Subcellular Proteomics : From Cell Deconstruction to System Reconstruction / Eric Bertrand

deal with the second major issue, the resolving of the hydrophobic proteinsfound in biological membrane samples, which they solve through two-dimensionalBAC/SDS-PAGE gel electrophoresis.

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Subcellular Proteomics

Subcellular Biochemistry

Volume 43

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SUBCELLULAR BIOCHEMISTRY

SERIES EDITOR

J ROBIN HARRIS, University of Mainz, Mainz, Germany

ASSISTANT EDITORS

B.B BISWAS, University of Calcutta, Calcutta, India

P QUINN, King’s College London, London, U.K

Recent Volumes in this Series

Volume 31 Intermediate Filaments

Edited by Harald Herrmann and J Robin Harris

Volume 32 alpha-Gal and Anti-Gal: alpha-1,3-Galactosyltransferase, alpha-Gal

Epitopes and the Natural Anti-Gal Antibody

Edited by Uri Galili and Jos-Luis Avila

Volume 33 Bacterial Invasion into Eukaryotic Cells

Tobias A Oelschlaeger and Jorg Hacker

Volume 34 Fusion of Biological Membranes and Related Problems

Edited by Herwig Hilderson and Stefan Fuller

Volume 35 Enzyme-Catalyzed Electron and Radical Transfer

Andreas Holzenburg and Nigel S Scrutton

Volume 36 Phospholipid Metabolism in Apoptosis

Edited by Peter J Quinn and Valerian E Kagan

Volume 37 Membrane Dynamics and Domains

Edited by P.J Quinn

Volume 38 Alzheimer’s Disease: Cellular and Molecular Aspects of Amyloid beta

Edited by R Harris and F Fahrenholz

Volume 39 Biology of Inositols and Phosphoinositides

Edited by Lahiri Majumder and B.B Biswas

Volume 40 Reviews and Protocols in DT40 Research

Edited by Jean-Marie Buerstedde and Shunichi Takeda

Volume 41 Chromatin and Disease

Edited by Tapas K Kundu and Dipak Dasgupta

Volume 42 Inflammation in the Pathogenesis of Chronic Diseases

Edited by Randall E Harris

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Subcellular

Proteomics

From Cell Deconstruction

to System Reconstruction Subcellular Biochemistry

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A C.I.P Catalogue record for this book is available from the Library of Congress.

Printed on acid-free paper

All Rights Reserved

© 2007 Springer

No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose

of being entered and executed on a computer system, for exclusive use by the purchaser of the work.

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INTERNATIONAL ADVISORY EDITORIAL BOARD

R BITTMAN, Queens College, City University of New York, New York, USA

D DASGUPTA, Saha Institute of Nuclear Physics, Calcutta, India

H ENGELHARDT, Max-Planck-Institute for Biochemistry, Munich, Germany

L FLOHE, MOLISA GmbH, Magdeburg, Germany

H HERRMANN, German Cancer Research Center, Heidelberg, Germany

A HOLZENBURG, Texas A & M University, Texas, USA

H-P NASHEUER, National University of Ireland, Galway, Ireland

S ROTTEM, The Hebrew University, Jerusalem, Israel

M WYSS, DSM Nutritional Products Ltd., Basel, Switzerland

P ZWICKL, Max-Planck-Institute for Biochemistry, Munich, Germany

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To Michel and his speedy recovery His experience and enthusiasm have been sorelymissed in the final stages of preparing this volume

To my son, Alexandre, far from me now, but still close to my heart

To my mother, who has been a source of inspiration in many more ways than shecould have imagined

Eric

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TABLE OF CONTENTS

Thierry Rabilloud

René P Zahedi, Jan Moebius and Albert Sickmann

3 Microparticles: A New Tool for Plasma Membrane

Laurent Miguet, Sarah Sanglier, Christine Schaeffer, Noelle Potier,

Laurent Mauvieux and Alain Van Dorsselaer

4 Lipid Raft Proteomics: More than Just

Leonard J Foster and Queenie W T Chan

5 Organelle Proteome Variation Among Different Cell Types:

Deirdre M Kavanagh, William E Powell, Poonam Malik,

Vassiliki Lazou and Eric C Schirmer

Fengju Bai and Frank A Witzmann

ix

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Christine Olver and Michel Vidal

Vincent Collura and Guillaume Boissy

M.O Collins and S.G.N Grant

Ronald Roepman and Uwe Wolfrum

11 Systems Biology and the Reconstruction of the Cell:

Frank J Bruggeman, Sergio Rossell, Karen van Eunen,

Jildau Bouwman, Hans V Westerhoff and Barbara Bakker

12 Automated, Systematic Determination of Protein

Elvira García Osuna and Robert F Murphy

13 Systems Biology of the Endoplasmic Reticulum

Marie-Elaine Caruso and Eric Chevet

14 Systems Nanobiology: From Quantitative Single Molecule

Joerg Martini, Wibke Hellmich, Dominik Greif, Anke Becker,

Thomas Merkle, Robert Ros, Alexandra Ros,

Katja Toensing and Dario Anselmetti

Michel Faupel, Débora Bonenfant, Patrick Schindler,

Eric Bertrand, Dieter Mueller, Markus Stoeckli, Francis Bitsch,

Tatiana Rohner, Dieter Staab and Jan Van Oostrum

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16 Differential Epitope Identification of Antibodies Against

Intracellular Domains of Alzheimer’s Amyloid Precursor

Xiaodan Tian, Madalina Maftei, Markus Kohlmann,

Bernadette Allinquant and Michael Przybylski

17 LC-MALDI MS and MS/MS – An Efficient Tool in

Dieter R Mueller, Hans Voshol, Annick Waldt, Brigitte Wiedmann

and Jan van Oostrum

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ACKNOWLEDGEMENTS

In light of the somewhat eventful circumstances in which the current volume cameinto being, we are sincerely grateful to the people who contributed to its preparation.Their hard work as well as their patience and kind understanding toward the editorswere greatly appreciated We are most grateful to Mary Johnson, who acted as thepublishing editor for this volume We wish to thank all of the contributors and theirstaff for submitting their respective chapters as needed, despite busy teaching andresearch schedules We would also like to acknowledge several very good friendsfor their voluntary editorial assistance and even more importantly their unfailingmoral support: Eileen Rojo, Hugues Ryckelynck and Claire Mc Donack Last, but

by no mean least, we are particularly grateful to Dr Robin Harris, who gave us theopportunity to edit this volume for the “Subcellular Biochemistry” book series

xiii

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Frank J Bruggeman

BioCentre Amsterdam, Free University Amsterdam, Faculty of Earth and Life ences, Department of Molecular Cell Physiology, Amsterdam, The Netherlandsand also Manchester Centre for Integrative Systems Biology, Manchester Inter-disciplinary BioCentre, University of Manchester, United Kingdom

Elvira García Osuna

Center for Bioimage Informatics and Department of Biomedical Engineering,Carnegie Mellon University, Pittsburgh, Pennsylvania, United States

Uni-Wibke Hellmich

Experimental Biophysics and Applied Nanoscience, Physics Faculty, Bielefeld versity and Bielefeld Institute for Biophysics and Nanoscience (BINAS), Center forBiotechnology (CeBiTec), Bielefeld, Germany

Uni-BERTRAND: “BERTRAND_FM” — 2007/6/20 — 15:16 — PAGE xvii — #17

Jan Moebius

Protein Mass Spectrometry and Functional Proteomics Group, Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg,Germany

