst, Kerstin David, Thorsten Schlomm, and Ferdinand von Eggeling

6.4 Sample Preparation for Flow CytometryDerek C. Davies

References

7 Chromatography

7.1 Sample Preparation for HPLC-Based Proteome AnalysisEgidijus Machtejevas and Klaus K. Unger

7.2 Sample Preparation for Two-Dimensional Phosphopeptide Mapping and Phosphoamino Acid AnalysisAnamarija Kruljac-Letunic and Andree Blaukat

References

8 Structural Proteomics

8.1 Exploring Protein–Ligand Interactions by Solution NMRRudolf Hartmann, Thomas Stangler, Bernd W.König, and Dieter Willbold

8.2 Sample Preparation for CrystallographyDjordje Musil

References

9 Interaction Analysis

9.1 Sample Preparation for Protein Complex Analysis by the Tandem Affinity Purification (TAP) MethodBertrand Séraphin and Andrzej Dziembowski

9.2 Exploring Membrane ProteomesFilippa Stenberg and Daniel O. Daley

References

10 Post-Translational Modifications

10.1 Sample Preparation for Phosphoproteome AnalysisRené P. Zahedi and Albert Sickmann

10.2 Sample Preparation for Analysis of Post-Translational Modifications: GlycosylationDavid S. Selby, Martin R. Larsen, Miren J. Omaetxebarria, and Peter Roepstorff

References

11 Species-Dependent Proteomics

11.1 Sample Preparation and Data Processing in Plant ProteomicsKatja Baerenfaller, Wilhelm Gruissem, and Sacha Baginsky

11.2 Sample Preparation for MudPIT with Bacterial Protein SamplesAnsgar Poetsch and Dirk Wolters

11.3 Sample Preparation for the Cell-Wall Proteome Analysis of Yeast and FungiKai Sohn, Ekkehard Hiller, and Steffen Rupp

References

12 The Human Proteosome

12.1 Clinical Proteomics: Sample Preparation and StandardizationGerd Schmitz and Carsten Gnewuch

12.2 Stem Cell ProteomicsRegina Ebert, Gabriele Möller, Jerzy Adamski, and Franz Jakob

References

13 Bioinformatics

13.1 Bioinformatics Support for Mass Spectrometric Quality ControlKnut Reinert, Tim Conrad, and Oliver Kohlbacher

13.2 Use of Physico-Chemical Properties in Peptide and Protein IdentificationAnastasia K. Yocum, PeterJ. Ulintz, and Philip C. Andrews

References

Index

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The Editor

Dr. Jörg von Hagen

Merck KGaAChromatography and BioscienceFrankfurter Strasse 25064271 DarmstadtGermany

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de.

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Cover WMXDesign GmbH, Heidelberg

ISBN: 978-3-527-31796-7

Preface

Why is there a need to consider Sample preparation in proteomics? Following the successes of the genome era, researchers have switched their efforts to analyzing complex protein mixtures, hopefully to obtain deeper insights into the molecular development of diseases by comparing whole proteomes from healthy versus disease tissues, body fluid samples, or other sources. Proteomics was born on the waves of these advances and, as a consequence, enormous investments were made in many attempts to unravel the proteome for biomarker identification. The first wave of proteomics resulted in a re-arming of the laboratories which, by this time, no longer required vastly expensive equipment such as mass spectrometers. Inevitably, this surge of interest led to a vast number of reports in which biomarkers had, supposedly, been identified. The second wave of proteomics has been characterized more by the establishment of diverse methods and their combination, as so-called “standard proteomic workflows”. Today, this subset of methodologies, databases and workflows appears largely to have been optimized, and the numbers of applications for the funding of studies and grants which include the catchword “proteomics” are rapidly increasing as the research teams continue their quests for meaningful data. Yet, the best way to obtain high-quality data and ensure consistency is not only to perform analyses in replicate but also – and more importantly – to standardize the methods of sample preparation.

