Proteomics: a new approach to the study of disease

Review Article

Proteomics: a new approach to the study of disease

George Chambers1, Laura Lawrie2, Phil Cash3 and Graeme I. Murray1*1 Department of Pathology, University of Aberdeen, Aberdeen, UK2 Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen, UK3 Department of Medical Microbiology, University of Aberdeen, Aberdeen, UK

*Correspondence to:Dr Graeme I. Murray,Department of Pathology,University of Aberdeen,Foresterhill, AberdeenAB25 2ZD, UK.E-mail: [email protected]

Received: 22 March 2000

Accepted: 5 June 2000

Abstract

The global analysis of cellular proteins has recently been termed proteomics and is a key area of

research that is developing in the post-genome era. Proteomics uses a combination of sophisticated

techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry,

amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to

characterize proteins. The application of proteomics provides major opportunities to elucidate

disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review

aims to explain brie¯y the background to proteomics and then to outline proteomic techniques.

Applications to the study of human disease conditions ranging from cancer to infectious diseases

are reviewed. Finally, possible future advances are brie¯y considered, especially those which may

lead to faster sample throughput and increased sensitivity for the detection of individual proteins.

Copyright # 2000 John Wiley & Sons, Ltd.

Keywords: proteomics; gel electrophoresis; tumour; Alzheimer's disease; cardiomyopathy

Introduction

The human genome project is providing a wealth ofinformation about the sequences of individual genes,but as this project reaches completion, the focusof research is now moving to the immense task ofidentifying the structure, function, and interactions ofthe proteins produced by individual genes and theirroles in speci®c disease processes. The global analysisof cellular proteins has recently been termed proteo-mics and is a key area of developing research in thepost-genome era. Proteomics uses a combination ofsophisticated techniques including 2D-gel electrophor-esis, image analysis, mass spectrometry, amino acidsequencing, and bio-informatics to resolve comprehen-sively, to quantify, and to characterize proteins [1].

There are several reasons for focusing on the analysisof proteins: the level of mRNA expression frequentlydoes not represent the amount of active protein in a cell[2]; the gene sequence does not describe post-translational modi®cations, which may be essential forprotein function and activity; and the study of thegenome does not describe dynamic cellular processes [3].The application of proteomics can be expected to have amajor impact by providing an integrated view ofindividual disease processes at the protein level.

Recent improvements in the techniques of proteinanalysis, and in particular the development ofadvanced bio-informatic databases and analysis soft-ware, have allowed the development of proteomics.The techniques used are now suf®ciently robust andreliable to allow speci®c questions to be addressedregarding protein expression in individual diseases. Inparticular, proteomics offers pathologists the possibi-

lity of identifying disease-associated protein markers toassist in diagnosis or prognosis and to select potentialtargets for speci®c drug therapy.

The purpose of this review is to provide an outline ofcurrent proteomic techniques and to discuss theapplications of proteomics to the study of individualdisease processes.

Proteomics techniques

The key steps in proteomics are the separation andvisualization of complex protein mixtures, most com-monly by 2D-gel electrophoresis, and the identi®cationof proteins by mass spectrometry. The overall strategyfor proteomics is outlined in Figure 1.

Protein solubilization

Effective 2D-gel electrophoresis requires that proteins besolubilized. The method used to do this must becompatible with iso-electric focusing (IEF) and shouldnot introduce modi®cations to proteins that could affectmass spectrometry. The most common means of proteinsolubilization employed in proteomic studies is to lysethe tissue of interest, either chemically or mechanically.Often centrifugation is necessary to separate the solubi-lized protein from non-solubilized material. Sub-cellularfractions may also be prepared to permit the analysis ofspeci®c cellular compartments such as the nuclear ormitochondrial fractions. The extract can be incubatedwith nucleases to remove DNA or RNA molecules,which can interfere with electrophoresis. It is particu-larly dif®cult to solubilize hydrophobic and membrane-bound proteins. Sodium dodecyl sulphate (SDS) is the

Journal of PathologyJ Pathol 2000; 192: 280±288.