Biomed-BERTRAND: “BERTRAND_FM” — 2007/6/20 — 15:16 — PAGE xviii — #18

Alexandra Ros

Experimental Biophysics and Applied Nanoscience, Physics Faculty, BielefeldUniversity and Bielefeld Institute for Biophysics and Nanoscience (BINAS), Centerfor Biotechnology (CeBiTec), Bielefeld, Germany

Christine Schaeffer

Laboratoire de Spectrométrie de Masse Bio-Organique, ECPM, UMR/CNRS 7178,Institut Pluridisciplinaire Hubert CURIEN, Université Louis Pasteur, Strasbourg,France

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Alain Van Dorsselaer

Laboratoire de Spectrométrie de Masse Bio-Organique, ECPM, UMR/CNRS 7178,Institut Pluridisciplinaire Hubert CURIEN, Université Louis Pasteur, Strasbourg,France

Karen van Eunen

BioCentre Amsterdam, Free University Amsterdam, Faculty of Earth and LifeSciences, Department of Molecular Cell Physiology, Amsterdam, The Netherlands

Jan van Oostrum

Novartis Institutes for BioMedical Research, Genome and Proteome Sciences/Systems Biology, Basel, Switzerland

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Rudolf-Virchow-BERTRAND: “BERTRAND_FM” — 2007/6/20 — 15:16 — PAGE xxi — #21

INTRODUCTION

As proteomics technologies are reaching a plateau in the number of proteins that can

be resolved and detected, pre-fractionation steps have become essential to increase thedepth of proteomic analysis So far, many pre-fractionation steps have been based onchromatography methods where the proteins are separated according to their individ-ual physicochemical properties Subcellular fractionation methods proved to be verypotent protein pre-fractionation steps: they allow the representation of low abundanceproteins, and they can be combined with chromatography steps Moreover, as the iso-lated subcellular components also represent functional units, subcellular fractionationallows the proteomic analysis of protein subsets that are functionally related in a bio-logically relevant manner The first three sections of this volume deal with differentlevels of subcellular organization that also correspond to specific methodologicalapproaches

In his keynote chapter, Thierry Rabilloud superbly introduces the first section with

a thorough definition of membrane proteomics where he pinpoints key theoretical andpractical issues of this field, thereby setting the stage for the next contributions Miguet

et al address the first key issue: the quality of the membrane preparation; they duce and validate a microparticle strategy for plasma membrane purification Zahedi

intro-et al deal with the second major issue, the resolving of the hydrophobic proteinsfound in biological membrane samples, which they solve through two-dimensionalBAC/SDS-PAGE gel electrophoresis To close the first section, Foster and Chanreview the proteomics of lipid rafts, membrane structures that are involved in intra-cellular trafficking and signal transduction They describe a clever validation schemebased on the sensitivity of lipid rafts to cholesterol disruption

A central theme in the second section on organelle subproteomes is the variability

of their composition and how it can be exploited and interpreted Kavanagh et al.describe a state-of-the-art substractive proteomics scheme that relies on an in sil-ico purification step based on the comparison of organelle subproteomes With thisapproach, they could demonstrate variations in subproteome content across tissues

In the next chapter on synatosome proteomics, Bai and Witzmann review the currentefforts to correlate synaptic plasticity and variations in synaptic subproteome content

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with a special emphasis on post-translational modifications Finally, Olver and Vidaldiscuss how the proteomic analysis of exosomes would give clues to the molecularbasis of their biogenesis and contribute to a better understanding of their function.Moreover, they propose that the variations observed in exosome protein content areuseful for biomarker discovery

The third section deals with protein complexes, which are considered as the ular machinery that performs most cell functions This area is certainly not a trivialone: there are several types of protein complexes and protein-protein interactions and

molec-it is not always clear which methodology is most sumolec-itable to use in emolec-ither context Intheir chapter, Boissy and Collura sort out for us the concepts and methods encountered

in interactomics, guide us through data interpretation issues and share with us theirinsight on the very nature of interactions They plead for a systematic integration ofinteraction maps with functional genomics and molecular genetics data: the potential

of such an approach is strikingly demonstrated in the next two chapters Collins andGrant examine the molecular architecture of membrane associated signalling com-plexes in the nervous system, they highlight the role scaffolding proteins within thesecomplexes and point out to a few much needed construction rules Aligning interac-tion and functional genomics data, they build a case for a modular organisation oflarge complexes into functional sub-networks To complete the picture, based on athorough review on the complexes of the photoreceptor cilia, Roepman and Wolfrumsketch out an approach to organize complexes in functional modules and investigatetheir interactions

Assuming that the protein content of an organelle has been inventoried and itsprotein complexes characterized, the next step is to translate this knowledge intofunctionally relevant interpretations This is the purpose of systems biology: if weconsider organelles as systems that function and communicate with each other throughtheir protein machinery, it makes sense to apply such an approach at the subcellularlevel In their chapter, Caruso and Chevet prove that this concept can actually beapplied to reconstruct the stress signalling network of the endoplasmic reticulum.They build an integrative signalling map that qualitatively accounts for the interac-tions of the stress network with other endoplasmic reticulum machineries and alsowith other organelles On the quantitative side, Bruggeman et al introduce a theoret-ical framework for subcellular systems biology and thoroughly review the relevantmathematical approaches Drawing on their extensive experience of metabolic net-works, they argue that a direct translation of subcellular units as modules within amathematical model of the cell can be advantageous both for solving the problemand interpreting the results Garcia Osuna and Murphy survey the current automatedmethods for high-throughput determination of protein subcellular location that areused to reconstruct subcellular anatomy at high resolution These methods provideessential information on the dynamic aspect of subcellular events in individual cells:

it would also be extremely interesting to combine them with the molecular switchesdescribed by Martini et al in the next section

This brings us to the fifth and last section of this volume, where the most recenttechnological developments in proteomics are reviewed Martini et al introduce

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the emerging field of systems nanobiology that relies on ultrasensitive methods andinstruments to investigate cellular processes at the single molecule level An appli-cation, among others, is to monitor the dynamics of protein translocation from onesubcellular compartment to another using two-photon laser scanning microscopy and

a photoactivable GFP as a molecular switch Faupel et al review the current tions of biophotonic technologies to proteomics with a focus on mass spectrometrybased molecular imaging Tian et al describe a fast-track approach for the character-ization of antibody epitopes using Fourier transform ion cyclotron resonance massspectrometry (FTICR-MS) The application of this method to the Amyloid PrecursorProtein has important consequences for the study of intracellular processing path-ways relevant to Alzheimer’s disease Finally, Mueller et al describe the interfacing

applica-of LC and MALDI-MS and – MS/MS, discuss its performance, and present selectedapplications in the proteomics field including the analyses of membrane proteins andprotein interactions

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SECTION 1

MEMBRANE PROTEOMICS

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2 Sample Preparation Issues for Membrane Proteomics 5

3 Issues linked to the separation process 63.1 Constraints in Proteomics Based on Zone Electrophoresis 73.2 Constraints Proteomics Based on Two-dimensional Gel

Electrophoresis 83.3 Constraints in Peptide Separation-Based Approaches 93.4 Constraints in Protein Chromatography-Based Approaches 9

4 Concluding remarks 10References 10

1 INTRODUCTION

Before addressing the key issues pertaining to proteomics of membrane proteins,which is an important subject in the wider topic of subcellular proteomics, the firstquestion that must be asked is the definition of a membrane protein While this mightseem trivial at the first glance, the answer is far from being obvious A commonlyaccepted definition for a membrane protein is a protein associated with a membrane,that is, a lipid bilayer However, the meaning of the word “associated” in this case isthe subject of almost infinite debate In fact, two different situations arise In the first,simple situation, the polypeptide chain spans the lipid bilayer a certain number oftimes These proteins are defined as integral or intrinsic membrane proteins, as theirassociation with the lipid bilayer is doubtless The second, much more complicatedcase, is when the association with the lipid bilayer is not achieved by transmembranesegments or by transmembrane barrels (e.g in porins), because many other types ofassociation with membranes are encountered In one particular type, the association ismediated by a post translational modification of the polypeptide, namely the grafting

of an fatty acid or polyisoprenyl chain Polyisoprenylation has been demonstrated

to result in physical association with the membrane, as shown for small G proteins

3

E Bertrand and M Faupel (eds.), Subcellular Proteomics, 3–11.