What is meant by the term “proteomics”? Whilst this is to some extent a philosophical question, the answer depends heavily on an individual’s point of view. Some researchers describe proteomics as a unique scientific area for the analysis of whole proteomes, as notably do clinical proteome scientists. Others define proteomics as a subset of methodologies that are valuable in the analysis of proteins, as proteins represent the most common drug targets today and are the molecules closest to the point of invention in living cells. Despite these differences of opinion, common sense among the scientific community decrees that sample preparation procedures must be kept as simple as possible. In this way, such procedures will go hand in hand with high accuracy and standardization. Clearly, proteomics – in contrast to genomics, which embraces sensitivity, abundance and a combination of different methods – depends on the state of the biological sample itself. The main question, therefore, is how to create an optimal workflow for each particular experimental set-up.

This book will provide those scientists on the third wave of proteomics – whether researchers or simply users of protein biochemical methodologies – with a comprehensive overview of the different requirements for sample preparation when using today’s technologies. Hopefully, it will also provide any “beginners” in proteomics with some very brief “recipes” designed by well-known experts in each particular field.

I believe that this book will “sensitize” the need for sample preparation in proteomics, and will illustrate – with many useful practical applications – the problems which stem from the complexity of whole proteome samples. In this way it will provide solutions for those scientists who are new to this intriguing field of proteomics.

Jörg von Hagen

List of Contributors

Afsaneh Abdolzade-Bavil

BD Diagnostics – Preanalytical

Systems

Am Klopferspitz 19a

82152 Planegg/Martinsried

Germany

Jerzy Adamski

German Research Center for

Environmental Health

Institute of Experimental Genetics

Ingolst,ädter Landstrasse 1

85764 Neuherberg

Germany

N. Leigh Anderson

CEO, Plasma Proteome Institute

P.O. Box 53450

Washington, DC 20009-3450

USA

Sven Andrecht

Merck KGaA

Chromatography and Bioscience

Frankfurter Strasse 250

64271 Darmstadt

Germany

Philip C. Andrews

University of Michigan Medical School

Department of Biological Chemistry

National Resource for Proteomics

and Pathways

300 North Ingalls Street

Ann Arbor, MI 48109-0606

USA

Sacha Baginsky

ETH Zürich

LFW E51.1

Universitätsstrasse 2

8092 Zürich

Switzerland

Jon Barbour

Ruhr-University Bochum

Medical Proteome-Center

Universitätsstrasse 150

44801 Bochum

Germany

Katja Bärenfaller

ETH Zürich

Institute of Plant Sciences

Universitätsstrasse 2

8092 Zürich

Switzerland

Philippe Bastiaens

EMBL Heidelberg

Meyerhofstrasse 1

69117 Heidelberg

Germany

Andree Blaukat

Merck KGaA

Oncology – Biochemistry & Cellular

Pharmacology

Frankfurter Strasse 250

64293 Darmstadt

Germany

Andrea Buchstaller

Ludwig Maximilians University

Institute of Pathology

Thalkirchener Strasse 36

80337 Munich

Germany

Ross Burn

PR&D, AstraZeneca

Avlon Works

Severn Road

Hallen, Bristol BS10 7ZE

United Kingdom

Tim Conrad

Institut für Mathematik

Fachbereich Mathematik und

Informatik

AG Bio Computing Group

Arnimallee 6

14195 Berlin

Germany

Daniel O. Daley

Stockholm University

Department of Biochemistry

and Biophysics

Svante Arrhenius väg 12

10691 Stockholm

Sweden

Kerstin David

Indivumed GmbH

Center for Cancer Research

Israelitisches Krankenhaus Hamburg

Orchideenstieg 14

22297 Hamburg

Germany

Derek C. Davies

London Research Institute

Cancer Research UK

FACS Laboratory

44 Lincoln’s Inn Fields

London WC2A 3PX

United Kingdom

Tom Dunkley

University of Cambridge

Cambridge Centre for Proteomics

Department of Biochemistry

Downing Site

Cambridge CB2 1QW

United Kingdom

Andrzej Dziembowski

Equipe Labellisée La Ligue

CGM, CNRS UPR2167

Avenue de la Terrasse

91198 Gif sur Yvette Cedex

France

and

Present address:

Warsaw University

Department of Genetics

Pawinskiego 5a

02-106 Warsaw

Poland

Regina Ebert

University of Würzburg

Orthopedic Center for

Musculoskeletal Research

Orthopedic Department

Brettreichstrasse 11

97074 Würzburg

Germany

Christoph Eckerskorn

BD Diagnostics

Preanalytical Systems

Innovationszentrum Biotechnologie

Am Klopferspitz 19

82152 Martinsried/Planegg

Germany

Ferdinand von Eggeling

Medical Faculty at the

Friedrich Schiller University

Institute of Human Genetics and

Anthropology

07740 Jena

Germany

Günther Ernst

Medical Faculty at the

Friedrich Schiller University

Institute of Human Genetics and

Anthropology

07740 Jena

Germany

Patricio Espinoza Vallejos

Quadraspec Inc.

