Protein Arrays, Biochips, and Proteomics

423 Pages·2003·4.79 MB·English

by  Joanna S. Albala

Protein Arrays,
Biochips,
and Proteomics
The Next Phase of Genomic Discovery
edited by
Joanna S. Albala
Lawrence Livermore National Laboratory
Livermore, California, U.S.A.
Ian Humpheiy-Smith
University of Utrecht
Utrecht, The Netherlands
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Foreword
Duringtheleanyearsofproteomics,thefieldwaslargelydominatedbytechniques
suchastwo-dimensionalgelelectrophoresis.Morerecently,thespectacularinno-
vationsinmassspectrometryhavegivenproteomicsashotinthearmandtrans-
formed the discipline. The complete sequencing of the human genome and that
of other model organisms has further boosted proteomics in many ways, not
leastbyprovidingasequence-basedframeworkforminingthehumanandother
proteomes.Clearly,however,tomakeasubstantialimpactinbiomedicine,from
disease-marker identification to accelerating drug development, proteomics has
toevolvemuchfurtherinthedirectionofprovidinghigh-throughput,high-sensi-
tivity, proteome-scale profiling. Unlike genomic-type profiling, which tends to
be unidimensional, as exemplified by DNA microarrays that allow RNA abun-
danceto bemeasured,there isaneed attheprotein leveltocapture amultitude
ofproteinattributes.Thereisalsoaneedtodetermineinacellandtissuecontext
not just the abundance of protein constituents but also their posttranslational
modifications, aswell as their functionalstates and theirinteractions with other
proteins and molecules, all with requisite high-throughput and high-sensitivity.
The emerging field of protein biochips and microarrays is intended to address
suchneedsandwilllikelymarkyetanotherevolutioninproteomics.Thestakes
are high and the challenges are enormous.
Themilestonesinanyemergingfieldsoonerorlaterincludethepublication
ofbooksthatreviewprogressandprovidebothcriticalandforward-lookingper-
spectives.ThisisthecaseforthistimelybookwiththecatchytitleProteinArrays,
Biochips, and Proteomics: The Next Phase of Genomic Discovery. The editors
have all the desired credentials and are well-suited for the task of assembling
contributingauthorswhoareexpertsinthefield.Theeditorshavedevotedmuch
iii
iv Foreword
effort in their careers to activities that define the current status of protein chips
and microarrays. They are very well connected and are prominently featured in
meetings devoted to the subject.
Commensurate with the need to assay a wide range of protein attributes,
anequallywiderangeofchiptypeshavebecome availablethatarereviewedin
thisbookwithrespecttotheirmeritsandlimitations.Innovativetechnologiesin
this field have been developed by academics and by biotechnology companies,
thuscontributingcreativesolutionstochallengingproblems.However,themost
challengingproblemofall—deliveringcontentonaproteomescale—isbeyond
thereachofbothacademicsandmostbiotechcompanies,simplybecauseofthe
very high costs involved in producing the tens—and more likely hundreds—of
thousands ofproteins encodedjust in thehuman genome,or to producecapture
agents directed against these proteins and their various epitopes. A consortium
approachnotunlikethatputtogetherforsequencingthegenomeorforcataloging
genome-wide single-nucleotide polymorphisms may need to be implemented to
meetthischallenge.Strategicconsiderationssuchasthesearebeingpursued,for
example,bytheHumanProteomeOrganizationwithitsproteome-scaleantibody
initiative.
So what is in this book for the reader? Obviously, not all applications of
protein chips need to be on a proteome scale. Much could be accomplished,
particularlybyacademic investigators,throughfocusedapproaches thattargeta
familyofproteins,aspecificsignalingpathway,oraparticularposttranslational
modification. This book contains a wealth of information that brings the reader
up to date in the field of proteomics, protein biochips, and array-based protein
strategies,fromthetheoreticaltothepracticalaspects,withtopicsrangingfrom
functionalizedchip surfacesand theperformance ofultrasensitiveligand assays
using microarrays to strategies for expressing proteins. There is even a chapter
thatreviewstheproteomicsmarketinitsvariousaspects.Thetextiseasytoread,
asarethenumerousfiguresandchartsbefittingabookonchipsandmicroarrays.
Itisrathergratifying toseethatthefieldof proteomicsnowencompasses
chemical engineers, analytical chemists, biochemists, cell and molecular biolo-
gists,clinicalscientists,andbioinformaticians,justtolistafewofthesubspecial-
ties. I am confident that people in the field of proteomics or those who are
contemplatingusingproteomics,howevervariedtheirinterests,willderivevalua-
ble knowledge from reading this book.
Sam Hanash
President, Human Proteome Organization
Professor of Pediatrics
University of Michigan
Ann Arbor, Michigan, U.S.A.
Preface
Wasinger and colleagues (Electrophoresis, 1995, 16: 1090–1094) first defined
the term proteome as: ‘‘the total protein complement able to be encoded by a
given genome.’’ It is important to note that this encoded complement can vary
significantly,temporally,andwithrespecttocellandtissuetype,whilethetem-
poral variation can occur over very short time intervals. In an immunological
context it is this antigenic diversity (temporal, cellular, and tissue-specific) that
constitutesself.Acentraltenetofmodernimmunologyisthathealthyindividuals
withdevelopinglymphocytesmustbeexposedtomostofself,soastoavoidthe
dysfunctional state of autoimmunity. Thus, on a daily basis, the human body