© 2007 Springer.

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(Verma et al 1994) Strong association of proteins with lipid membranes is alsoachieved by glycolipid anchors such as the glycosylphosphatidyl inositol common in

eukaryotic species, or, as in bacterial lipoproteins, by an N -acyl diglyceride linkage

to an N -terminal cysteine residue that becomes available after cleavage of a signal

sequence (Cross 1991) Last but not least, the protein can also be acylated by a simplefatty acid chain In this case, the reality of the anchoring to the lipid bilayer is muchless clear

Protein-membrane association via a post-translational modification introducesthe notion of dynamic association and partitioning of proteins between the membranephase of the cells and the aqueous phase (cytosol or inner phase of organelles) Con-sequently, such proteins can be found both as membrane-associated and membrane-free, which is not the case with intrinsic membrane proteins which are strictlymembrane embedded Another type of association to membrane is mediated byprotein–protein interactions with other membrane proteins A typical example ofthis situation is provided by the respiratory complexes In the case of ubiquinol-cytochrome c oxidoreductase, core proteins 1 and 2 does not show any interactionwith the lipid membrane, but only with the protein subunits spanning the membrane(e.g cytochrome b) (Iwata et al 1998)

However, the situation is often much less clear than this one, and drives quitefast into the infinite debate of what is a membrane protein apart from the integralones In addition to the dynamic view of association to membranes, this complicatedsituation arises mainly from the operational, biochemical, definition of membranes.When a cell is lysed in an aqueous, detergent-free, medium, the cell-limiting mem-brane and the network of inner membranes (when applicable) will fragment intovesicles which can be separated from the bulk of cytosol by sedimentation orpartition techniques These techniques are also able to separate membrane-boundorganelles (e.g mitochondria and plasts) from other cellular components, providinggood purity However, not every protein present in such preparations can be con-sidered as a membrane protein For example, when vesicles will form from largerstructures upon lysis, simple physical entrapment will bring soluble proteins in thelumen of the vesicles Furthermore, it appears more and more clearly that someclasses of proteins (e.g cytoskeletal proteins or ribosomal proteins) are directly orindirectly associated to bona fide transmembrane proteins or to the lipid bilayer.Should these proteins be classified as membrane proteins? Should the non-membranespanning subunits of respiratory complexes be considered as membrane proteins?This gives an example of the debate that can take place on the notion of mem-brane proteins

This could be seen as a very theoretical debate, but this has indeed very practicalimplications Let us take the example of the analysis of a reticulum preparation.Endoplasmic reticulum is the place of synthesis of most secreted proteins and ofmost transmembrane proteins As such, it contains many ribosomes (the Rough ER)and the reticulum vesicles are known to be associated with cytoskeletal filaments.The problem arises from the fact that the protein content of these cytoskeletal andribosomal “contaminants” is concentrated in a few proteins This means in turn that

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even if these “contaminants” represent a few percent of the total protein mass of thepreparation, the few proteins represented will be easily detected by any proteomicsanalysis method

Conversely, the “real” reticulum proteins are scattered among almost all thesecreted proteins and transmembrane proteins of the cells, plus the resident pro-teins of the ER This means in turn that these proteins will appear as less prominentupon proteomics analysis, just because each protein species is more diluted than thefew “contaminating” ones

2 SAMPLE PREPARATION ISSUES FOR

MEMBRANE PROTEOMICS

A consequence of the above considerations is that sample preparation is very critical

in membrane proteomics As the volume occupied by the lipid bilayer in a membranesample is very small in comparison to the volume of the aqueous phase, the transmem-brane proteins must be considered as rare components of the sample, although theyoften drive most of the interest of the researchers In addition to that, these transmem-brane proteins often pose a very difficult solubilization problem Transmembraneproteins usually contain domains protruding in the extracellular environment or inthe cytosol These domains behave as classical protein domains and are therefore sta-ble in a water-based solvent However, membrane proteins also contain domains thatare embedded more or less deep in the lipid bilayer Consequently, these domains arestable in a hydrocarbon-like environment If a membrane protein is to be solubilizedprior to its purification, this means that the solvent which is used to this purpose mustshow at the same time water-like and hydrocarbon-like properties A water-based sol-vent will induce aggregation of the membrane domains via hydrophobic interactions,and thus protein precipitation However, an organic solvent will induce in most casesprecipitation of the membrane proteins via their water-soluble domains, which aredenatured and coalesce in such a solvent Some very hydrophobic proteins, however,are soluble in organic solvents (e.g Molloy et al 1999; Blonder et al 2003) This

is due to the reduced size of their non-membrane domains, and to the fact that allwater-soluble protein domains contain a hydrophobic core, which can be soluble inorganic solvents In some cases, these positive solubilization forces can overcomethe precipitation-deriving forces and make the protein soluble in organic solvents.This is however not a general case, and the general rule for membrane proteins isthat they require a solvent that has as the same time strong water-like and stronghydrocarbon-like properties This is not the situation of mixed solvents, that ismixtures of water and water-miscible solvents, which only offer average proper-ties and not a combination of both properties Such a combination is only offered by astable dispersion of hydrocarbon chains in a water-based solvent This is the definition

of lipid membranes, but such assemblies are very difficult to handle along a cation process Hopefully, this definition is also the one of detergent micelles, whichare much easier to handle along a purification process These constraints explain why

purifi-BERTRAND: “BERTRAND_CH01” — 2007/6/8 — 20:07 — PAGE 6 — #6

Conversely, detergent having a flexible tail and a strongly polar (i.e ionic) headare viewed as “strong” detergents In addition of their lipid solubilizing properties,these detergents are able to disrupt the hydrophobic interactions maintaining thestructure of the proteins They are therefore denaturing However, because of theirionic nature, the bound detergent imparts a net electrical charge to the denaturedproteins and induces a strong electrostatic repulsion between protein molecules Thus,even denatured proteins can no longer aggregate, and these ionic detergents are verypowerful protein solubilizing agents

These protein solubilization conditions have a key impact on the protein ation that can be carried out afterwards, in the sense that they will restrict the choice

fraction-to techniques with which they are compatible As an example, ionic detergents arenot compatible with any technique using protein charge or pI as the fractionationparameter