Copyright # 2000 John Wiley & Sons, Ltd.

detergent of choice for one-dimensional polyacrylamidegel electrophoresis (PAGE), but a high concentration ofSDS is not compatible with IEF. Usually bufferscontaining zwitterionic detergents are used, such as 3-(3-chloramidopropyl)dimethylammonio-1-propanesul-phonate (CHAPS) or non-ionic detergents such asNonidet P-40 (NP40), rather than ionic detergents suchas SDS. A high concentration of urea is used to denaturethe proteins. However, many new reagents developed inthe past few years can improve protein solubilization,and in particular permit the solubilization of hydro-phobic proteins. These reagents include chaotropes suchas thiourea; improved surfactants which have greatercompatibility with chaotropes, such as sulphobetaines;and reducing agents such as tributyl phosphine. Use ofall three types of reagent in a single solution greatlyassists the solubilization of hydrophobic proteins, butthe enhanced solubility can cause problems with 2D-gelelectrophoresis by overloading the gels with proteins ofoverlapping charges and masses. To solve this problem,the technique of sequential extraction has been devel-oped. The ®rst step in this process is an initial proteinextraction using Tris base. The insoluble fraction isthen extracted using a solution containing 8 M urea, 4%CHAPS, and dithiothreitol. The remaining insolublepellet is very rich in membrane proteins and is thenextracted with 5 M urea, 2 M thiourea, 2% CHAPS, 2%SB 3±10 (a sulphobetaine), and 2 mM tributyl phos-phine. This method, when tested on protein extractsfrom Escherichia coli, yielded 11 membrane proteins,including ®ve that had not been previously identi®ed[4].

Protein separation

Currently the most effective and widely used methodfor protein separation is two-dimensional polyacryl-

amide gel electrophoresis (2D-gel electrophoresis), ®rst

described in 1975 [5,6]. With this technique, proteins

are ®rst separated by their charge in the IEF step and

then separated by their molecular size using a

polyacrylamide gel. There is also a variation of the

isoelectric focusing method which uses immobilized pH

gradients (IPGs) in the ®rst dimension. This technol-

ogy was developed to assist in overcoming the

problems of pH gradient instability, discontinuous

and restricted pH gradients, and the dif®culty of

standardizing batches of carrier ampholytes. The

recent introduction of commercially available IPG

strips has greatly facilitated the reliability and consis-

tency of 2D-gel electrophoresis [3].Visualization of proteins following 2D-gel electro-

phoresis is usually done by staining with either

Coomassie brilliant blue stain, which will detect

proteins present in amounts greater than 100 ng, or,

for greater sensitivity, by silver staining, which can

detect proteins in the 2±5 ng range [3]. However, care

must be taken with silver staining to avoid cross-

linking reagents such as glutaraldehyde [7], as the

use of aldehydes makes the proteins less susceptible

to protease digestion and reduces the ef®ciency of

subsequent mass spectrometry. The recent develop-

ment of new ¯uorescent dyes such as the SYPRO series

of protein dyes may improve the sensitivity of protein

detection without compromising mass spectrometric

analysis. These dyes are effective for staining 2D-gels

and offer a similar level of sensitivity to silver staining

[8,9].Other protein separation methods have also been

developed, particularly two-dimensional high-perfor-

mance liquid chromatography (HPLC). It has been

shown that this system can successfully resolve the

mixture of proteins obtained from the lysis of E. coli

and allow the isolation of individual proteins [10]. In

the ®rst dimension, proteins are separated by size-

exclusion chromatography, and in the second dimen-

sion by reverse-phase HPLC. This system produces 2D

chromatograms that are analogous to 2D electrophor-

esis gels, with discrete spots separated on an X±Y axis.