3000 Kent Avenue

West Lafayette, IN 47906

USA

Andreas Girod

EMBL Heidelberg

Meyerhofstrasse 1

69117 Heidelberg

Germany

Matthias Glückmann

Applied Biosystems

Mass Spectrometry and Proteomics

64293 Darmstadt

Germany

Carsten Gnewuch

Klinikum der Universität Regensburg

Institut für Klinische Chemie und

Laboratoriumschemie

Franz-Josef-Strauss-Allee 11

93053 Regensburg

Germany

Angelika Görg

TU München

Proteomics Department

Am Forum 2

85354 Freising-Weihenstephan

Germany

Wilhelm Gruissem

ETH Zürich

Institute of Plant Sciences

Universitätsstrasse 2

8092 Zürich

Switzerland

Jörg von Hagen

Merck KGaA

Chromatography and Bioscience

Frankfurter Strasse 250

64271 Darmstadt

Germany

Erich Hamberger

Universität Salzburg

Fachbereich Kommunikations-wissenschaft

Rudolfskai 42

5020 Salzburg

Austria

Rudolf Hartmann

Heinrich-Heine-Universität Düsseldorf

Institut für Physikalische Biologie

40225 Düsseldorf

Germany

Ronal C. Hendrickson

Merck & CO.

Merck Research Laboratories

Rahway, NJ

USA

Ekkehard Hiller

Fraunhofer Institute for Interfacial

Engineering and Biotechnology (IGB)

Department of Molecular

Biotechnology

Nobelstrasse 12

70569 Stuttgart

Germany

Franz jakob

University of Würzburg

Orthopedic Center for

Musculoskeletal Research

Orthopedic Department

Brettreichstrasse 11

97074 Würzburg

Germany

Michael Karas

Johann Wolfgang Goethe University

Institute of Pharmaceutical Chemistry

Max-von-Laue-Strasse 91

60438 Frankfurt

Germany

Josef Kellermann

MPI für Biochemie

Proteinanalysis

Am Klopferspitz 19 a

82152 Martinsried/Planegg

Oliver Kohlbacher

Wilhelm-Schickard-Institut

für Informatik

Sand 14

72076 Tübingen

Germany

Bernd W. König

Heinrich-Heine-Universität Düsseldorf

Institut für Physikalische Biologie

40225 Düsseldorf

Germany

and

Forschungszentrum Jülich

Institut für Neurowissenschaften

und Biophysik, Biomolekulare NMR

52425 Jülich

Germany

Anamarija Kruljac-Letunic

EMBL Heidelberg

Cell Biology and Biophysics

Meyerhofstrasse 1

69117 Heidelberg

Germany

Sabine Kuhfuss

BD Diagnostics – Preanalytical Systems

Am Klopferspitz 19a

82152 Martinsried/Planegg

Germany

Martin R. Larsen

University of Southern Denmark

Department of Biochemistry

and Molecular Biology

Campusvej 55

5230 Odense M

Denmark

Greg Lawrence

Quadraspec Inc.

3000 Kent Avenue

West Lafayette, IN 47906

USA

Kathryn S. Lilley

University of Cambridge

Department of Biochemistry

Downing Site

Cambridge CB2 1QW

United Kingdom

Egidijus Machtejevas

Johannes Gutenberg University

Institute of Inorganic and

Analytical Chemistry

Duesbergweg 10–14

55099 Mainz

Germany

Christian Melle

Medical Faculty at the

Friedrich Schiller University

Core Unit Chip Application (CUCA)