All of the above is mainly true when some fractionation is to be carried out at theprotein level In some approaches, the fractionation is carried out only on peptidesarising from the digestion of the membrane preparation In this case too, the choice ofthe solubilization media will be dictated by the constraints imposed by the subsequentpeptide fractionation process However, the use of chromatographic peptide fraction-ation tools, and especially those based on reverse-phase chromatography, will makethe use of detergents more problematic, leading to specially designed, detergent-freeprotocols (Wu et al 2003; Fischer et al 2006)

3 ISSUES LINKED TO THE SEPARATION PROCESS

Once the membrane proteins have been solubilized, usually in a water-detergentmedium, they must be fractionated As each fractionation method brings its ownconstraints, this will further restrict the scope of possibilities that can be used for

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sample solubilization The following sections will exemplify some constraints linked

to the most commonly used fractionation processes in proteomics

3.1 Constraints in Proteomics Based on Zone Electrophoresis

In these proteomics methods, the separation process is split in two phases (e.g in Bell

et al 2001) The first phase is a protein separation by denaturing zone electrophoresis,that is in the presence of denaturing detergents, most often sodium dodecyl sulfate(SDS) The second phase is carried out by chromatography on the peptides produced

by digestion of the separated proteins This has no impact on the sample preparationitself, which just needs to be compatible with the initial zone electrophoresis.This is by far the simplest case Sample preparation is achieved by mixing theinitial sample with a buffered, concentrated solution of an ionic detergent, usuallycontaining a reducer to break disulfide bridges and sometimes an additional nonionicchaotrope such as urea Ionic detergents are among the most powerful protein dena-turing solubilizing agents Their strong binding to proteins make all proteins to bear

an electric charge of the same type, whatever their initial charge may be This induces

in turn a strong electrostatic repulsion between protein molecules, and thus mal solubility The system of choice is based on SDS, as this detergent binds ratheruniformly to proteins However, SDS alone at room temperature, even at high con-centrations, may not be powerful enough to denature all proteins This is why heating

maxi-of the sample in the presence maxi-of SDS is usually recommended The additional uration brought by heat synergizes with SDS to produce maximal solubilization anddenaturation However, some hydrophobic transmembrane proteins do not withstandthis heating step In this case, their hydrophobic parts coagulate through the effect ofheat much faster than binding of SDS can solubilize them This leads to precipitation

denat-of these proteins

The use of SDS is not always without drawbacks One of the most important isencountered when the sample is rich in DNA A terrible viscosity results, which canhamper the electrophoresis process Moreover, some protein classes (e.g glycopro-teins) bind SDS poorly and are thus poorly separated in the subsequent electrophoresis

In such cases, it is advisable to use cationic detergents They are usually less potentthan SDS, so that a urea-detergent mixture must be used for optimal solubilization(MacFarlane 1989) Moreover, electrophoresis in the presence of cationic detergentsmust be carried out at a very acidic pH, which is not technically simple but stillfeasible (MacFarlane 1989) This technique has however gained recent popularity

as a double zone electrophoresis method able to separate even membrane proteins(Hartinger et al 1996), and showing more separation power than SDS electrophoresisalone

An important and often overlooked variegation of zone electrophoresis-basedseparations is the mixed native-denaturing two-dimensional (2D) electrophoresisdeveloped mainly by Schägger and coworkers (Schägger and Von Jagow 1991).Because the first dimension is a native electrophoresis, this approach provides invalu-able information upon the assembly of membrane complexes However, it poses

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in turn tricky solubilization problems, as the powerful ionic detergents cannot beused because of their denaturing power The answer to this difficult problem is thecombined used of a salt (or a dielectric compound such as aminocaproic acid), aneutral, non-denaturing detergent and a reagent bringing additional electrical charges

to the protein complexes to increase their solubility and prevent their aggregationduring electrophoresis This charge-transfer reagent is usually a protein dye such asCoomassie Blue

Because of the complexity of this solubilization problem, the optimization of thesystem is usually difficult, and this system has been most often used for mitochondrialand chloroplastic membrane complexes (reviewed in Nijtmans et al 2002), where ithas shown the ability to analyze even very hydrophobic membrane proteins (Devreese

et al 2002) However, this separation approach has been recently applied to wholecell extracts (Camacho-Carjaval et al 2004) and also to nuclear proteins (Novakova

et al 2006)

3.2 Constraints Proteomics Based on Two-dimensional

Gel Electrophoresis

In this scheme, the proteins are first separated by isoelectric focusing (IEF) followed

by SDS electrophoresis The constraints made on sample preparation are thus thoseinduced by the IEF step One of these constraints is the impossibility to use ionicdetergents at high concentrations, as they would mask the protein charge and thusdramatically alter its isoelectric point (pI) Ionic detergents can however be used atlow doses to enhance initial solubilization (Wilson et al 1977), but their amount

is limited by the capacity of the IEF system (in terms of ions tolerated) and by theefficiency of the detergent exchange process with takes place during the IEF step.Another major constraint induced by IEF is the requirement for low ionic strength,induced by the high electric fields required for pushing the proteins to their isoelectricpoints This means in turn that only uncharged compounds can be used to solubilizeproteins, that is neutral chaotropes and detergents The basic solubilization solutionsfor IEF thus contain high concentrations of a non-ionic chaotrope, historically ureabut now more and more a mixture of urea and thiourea (Rabilloud et al 1997),together with a reducer and a nonionic detergent While CHAPS and Triton X-100are the most popular detergents, it has been recently shown that other detergents canenhance the solubility of proteins and give better performances (Chevallet et al 1998;Luche et al 2003) In this solubilization process, detergents play a multiple role Theybind to proteins and help to keep them in solution, but they also break protein-lipidsinteractions and promote lipid solubilization

Last but certainly not least, it must be recalled that proteins are at their pI at the end

of the IEF process, and the pI is the solubility minimum This means that solubilityproblems are very important in IEF This is especially true with native proteins, andIEF is thus best performed with denatured proteins However, even in this case,many proteins such as membrane proteins are poorly soluble under IEF conditions.Compared with detergent-based electrophoresis, this problem is further enhanced by

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the fact that only electrically neutral detergents can be used Ionic detergents wouldmodify the pI of the proteins and thus prevent any correct separation through thisparameter This however means in turn that the beneficial electrostatic repulsion effectcannot be used in isoelectric focusing, thereby leaving proteins under conditions inwhich their solubility is minimal While this is not so much a problem for proteinsthat are normally water-soluble, this turns to be a major problem for proteins that arepoorly water-soluble, and especially transmembrane proteins This explains why thisclass of proteins is strongly under-represented in this type of separation (for reviewsee Santoni et al 2000)

3.3 Constraints in Peptide Separation-Based Approaches

In these methods, the sample preparation process is designed to offer optimal digestion

of the proteins into peptides This means that proteins must be extracted from thesample and denatured to maximize exposure of the protease cleavage sites This alsomeans that the protease used for peptide production must be active in the separationmethod In this case, the robustness of many proteases is a clear advantage Classicalextraction media usually contain either multimolar concentrations of chaotropes ordetergents In the latter case, the sample is usually solubilized and denatured in highconcentrations of ionic detergents, and simple dilution is used to bring the detergentconcentration down to a point compatible with other steps such as chemical labeling

or proteolysis

The choice between chaotropes and detergent is driven mainly by the constraintsimposed by the peptide separation method In the wide-scope approach based ononline two-dimensional chromatography of complex peptide mixtures (Washburn

et al 2001), both the ion exchange and reverse phase steps are very sensitive todetergents It must be mentioned that these online two-dimensional chromatographicmethods are one of the rare cases where the interface between the two separationmethods does not bring extra robustness, so that the sample preparation must be com-patible with both chromatographic methods The modification approach (Gevaert

et al 2003), which uses intensively reverse phase chromatography, is also very tive to detergent interference This rules out the use of detergent and favors the use ofchatropes, generally nonionic ones because of the ion exchange step Urea is used forthese methods, with possible artefacts induced by urea-driven carbamylation of thesample during the lengthy digestion process Inclusion of thiourea and lowering of theurea concentration could decrease the incidence of carbamylation in these methods

sensi-In methods where a detergent-resistant method is used, for example avidin selection

of biotinylated peptides (Gygi et al 1998), extraction by SDS is clearly the method

of choice, as the above-mentioned drawbacks are absent

3.4 Constraints in Protein Chromatography-Based Approaches

In these methods, the separation is carried out by a chromatographic setup Thissetup may use various chromatographic principles for protein separation, and this