is faced with—and presumably succeeds at—the task of teaching developing
lymphocytesthenatureofselfantigens,i.e.,thehumanproteomeinitsinnumera-
bleiterations.Currently,however,experimentalproteomicsisfarfromachieving
similaranalyticalsuccess;thetaskofaccessinganddetectingallelementswithin
an entire mammalian proteome looms as an almost insurmountable charge, due
mostly to the predominance of low-abundance gene products that continue to
defydetection.Aproteomeofalivingcellororganismisahighlydynamicentity,
andfollowingitsmanyfacetsinhealthanddiseaseconstitutesamajorchallenge
to the biomedical and scientific community as we collectively attempt to build
uponthewealthofunderstandingaffordedbycompletionoftheHumanGenome
Project. A variety of technologies will be required to come to grips with this
technological challenge.
Herein, we have attempted to bring together authors at the forefront of
theirdisciplinetoprovideanoverviewofcurrentandemergingtrendsandtheir
applicationstothestudyof proteomics,particularlyarray-basedproceduresthat
offer the promise of ‘‘near-to-total’’ proteomic screening in a high-throughput
v
vi Preface
microenvironmment,includinganalysisofcomplexmammalianproteomes,ina
mannersimilartothatachievedforentiregenomesandtranscriptomes.Ofnote-
worthy importance, however, are the associated financial and infrastructural re-
sources likely to be required. They are no less daunting than was the initiation
of the Human Genome Project more than a decade ago; the Human Proteome
Project will require equally grandiose means on a global scale, if success is to
be forthcoming over the next decade. For both the pharmaceutical industry and
academics, the stimulus to proceed remains paramount in that it is the proteins,
and not the nucleic acids, that are the molecular workhorses of the cell, that is,
the physical players that decide physiological fates in action-packed scenarios
with multiple possible endpoints more complex and perverse than the greatest
suspensethrillerofAlfredHitchcockorAgathaChristie.Whethertheknivesand
forksareemployedforabanquetoramassacredependsontheorderedpermuta-
tionsofproteinisoforms,allofwhichawaitdecipheringwithintheinfiniteworld
ofthemultidimensionalcomplexityassociatedwithintracellularmolecularinter-
actions.
The study of proteomics combines biochemistry, genetics, genomics, and
molecularbiologytoexplorecellularnetworksinaparallelized,high-throughput,
global format. Proteomics has its roots in protein profiling by two-dimensional
gel electrophoresis and yet appears to some as a newcomer on the scientific
scene, a logical next phase in genomic research. Because the nature of science
isdynamic,thistextbookattemptstoaddressproteomicspast,present,andfuture.
The aim is to present a variety of technologies and applications for proteomics
research that will have broad application for the individual researcher and that
should assist in the introduction of important concepts to newcomers.
Thefirstfivechaptersfocusontheemergingtechnologyofproteinarrays
and biochips in proteomic research and advances in their application to protein
diagnosticsandtherapeutics.Chapters1and 2provideaglobaloverviewofthe
emergingproteinarrayfieldaswellasathoroughhistoricalperspective.Chapters
3–5 expand on the details of generating and developing protein array technolo-
gies.
Chapters 6 and 7 explore array-based proteomics focusing on the use of
resourcesfromgenomicstrategies,particularlyESTs(expressedsequencetags),
cDNA databases, and robotics for generating protein content through high-
throughputrecombinantexpressiontechniques.Thechapterthatfollowsexamines
second-generationproteomicsanddescribesmethodsthatintegrateproteinprofil-
ing by mass spectrometry with protein biochips. Chapter 9 describes shotgun
proteomics applications using several mass spectrometry techniques.
Chapters10and11examineanalysisofproteinfunction,specificallypro-
tein–protein interaction assays, and explore unique applications in proteomics
relatingvariousspecies,movingthroughthephylogenetictree,exemplifyinghow
proteomicscanbeexploitedinmodelorganismsforapplicationtomorecomplex
Preface vii
biologicalsystems.Chapter12exploresadvancesinstructuralproteomicsaimed
atprovidingagreaterunderstandingofproteinbiochemistryandcellularfunction.
Then,reflectinganageinwhichweareinundatedwithinformation,Chapter13
focusesontheintegrationofgenomicsandproteomicsinformation.Finally,Chap-
ter 14 provides an educated insight into the growing proteomics market and its
emerging biotech sector.
This text aims to be the first to present a variety of genomic-based, high-
throughputstrategiesforthestudyofproteinsbythescientistswhoaredefining
proteomics.Itprovidesafoundationfromwhichtoexaminethefieldofproteo-
mics as it evolves, to broaden our collective scientific outlook on the future
direction of biological research.
Joanna S. Albala
Ian Humphery-Smith

Contents
Foreword Sam Hanash iii
Preface v
Contributors xi
1. Protein Biochips and Array-Based Proteomics 1
Ian Humphery-Smith
2. Ultrasensitive Microarray-Based Ligand Assay Technology 81
Roger Ekins and Frederick Chu
3. Practical Approaches to Protein Microarrays 127
Brian Haab
4. Protein Biochips: Powerful New Tools to Unravel the
Complexity of Proteomics? 145
Steffen Nock and Peter Wagner
5. Functionalized Surfaces for Protein Microarrays: State of the
Art, Challenges, and Perspectives 159
Erik Wischerhoff
6. High-Throughput Protein Expression, Purification, and
Characterization Technologies 173
Stefan R. Schmidt
ix