Institute of Human Genetics and

Anthropology

07740 Jena

Germany

Helmut E. Meyer

Ruhr-University Bochum

Medical Proteome-Center

Universitätsstrasse 150

44801 Bochum

Germany

Gabriele Möller

German Research Center

for Environmental Health

Institute of Experimental Genetics

Ingolstädter Landstrasse 1

85764 Neuherberg

Germany

Djordje Musil

Merck KGaA

Merck Serono Research

NCE-Tech / LDT/ MIB

Frankfurter Strasse 250

64293 Darmstadt

Germany

Mikkel Nissum

BD Diagnostics – Preanalytical Systems

Am Klopferspitz 19a

82152 Martinsried/Planegg

Germany

Yilmaz Niyaz

P.A.L.M. Microlaser Technologies

GmcB

Am Neuland 9

82147 Bernried

Germany

David Nolte

Purdue University

Department of Physics

525 Northwestern Avenue

West Lafayette, IN 47907-2036

USA

Miren J. Omaetxebarria

University of The Basque Country

Department of Biochemistry and

Molecular Biology

Faculty of Science and Technology

Sarriena z/g

48940 Leioa

Spain

Cloud P. Paweletz

Merck & CO.

Merck Research Laboratories

Rahway NJ

USA

David Perrett

Queen Mary University London

St. Bartholomew’s Hospital Medial

College

West Smithfield

London EC1A 7BE

United Kingdom

Ansgar Poetsch

Ruhr-University Bochum

Medical Proteome-Center

Universitätsstrasse 150

Germany

Fred Regnier

Purdue University

Department of Chemistry

560 Oval Drive

West Lafayette, IN 47907-2084

USA

Knut Reinert

Freie Universität Berlin

Institut für Informatik

Takustrasse 9

14195 Berlin

Germany

Peter Roepstorff

University of Southern Denmark

Department of Biochemistry

and Molecular Biology

Campusvej 55

5230 Odense M

Denmark

Steffen Rupp

Fraunhofer Institute for Interfacial

Engineering and Biotechnology (IGB)

Department of Molecular Biotechnology

Nobelstrasse 12

70569 Stuttgart

Germany

Pawel Sadowski

University of Cambridge

Cambridge Centre for Proteomics

Department of Biochemistry

Downing Site

Cambridge CB2 1QW

United Kingdom

Hermann Schägger

Universitätsklinikum

Zentrum der Biologischen Chemie

Molekulare Biochemie

Theodor-Stern-Kai 7, Haus 26

60590 Frankfurt

Germany

Thorsten Schlomm

University Clinic Hamburg-Eppendorf

Department of Urology

20246 Hamburg

Germany

Gerd Schmitz

University Hospital Regensburg

Institute for Clinical Chemistry

and Laboratory Medicine

Franz-Josef-Strauss-Allee 11

93053 Regensburg

Germany

Joerg Schreiber

Quadraspec Inc.

3000 Kent Avenue

West Lafayette, IN 47906

USA

Karin Schütze

Carl Zeiss Microlmaging GmbH

Am Neuland 9

82347 Bernried

Kerstin Seemann

Merck KGaA

Analytical Development and

Bioanalytics

64293 Darmstadt

Germany

David S. Selby

Harrison Goddard Foote

Belgrave Hall, Belgrave Street

Leeds LS2 8DD

United Kingdom

Bertrand Séraphin

CGM–CNRS UPR2167

Equipe Labellisée La Ligue

Avenue de la Terrasse

91198 Gif sur Yvette Cedex

France

Albert Sickmann

Rudolf-Virchow-Center for

Experimental Biomedicine

Protein Mass Spectrometry and

Functional Proteomics

Versbacher Strasse 9

97078 Würzburg

Germany

Kai Sohn

Fraunhofer Institute for Interfacial

Engineering and Biotechnology (IGB)