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choice will of course alter the possibilities for protein solubilization For example,the use of an ion-exchange step (Szponarski et al 2004) will preclude the use of ionicdetergents and thus produce a solubilization situation close to the one observed in 2Dgels However, an important advantage of ion exchange over chromatofocusing or IEF

is that the proteins do not need to reach their pI in this separation scheme This allows

in turn to increase protein solubility, which is a major concern in transmembraneprotein analysis

Here again, not all chromatographic setups are usable for any proteomics question.The use of protein reverse phase chromatography, which has been advocated forplasma proteomics (Moritz et al 2005), precludes in turn the use of any detergent

of any type This prevents the use of this chromatographic setup in most subcellularproteomics experiments, where detergents must be used to solubilize the membranelimiting the subcellular compartments

4 CONCLUDING REMARKS

It should be obvious from the above that membrane proteomics strictly followsMurphy’s law This is due to the fact that there is a mutual exclusion, for physico-chemical reasons, between on the one hand the conditions that must be used tosolubilize in water all membrane proteins, including the most hydrophobic ones, andthus give a fair representation of the protein population in the sample, and on the otherhand the conditions prevailing in the high resolution peptide separation methods Ontop of this problem, there is a second problem linked to the membrane versus aque-ous phase volume in many subcellular preparations, which makes transmembraneproteins rare compared to water-soluble proteins

Thus, everything concurs in making membrane proteomics probably one of themost difficult sub-field in proteomics Up to now, the proteomics toolbox that we canuse has proven quite imperfect to provide a thorough, quantitative and precise (i.e.including post-translational modifications analysis) analysis of membrane proteins

REFERENCES

Bell, A.W., Ward, M.A., Blackstock, W.P., Freeman, H.N., Choudhary, J.S., Lewis, A.P., Chotai, D., Fazel, A., Gushue, J.N., Paiement, J., Palcy, S., Chevet, E., Lafreniere-Roula, M., Solari, R., Thomas, D.Y., Rowley, A and Bergeron, J.J (2001) Proteomics characterization of abundant Golgi

membrane proteins J Biol Chem 276, 5152–5165.

Blonder, J., Conrads, T.P., Yu, L.R., Terumuma, A., Janini, G.M., Issaq, H.J., Vogel, J and Veenstra, T.D (2004) A detergent- and cyanogen bromide-free method for integral membrane proteomics: application

to Halobacterium purple membranes and human epidermis Proteomics 4, 31–45.

Camacho-Carvajal, M.M., Wollscheid, B., Aebersold, R., Steimle, V and Schamel, W.W (2004) dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular

Two-lysates: a proteomics approach Mol Cell Proteomics 3, 176–182.

Chevallet, M., Santoni, V., Poinas, A., Rouquié, D., Fuchs, A., Kieffer, S., Rossignol, M., Lunardi, J., Garin, J and Rabilloud, T (1998) New zwitterionic detergents improve the analysis of membrane

proteins by two-dimensional electrophoresis Electrophoresis 19, 1901–1909.

Cross, G.A (1990) Glycolipid anchoring of plasma membrane proteins Ann Rev Cell Biol 6, 1–39.

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Devreese, B., Vanrobaeys, F., Smet, J., Van Beeumen, J and Van Coster, R (2002) Mass ric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional

spectromet-blue-native polyacrylamide gel electrophoresis Electrophoresis 23, 2525–2533.

Fischer, F., Wolters, D., Rogner, M and Poetsch, A (2006) Toward the complete membrane proteome: high coverage of integral membrane proteins through transmembrane peptide detection Mol Cell.

Proteomics 5, 444–453.

Gevaert, K., Goethals, M., Martens, L., Van Damme, J., Staes, A., Thomas, G.R and Vandekerckhove, J (2003) Exploring proteomes and analyzing protein processing by mass spectrometric identification of

sorted N -terminal peptides Nat Biotechnol 21, 566–569.

Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H and Aebersold, R (1999) Quantitative analysis

of complex protein mixtures using isotope-coded affinity tags Nat Biotechnol 17, 994–999.

Hartinger, J., Stenius, K., Hogemann, D and Jahn, R (1996) 16-BAC/SDS-PAGE: a two-dimensional gel

electrophoresis system suitable for the separation of integral membrane proteins Anal Biochem 124,

126–133.

Iwata, S., Lee, J.W., Okada, K., Lee, J.K., Iwata, M., Rasmussen, B., Link, T.A., Ramaswamy, S and Jap, B.K (1998) Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex.

Science 281, 64–71.

Luche, S., Santoni, V and Rabilloud, T (2003) Evaluation of nonionic and zwitterionic detergents as

membrane protein solubilizers in two-dimensional electrophoresis Proteomics 3, 249–253.

Macfarlane, D.E (1989) Two dimensional benzyldimethyl-n-hexadecylammonium chloride-sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis: a high capacity high resolution

technique for the purification of proteins from complex mixtures Anal Biochem 176, 457–463 Molloy, M.P., Herbert, B.R., Williams, K.L and Gooley, A.A (1999) Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis Electrophoresis 20, 701–704.

Moritz, R.L., Clippingdale, A.B., Kapp, E.A., Eddes, J.S., Ji, H., Gilbert, S., Connolly, L.M and Simpson, R.J (2005) Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis

of human plasma depleted of multi high-abundance proteins Proteomics 5, 3402–3413.

Nijtmans, L.G., Henderson, N.S and Holt, I.J (2002) Blue Native electrophoresis to study mitochondrial

and other protein complexes Methods 26, 327–334.

Novakova, Z., Man, P., Novak, P., Hozak, P and Hodny, Z (2006) Separation of nuclear protein complexes

by blue native polyacrylamide gel electrophoresis Electrophoresis 27, 1277–1287.

Rabilloud, T., Adessi, C., Giraudel, A and Lunardi, J (1997) Improvement of the solubilization of proteins

in two-dimensional electrophoresis with immobilized pH gradients Electrophoresis 18, 307–316.

Santoni, V., Molloy, M.P and Rabilloud, T (2000) Membrane proteins and proteomics: un amour

imposssible? Electrophoresis 21, 1054–1070.

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complexes in enzymatically active form Anal Biochem 199, 223–231.