Department of Molecular

Biotechnology

Nobelstrasse 12

70569 Stuttgart

Germany

Thomas Stangler

Heinrich-Heine-Universität Düsseldorf

Institut für Physikalische Biologie

40225 Düsseldorf

Germany

and

Forschungszentrum Jülich

IBI-2

Institut für Naturwissenschaften

52425 Jülich

Germany

Filippa Stenberg

Stockholm University

Department of Biochemistry and

Biophysics

Svante Arrhenius väg 12

10691 Stockholm

Sweden

Andreas Tholey

Universität des Saarlandes

Technische Biochemie

Functional Proteomics Group

Campus A 1-5

66123 Saarbrücken

Germany

PeterJ. Ulintz

University of Michigan Medical School

Bioinformatics Graduate Program

National Resource for Proteomics

and Pathways

300 North Ingalls Street

Ann Arbor, MI 48109-0606

USA

Klaus K. Unger

Johannes Gutenberg University

Institute of Inorganic and Analytical

Chemistry

Duesbergweg 10-14

55099 Mainz

Germany

Bettina Warscheid

Ruhr-University Bochum

Medical Proteome-Center

Universitätsstrasse 150

44780 Bochum

Germany

Gerhard Weber

BD Diagnostics

Am Klopferspitz 19a

82152 Martinsried/Planegg

Germany

Walter Weiss

Technical University Munich

Proteomics Department

Am Forum 2

85350 Freising-Weihenstephan

Germany

Sebastian Wiese

Ruhr-University Bochum

Medical Proteome-Center

Universitätsstrasse 150

44801 Bochum

Germany

Robert Wildgruber

BD Diagnostics

Am Klopferspitz 19a

82152 Martinsried/Planegg

Germany

Dieter Willbold

Heinrich-Heine-Universität Düsseldorf

Institut fiir Physikalische Biologie

40225 Düsseldorf

Germany

and

Forschungszentrum Jülich

Institut fiir Naturwissenschaften

IBI-2 / Molekulare Biophysik II

52425 Jülich

Germany

Ilka Wittig

Universitätsklinikum Frankfurt

Zentrum der Biologischen Chemie

Molekulare Bioenergetik

Theodor-Stern-Kai 7

60590 Frankfurt

Germany

Dirk Wolters

Ruhr-Universität Bochum

Analytische Chemie NC 4/72

Biomolekulare Massenspektrometrie

Universitätsstrasse 150

44801 Bochum

Germany

Nathan A. Yates

Merck & CO.

Merck Research Laboratories

Rahway, NJ

USA

Anastasia K. Yocum

University of Michigan Medical School

Michigan Center for Translational

Pathology

Department of Pathology

1150 West Medical Center Drive

Ann Arbor, MI 48109-0606

USA

René P. Zahedi

Rudolf-Virchow-Center for

Experimental Biomedicine

Protein Mass Spectrometry and

Functional Proteomics

Versbacher Strasse 9

97078 Würzburg

Germany

List of Abbreviations

2-DEtwo-dimensional electrophoresisBGEbackground electrolyteCEcapillary electrophoresisCGEcapillary gel electrophoresisCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydratecIEFcapillary isoelectric focusingcITPcapillary isotachophoresisCLODconcentration Limit of DetectionCZEcapillary zone electrophoresisDCdirect currentDMSOdimethyl sulfoxideEDTAethylenediaminetetraacetic acidEOFelectroosmotic flowFIAfield-amplified jnjectionHPLChigh-performance liquid chromatographyHUPOHuman Proteome OrganisationITPisotachophoresisLEleading electrolytLODlimit of detectionMEKCmicellar electrokinetic chromatographyMIPmolecular imprinted polymerMSmass spectrometryOPA/NACphthaldialdehyde/N-acety-L-cysteinePCpersonal computerpIisoelectric pointPVApolyvinyl alcoholRPLCreversed-phase liquid chromatographySDSsodium dodecyl sulfateSPEsolid-phase extractionSPMEsolid-phase micro-extractionTEterminating electrolyteTEMEDN,N,N′,N′ -tetramethylethylenediamineTrisTris(hydroxymethyl)aminomethaneUFultrafiltrationUVultravioletVisvisible

Part I

Perspectives in Proteomics Sample Preparation

1

Introduction

N. Leigh Anderson

A lot can happen to a protein in the time between its removal from an intact biological system and its introduction into an analytical instrument. Given the increasing sophistication of methods for characterizing many classes of post-translational modification, an increasing variety of protein-modifying processes need to be kept under control if we are to understand what is biology, and what is noise. Hence, the growing importance of sample preparation in proteomics. One might justifiably say that the generation of good samples is half the battle in this field.

Fortunately, proteomics provides us with good methods for studying sample preparation issues. Two-dimensional electrophoresis of plasma, for example, provides a visual protein fingerprint that allows the immediate recognition of sample handling issues such as clotting, platelet breakage, and extended storage at –20 °C (instead of–80 °C). A deeper exploration of plasma using mass spectrometry-based methods provides a more comprehensive picture, though perhaps more difficult to understand.