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Corresponding author: Dr Thierry Rabilloud (thierry.rabilloud@cea.fr)

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CHAPTER 2

TWO-DIMENSIONAL BAC/SDS-PAGE FOR

MEMBRANE PROTEOMICS

RENÉ P ZAHEDI, JAN MOEBIUS, and ALBERT SICKMANN

University of Wuerzburg, Germany

Abstract: Although often used in membrane proteome studies, conventional two-dimensional gel

elec-trophoresis (2-DE) is not well suited for resolving hydrophobic proteins Nevertheless, an alternative technique, two-dimensional BAC/SDS-PAGE (2-DB) using the cationic deter-

gent benzyldimethyl-n-hexadecylammonium chloride (BAC) in the first and the anionic

detergent SDS in the second dimension can be utilized as a powerful tool for the tion and analysis of membrane proteins Systematic studies demonstrated the advantage of 2-DB over one-dimensional SDS-PAGE and 2-DE with regard to membrane proteomics While in 2-DE gels, in particular proteins with more than one transmembrane domain (TMD) are underrepresented, one-dimensional SDS-PAGE lacks sufficient resolution for large scale analyses In contrast, 2-DB enabled the identification of extremely hydropho-

separa-bic proteins like cytochrome-c oxidase subunit I from S cerevisiae with a total of 12

known TMD Especially the application of tube gels in the first dimension as well as the recent introduction of improved buffer systems hold a great potential for future 2-DB-based membrane studies.

13

E Bertrand and M Faupel (eds.), Subcellular Proteomics, 13–20.

© 2007 Springer.

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1 INTRODUCTION

The separation of membrane proteins via conventional two-dimensional gel trophoresis (2-DE) consisting of a first dimension isoelectric focussing step and asubsequent second dimension SDS-PAGE is, although still widely used, stronglybiased against hydrophobic proteins One the one hand, IEF is limited to theusage of zwitterionic or non-ionic detergents, providing weak solubilizing capa-bilities with regard to hydrophobic proteins in contrast to strong ionic detergents,for example SDS On the other hand, proteins tend to precipitate upon reach-ing their isoelectric points (pI) or during the transfer to the second dimension –this is particularly true for membrane proteins Furthermore, membrane proteinsoften have pIs in the alkaline region, which in 2-DE is generally characterized byinferior resolution

elec-Although protocols have been constantly improved in recent years (Molloy 2000;Olsson et al 2002; Luche et al 2003), hydrophobic membrane proteins, especiallythose with multiple transmembrane domains (TMD) are generally underrepresented

in 2-DE based proteome studies (Santoni et al 2000) To account for these inherentlimitations alternative electrophoretic techniques have to be applied

While common one-dimensional SDS-PAGE is a powerful tool for separatinghydrophobic membrane proteins (Reinders et al 2006a), it only provides minorresolution of complex protein samples, and consequently is not suited for differ-ential analyses

However, alternative 2-DE methodologies, for instance combining SDS-PAGEwith PAGE systems based on the usage of cationic detergents like benzyldimethyl-

n-hexadecylammonium chloride (BAC) (Macfarlane 1983, 1989; Hartinger et al.

1996) or cetyltrimethylammonium bromide (CTAB) (Buxbaum 2003), enablingtwo-dimensional separation of hydrophobic membrane proteins, have gained moreattention in recent years Thereby, extremely hydrophobic membrane proteins withmultiple TMD can be separated with relatively high resolution for subsequentidentification by mass spectrometry

2 TWO-DIMENSIONAL BAC/SDS POLYACRYLAMIDE

GEL ELECTROPHORESIS

First introduced by Macfarlane et al for the separation of base labile tein methylation (Macfarlane, 1983), BAC-PAGE was then improved towards atwo-dimensional technique by combination with a subsequent second dimensionSDS-PAGE (Macfarlane, 1989) Although, both dimensions comprise a separationaccording to the molecular weight, slight differences in protein migration propertieswithin the two systems lead to an enhanced resolution, resulting in an elliptical sepa-ration area Already in 1996, Hartinger et al demonstrated the potential of combinedtwo-dimensional BAC/SDS-PAGE (2-DB) for the separation of membrane proteins(Hartinger et al 1996) The second dimension SDS-PAGE provides full compati-bility with downstream methods like Western Blotting, a broad variety of staining

pro-BERTRAND: “BERTRAND_CH02” — 2007/6/7 — 21:18 — PAGE 15 — #3

Two-dimensional BAC/SDS-PAGE for membrane proteomics 15

Figure 1 Scheme of a two-dimensional BAC/SDS PAGE using slab gels (A) Protein samples are

sep-arated on the first dimension BAC-PAGE After visualization of proteins, an entire gel lane is excised (B) The excised lane is re-buffered in 100 mM Tris, pH 6.8 for 30 min and afterwards incubated in 3 × SDS sample buffer for another 5-10 min in order to exchange BAC for SDS (C) The gel lane is transferred onto the second dimension SDS gel and fixed with a hot agarose solution After separation staining reveals

a characteristic spot pattern within an elliptical area.

techniques and mass spectrometric analysis Therefore, in recent years several studieshave revealed the great potential of this alternative technique for membrane pro-teomics (Otto et al 2001; Daub et al 2002; Diao et al 2003; Godl et al 2003;Coughenour et al 2004; Zahedi et al 2006)

3 GENERAL WORKFLOW

The general scheme of 2-DB is depicted in Figure 1: Samples are first separatedtowards the cathode in an acidic PAGE system based on the cationic detergentBAC Afterwards, protein lanes can be visualized by direct immersion into a col-loidal Coomassie staining solution However, since Coomassie tends to precipitate inpresence of cationic detergents which in turn leads to enhanced background staining,

an intermediate washing step of at least 2 h is recommended in order to remove BACfrom the gel surface

After visualization whole protein lanes are excised and prepared for the seconddimension Since SDS-PAGE utilizes a more alkaline buffer system than BAC-PAGE,first of all gel lanes have to be re-buffered Afterwards, BAC is exchanged for SDS

by incubation in 3× SDS sample buffer Finally, protein lanes are transferred onto asecond dimension gel for separation and fixed by a hot agarose solution

4 IMPROVEMENTS

While less complex samples can generally be separated using small gel sizes imately 7× 7 cm) in both dimensions, for samples of higher complexity switching

(approx-BERTRAND: “BERTRAND_CH02” — 2007/6/7 — 21:18 — PAGE 16 — #4

66 55

36 158

BAC

SDS

31 21 14

Figure 2 Two-dimensional BAC/SDS-PAGE of platelet membrane proteins While not only large gels are

recommended for complex samples, utilizing tube gels for the first dimension furthermore provides better resolution and more efficient transfer of proteins to the second dimension (less vertical smearing) (A) Small slab gel in the first dimension(7 × 7 cm) (B) Large tube gel in the first dimension (1 mm × 15 cm).

(C) Summary of the human platelet membrane proteome study (Moebius et al 2005) 158 proteins were exclusively identified from 2-DB gels, 65 from 1D-PAGE An overlap of 75 proteins was identified from both types of gels.

to larger high resolution gels is recommended (Figure 2) Here, slab gels as well astube gels can be utilized However, the usage of slab gels is much more complicated

in terms of handling (Figure 3)

In general, first dimension BAC-PAGE requires higher voltages and prolonged ning times than SDS-PAGE, resulting in an increased heat development – especially

run-in case of large dimension slab gels Therefore, coolrun-ing of runnrun-ing buffers durrun-ingseparation is mandatory

Besides, an incomplete transfer of proteins from the first to the second dimensioncan be noticed for slab gels The usage of tube gels with inner diameters of 1 mm andless totally abolishes this limitation and furthermore provides an improved resolutionleading to a higher number of identifications in proteome studies For this reason, theusage of tube gels is essential for differential studies Moreover, after first dimensionseparation, time-consuming staining procedures which might be accompanied by loss

of material can be omitted, as the entire gel can be transferred to the second dimensionwithout the need for prior excision

Resulting from our experience in the separation of a broad variety of samples with2-DB, resolution strongly depends on the nature of the separated sample Furthermore,upon long separation times, band broadening during first dimension separation can

be observed

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Two-dimensional BAC/SDS-PAGE for membrane proteomics 17

Figure 3 Processing of large slab (left) and tube (right) gels Since transferring whole gel lanes of

approx-imately 15–20 cm length onto polymerized second dimension gels mostly results in extensive damage of the gel, a more complicated but also safer strategy is recommended here First the excised gel lane is placed and fixed between two glass plates Then the separation gel is poured beneath the lane Finally, a stacking gel is poured on top, enclosing the first dimension gel lane In case of tube gels, the gel is transferred onto the already polymerized second dimension separation gel and fixed by hot agarose solution.

In general, after membrane purification, it is highly recommended to introduceadditional steps like carbonate- (Fujiki et al 1982) and/or Triton X114-extraction prior

to electrophoresis in order to reduce the amount of contaminating soluble proteins.Especially when separating plasma membrane enriched samples, resolution may beimpaired in both dimensions In that case protein precipitation prior to 2-DB mayresult in an improved separation, however it has to be kept in mind that precipitationprocedures are generally not quantitative and might lead to unspecific loss of material.Recently, an improved BAC-PAGE protocol was introduced, particularly compen-sating for the inferior efficiency of the stacking gel when compared to commonSDS-PAGE systems (Kramer 2006) By systematic studies the composition ofgel buffers, running buffers as well as the sample buffer were optimized, result-ing in a higher resolution and shorter separation time, comparable to SDS-PAGE(Kramer 2006) Although the impact of these improvements was only investigatedfor one-dimensional BAC-PAGE, they nevertheless hold a great potential for 2-DB

as well However, this remains to be demonstrated in future studies

5 POTENTIAL FOR MEMBRANE PROTEOME STUDIES

In systematic studies we demonstrated the potential of 2-DB compared to 2-DE(Zahedi et al 2005) and SDS-PAGE (Moebius et al 2005) regarding the separation

of membrane proteins

While 2-DE separation of purified endoplasmatic reticulum membranes from Canis

familiaris yielded only a few spots after visualization by silver staining, an unequal

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higher amount of protein spots could be visualized after 2-DB Among them, Sec61α

with a total of ten known TMD and a grand average hydrophobicity (GRAVY) index

of 0.558 could be identified by Western blotting as well as mass spectrometry thermore, 54 distinct ribosomal proteins were identified, which cannot be sufficientlyresolved by 2-DE since their pIs range from 9 to 12 Furthermore, the separation of

Fur-mitochondrial membranes from Saccharomyces cerevisiae yielded the subsequent

identification of the extremely hydrophobic cytochrome-c oxidase subunit I with atotal of 12 known TMD and a GRAVY index of 0.74 by mass spectrometry (MS)

In a comprehensive study of the human platelet membrane proteome we strated the need for a combined application of 2-DB and one-dimensional SDS-PAGEfor maximizing the amount of identified proteins Since both techniques addressdifferent subproteomes (Figure 2C) they may be utilized in a complementary way

demon-In 2-DB, a higher resolution increases the local protein concentration and tates identification However, in SDS-PAGE, whole lanes can be cut into equidistantslices, eliminating the need for protein visualization prior to excision Thereby, evenproteins, which cannot be visualized by staining procedures and therefore will escapedetection after 2-DB, can be identified by subsequent MS

facili-6 COMPARISON TO OTHER TECHNIQUES

Another two-dimensional PAGE technique, doubled SDS (dSDS), introduced by Rais

et al (2004), is capable of resolving hydrophobic proteins with GRAVY indices of

up to 0.86 – however it is has a lower resolution compared to 2-DB due to a smalleraccessible separation area

Since due to the lower resolution in 2-DB gels mostly several proteins co-localizewithin a single spot, in contrast to conventional 2-DE the usage of LC-MS/MS forprotein identification is recommended instead of MALDI-MS

Table 1 summarizes advantages and properties of the presented electrophoreticmethods regarding (membrane) proteome studies

7 OUTLOOK

Two-dimensional BAC/SDS polyacrylamide gel electrophoresis has been established

as further tool in the field of proteome research, especially regarding the separationand analysis of membrane proteins It is by far more efficient in resolving membraneproteins than common 2-DE and furthermore can be utilized in a complementary way

to one-dimensional SDS-PAGE Therefore, among other techniques, future proteomestudies focussing on membrane proteins should include 2-DB as well

Despite its lower resolution compared to 2-DE, 2-DB can also be applied incombination with the difference gel electrophoresis technique (DIGE) (Unlu et al.1997; Reinders et al 2006b), enabling the highly reproducible differential analysis

of biological membrane samples

However, for complex sample mixtures DIGE requires a very high resolution anddistinct protein spots Although the afore mentioned improvements by Kramer further

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Two-dimensional BAC/SDS-PAGE for membrane proteomics 19

Table 1 Comparison of different PAGE methods

Separation area Small lane Entire gel Elliptical area Elliptical area

Only visualized spots

Only visualized spots Recommended

MS strategy

enhance resolution during first dimension BAC-PAGE, additional prefractionation ofcomplex mixtures might be necessary in order to account for the high demands ofdifferential gel electrophoresis

Coughenour, H.D., Spaulding, R.S and Thompson, C.M (2004) The synaptic vesicle proteome: a

comparative study in membrane protein identification Proteomics 4, 3141–3155.

Daub, H., Blencke, S., Habenberger, P., Kurtenbach, A., Dennenmoser, J., Wissing, J., Ullrich, A and Cotten, M (2002) Identification of SRPK1 and SRPK2 as the major cellular protein kinases

phosphorylating hepatitis B virus core protein J Virol 76, 8124–8137.

Diao, A., Rahman, D., Pappin, D.J., Lucocq, J and Lowe, M (2003) The coiled-coil membrane protein

golgin-84 is a novel rab effector required for Golgi ribbon formation J Cell Biol 160, 201–212.

Fujiki, Y., Hubbard, A.L., Fowler, S and Lazarow, P.B (1982) Isolation of intracellular membranes by

means of sodium carbonate treatment: application to endoplasmic reticulum J Cell Biol 93, 97–102.

Godl, K., Wissing, J., Kurtenbach, A., Habenberger, P., Blencke, S., Gutbrod, H., Salassidis, K., Stein-Gerlach, M., Missio, A., Cotten, M and Daub, H (2003) An efficient proteomics method

to identify the cellular targets of protein kinase inhibitors Proc Natl Acad Sci U.S.A 100,

15434–15439.

Hartinger, J., Stenius, K., Hogemann, D and Jahn, R (1996) 16-BAC/SDS-PAGE: a two-dimensional gel

electrophoresis system suitable for the separation of integral membrane proteins Anal Biochem 240,

126–133.

BERTRAND: “BERTRAND_CH02” — 2007/6/7 — 21:18 — PAGE 20 — #8

Kramer, M.L (2006) A new multiphasic buffer system for benzyldimethyl-n-hexadecylammonium ride polyacrylamide gel electrophoresis of proteins providing efficient stacking Electrophoresis 27,

chlo-347–356.