Unfortunately, despite the power of these methods, we do not know as much about sample quality and sample processing as we need to. The general attitude to these issues in proteomics has been to focus on the standardization of a few obvious variables and hope that the power of the analytical methods allows the sought-for differences between sample groups to shine through. This short-cut approach is likely to be problematic. Not only do the unrecognized effects of sample preparation differences add noise to the background against which the biological signal must be detected, but the sample preparation effects themselves are occasionally confused with biology. Well-informed skeptics correctly suspect that variables as basic as how blood is drawn or stored can generate spurious biomarker signals if the case and control samples are not acquired in exactly the same way. At this point we do not have adequate definitions of what “in exactly the same way” actually means for any given analytical platform.

These problems point to a need to take sample preparation (including initial acquisition through all the steps leading up to analysis) as a mission-critical issue, worthy of time and effort with our best analytical systems. Published data on differences between serum and plasma protein composition, the effect of blood clotting, is interesting but very far from definitive – and in fact specialists in blood coagulation can offer a host of reasons why this process is not easily controllable (and hence not especially reproducible) in a clinical environment. Even a process as widely relied on as tryptic digestion is not really understood in terms of the time course of peptide release or the frequency with which “non-tryptic” peptides are generated – aspects which are critical for quantitative analysis. These and a host of similar issues can be attacked systematically using the tools of proteomics, with the aim of understanding how best to control and standardize sample preparation. In doing so, we will learn much about the tools themselves, and perhaps resolve the paradox surrounding the peptide profiling (originally SELDI) approach: that is, why it seems to be so successful in finding sample differences, but so unsuccessful in finding differences that are reproducible. Perhaps peptide profiling is the most sensitive method for detecting sample preparation artifacts: if it is, then it may be the best tool to support removal of these artifacts and ultimately the best way to classify and select samples for analysis by more robust methods.

Obviously, it is time to take a close look at sample preparation in proteomics. The reader is encouraged to weigh what is known against what is unknown in the following pages, and contemplate what might be done to improve our control over the complex processes entailed in generating the samples that we use.

2

General Aspects of Sample Preparation for Comprehensive Proteome Analysis

Sven Andrecht and jörg von Hagen

2.1 The Need for Standards in Proteomics Sample Preparation

Sample preparation is not the only step - but it is one of the most critical steps - in proteome research. The quality of protein samples is critical to generating accurate and informative data. As proteomic technologies move in the direction of higher throughput, upstream sample preparation becomes a potential bottleneck. Sample capture, transportation, storage, and handling are as critical as extraction and purification procedures. Obtaining homogeneous samples or isolating individual cells from clinical material is imperative, and for this standards are essential. Advances in microfluidic and microarray technologies have further amplified the need for higher-throughput, miniaturized, and automated sample preparation processes.

The need for consistency and standardization in proteomics has limited the success of solutions for proteomics sample preparation. Without effective standards, researchers use divergent methods to investigate their proteins, making it unrealistic to compare their data sets. Until standards emerge, the continual generation of randomized data sets is likely to contribute to the increasing complexity of proteomics research as well as sample preparation.

Proteomics aims to study dynamically changing proteins expressed by a whole organism, specific tissue or cellular compartment under certain conditions. Consequently, two main goals of proteomics research are to: (1) identify proteins derived from complex mixtures extracted from cells; and (2) quantify expression levels of those identified proteins. In recent years mass spectrometry (MS) has become one of the main tools to accomplish these goals by identifying proteins through information derived from tandem mass spectrometry (MS/MS) and measuring protein expression by quantitative MS methods. Recently, these approaches have been successfully applied in many studies, and can be used to identify 500 to 1000 proteins per experiment. Moreover, they can reliably detect and estimate the relative expression (proteins differentially expressed in different conditions) of high- and medium-abundance proteins, and can measure absolute protein expression (quantitation) of single proteins in complex mixtures. The ideal situation would be to catalogue all of the proteins present in a sample and their respective concentrations.However, this level of precision is not yet available because the number and concentration of proteins that can be resolved limits the proteomics methods.