Luche, S., Santoni, V and Rabilloud, T (2003) Evaluation of nonionic and zwitterionic detergents as

membrane protein solubilizers in two-dimensional electrophoresis Proteomics 3, 249–253.

Macfarlane, D.E (1983) Use of benzyldimethyl-n-hexadecylammonium chloride (“16-BAC”), a cationic

detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein

methylation in intact cells Anal Biochem 132, 231–235.

Macfarlane, D.E (1989) Two dimensional benzyldimethyl-n-hexadecylammonium chloride – sodium

dodecyl sulfate preparative polyacrylamide gel electrophoresis: a high capacity high resolution

technique for the purification of proteins from complex mixtures Anal Biochem 176, 457–463.

Moebius, J., Zahedi, R.P., Lewandrowski, U., Berger, C., Walter, U and Sickmann, A (2005) The human platelet membrane proteome reveals several new potential membrane proteins Mol Cell Proteomics

4, 1754–1761.

Molloy, M.P (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH

gradients Anal Biochem 280, 1–10.

Olsson, I., Larsson, K., Palmgren, R and Bjellqvist, B (2002) Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first

dimension Proteomics 2, 1630–1632.

Otto, H., Dreger, M., Bengtsson, L and Hucho, F (2001) Identification of tyrosine-phosphorylated proteins

associated with the nuclear envelope Eur J Biochem 268, 420–428.

Rais, I., Karas, M and Schagger, H (2004) Two-dimensional electrophoresis for the isolation of integral

membrane proteins and mass spectrometric identification Proteomics 4, 2567–2571.

Reinders, J., Zahedi, R.P., Pfanner, N., Meisinger, C and Sickmann, A (2006a) Toward the Complete Yeast Mitochondrial Proteome: Multidimensional Separation Techniques for Mitochondrial Proteomics.

J Proteome Res 5, 1543–1554.

Reinders, Y., Schulz, I., Graf, R and Sickmann, A (2006b) Identification of novel centrosomal proteins

in Dictyostelium discoideum by comparative proteomic approaches J Proteome Res 5, 589–598.

Santoni, V., Molloy, M and Rabilloud, T (2000) Membrane proteins and proteomics: un amour impossible?

Electrophoresis 21, 1054–1070.

Unlu, M., Morgan, M.E and Minden, J.S (1997) Difference gel electrophoresis: a single gel method for

detecting changes in protein extracts Electrophoresis 18, 2071–2077.

Zahedi, R.P., Meisinger, C and Sickmann, A (2005) Two-dimensional hexadecylammonium chloride/SDS-PAGE for membrane proteomics Proteomics 5, 3581–3588.

benzyldimethyl-n-Zahedi, R.P., Sickmann, A., Boehm, A.M., Winkler, C., Zufall, N., Schonfisch, B., Guiard, B., Pfanner, N and Meisinger, C (2006) Proteomic analysis of the yeast mitochondrial outer membrane reveals

accumulation of a subclass of preproteins Mol Biol Cell 17, 1436–1450.

Corresponding author: Dr Albert Sickmann

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CHAPTER 3

MICROPARTICLES: A NEW TOOL FOR PLASMA

MEMBRANE SUB-CELLULAR PROTEOMIC

LAURENT MIGUET1, SARAH SANGLIER1, CHRISTINE SCHAEFFER1,NOELLE POTIER1, LAURENT MAUVIEUX2, and

ALAIN VAN DORSSELAER1

1Laboratoire de Spectrométrie de Masse Bio-Organique, ECPM, UMR/CNRS 7178, Institut

Pluridisciplinaire Hubert CURIEN, Université Louis Pasteur, Strasbourg, France

2Institut d’Hématologie et d’Immunologie, Faculté de Médecine, Université Louis Pasteur

and Laboratoire d’Hématologie, Hôpital Hautepierre, Strasbourg, France

4 Validation of Microparticles as a New Tool

for Plasma Membrane Preparation 30

5 Perspectives 31References 33

1 INTRODUCTION

Membranes are critical components of cellular structure It has been reported thatplasma membrane proteins represent about 30% of all cellular proteins (Wallin andvon Heijne 1998) Even if plasma membrane was considered for a long time as asimple biological barrier between the cytosol of the cell and the extra-cellular envi-ronment, these membrane proteins have been demonstrated to play a crucial role inthe different fundamental biological processes as exchange of component or signaltransduction Also, more than half of all anticipated pharmacological drug targets arepredicted to be localized to the plasma membrane (Jang and Hanash 2003) Plasmamembrane can then clearly be considered as a sub-cellular compartment of first inter-est in regard to different diagnosis and/or therapeutic target proteins Indeed, foreach membrane protein there is potentially a specific antibody which can be used

21

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© 2007 Springer.

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for the diagnosis of several pathologies and also for treatment using armed ies (Harris 2004) Therefore, proteomic analysis of plasma membrane proteins is

antibod-of first importance Despite the importance antibod-of plasma membrane proteins, there isless understanding in this class of proteins due to the difficulty to obtain enrichedplasma membrane proteins preparation from eukaryotic cells Until now many dif-ferent strategies have been applied but are still laborious and imperfect In example

of these different approaches, biotinylation (Peirce et al 2004; Zhao et al 2004)silica coated (Rahbar and Fenselau 2004), partition phase repartition (Qoronfleh et

al 2003) or partial tryptic surface digestion have been tested, but they still remainunsatisfactory Although some studies of plasma membrane proteins using 2-DE havebeen reported (Galeva and Altermann 2002; Luche et al 2003), but the separation ofsuch hydrophobic proteins have been often poor (Santoni et al 2000) We describehere a new strategy in order to increase the proportions of plasma membrane proteinsidentified with the highest possible coverage percentage in the different membraneproteins preparations analyzed by mass spectrometry We will focus on microparticles

as a source of plasma membrane proteins

2 MICROPARTICLES

Each of the two leaflets of the plasma membrane bilayer has a specific lipid position Aminophospholipids (phosphatidylserine and phosphatidylethanolamine)are specifically segregated in the inner layer of the membrane, whereas the phos-phatidylcholine and the sphingomyelin are enriched in the external leaflet (Bevers

com-et al 1998) When cells are submitted to various stress conditions as mitogenic tions or apoptosis, the constitutive asymmetry between the inner and the outer leaflet

activa-of the plasma membrane is disrupted The major changes in the plasma membraneconstitution will be the delocalization of the phosphatidylserine to the outer leafletand an augmentation of the Ca2+ion concentration in the cytoplasm Such changesare going to disrupt the organisation of the cytoskeleton and drive to a blebbing of theplasma membrane and release microvesicles which are named microparticles (MPs)(Figure 1) (Hugel et al 2005; Miguet et al 2005)

Such microvesicles have size variable between 50 nm to 1µm and differ from othervesicles (like exosomes (30–100 nm)) In general, microparticles are phospholipidsvesicles derived from eukaryotic cells as a result of different types of stimulation.Microparticles can also be defined as phospholipids microvesicles containing certainmembrane proteins originating from the parental cell Microparticles circulate in theblood and contribute to numerous physiological processes MPs have been described

in various haematopoietic cells as platelets (Heijnen et al 1999), T-cells (Blanchard

et al 2002), polynuclear neutrophils (Mesri and Altieri 1999) or dendritic cells.After have been considered as cell dust, MPs are now considered to reflect cellactivation Platelet derived microparticles have been the most extensively studieduntil now They are now accepted to play an important role in the procoagulant