The method potentially offers higher throughput and

faster sample resolution than 2D-gel electrophoresis

and could be very amenable to automation. A simpler

single-dimension HPLC method has also been used to

resolve the individual proteins constituting the pro-

teome of recombinant adenovirus 5 [11].The latest development in the application of HPLC

to proteomics involves the coupling of an HPLC

column to an imaged capillary IEF system. Proteins

are ®rst separated by HPLC on a gel ®ltration

chromatography column and then transferred to an

integrated capillary IEF system. In tests, the system

was capable of separating and analysing a mixture of

two model proteins in 24 min. While this is not as fast

as the capillary micro-channel system discussed later, it

is still substantially faster than 2D-gel electrophoresis

[12].

Figure 1. An outline of a general strategy to performproteomics

Proteomics in the study of disease 281

Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 192: 280±288.

Image capture and analysis

The protein expression patterns generated from separa-tion techniques are usually complex and are bestanalysed by computer-based image analysis. In thisway, it is possible to create 2D databases. The ®rst forhuman cells included databases for transformedepithelial amnion cells, peripheral blood mononuclearcells, and embryonic lung MRC-5 ®broblasts [13±15],and 2D databases of a number of speci®c human tissuetypes and tumours have subsequently been developed.One of the best established is the SWISS-2D database,which was begun in 1993 [16]. The current versioncontains reference maps from human and mousetissues and also from micro-organisms, includingSaccharomyces cerevisiae and E. coli. A range ofother 2D databases are available, from human liver,to keratinocytes, to bladder carcinoma cells [17±19].Many 2D databases and proteomic resources areavailable on the World Wide Web (Table 1) [20].

A key part of many proteomic studies, especially inthe context of pathological investigations, is thecomparison of protein expression patterns betweennormal and abnormal tissue. Comparisons can bemade with established 2D databases if the sourcetissue is of the same type and if extraction and analysisconditions are similar. To facilitate such comparisons,it is necessary to use specialized image analysissoftware. While different 2D-gels are never perfectly

comparable, current gel analysis software can compen-

sate for this. Some systems such as ELISE and

HERMeS use a cluster-matching technique to compare

gels, whereas GELLABE relies on the operator

providing about 20 `landmark' matches which

`anchor' the two gel images under comparison. Soft-

ware systems also vary in the way their databases are

constructed and organized, from those using simple

models, like ELISE, to those using more complex ones,

like HERMes [21].The MELANIE system, ®rst introduced in 1988, is

based on correspondence analysis and ascendant

hierarchical classi®cation [22]. MELANIE II, the next

version of this system, and the one currently in most

widespread use, is capable of working on many

different operating systems. It is one of the latest and

most user-friendly 2D-gel analysis systems and can be

linked via the Internet to proteome databases available

on the World Wide Web [23]. These features have been

enhanced still further in MELANIE III, which has just

become available [24].Provided that electrophoresis conditions are similar,

good gel analysis software should be able to highlight

differences in protein expression between normal and

abnormal samples. Proteins of interest can be excised

directly from the gel or blotted onto a suitable

membrane [25±27] and they can then be subjected to

identi®cation techniques. Recent developments in

automated proteomic systems have allowed the possi-

Figure 2. A representative result of a proteomic investigation of colon carcinoma. Total cellular proteins from a colon carcinomahave been extracted using a CHAPS-based protein solubilization solution and then separated by 2D-gel electrophoresis. A singleprotein spot has been excised from the gel and characterized by MALDI-TOF mass spectrometry

282 G. Chambers et al.


bility of coupling spot-excising robots to imageanalysis software. The Proteomic Analyzer imageanalysis software system developed by GenomicsSolutions can be coupled to a Protein Picking2 roboticworkstation, which is capable of rapidly excisingthousands of protein spots from multiple gels andtransferring them to an automated protein digestionworkstation. This type of automated system is essentialfor high throughput proteomics.

Protein characterization and identi®cation

A variety of techniques are available to characterizeproteins. While 2D-gel electrophoresis can provide theisoelectric point and approximate mass of a protein,this information alone is currently insuf®cient toidentify the majority of proteins. One of the mostwidely used methods of identifying proteins is throughpeptide-mass ®ngerprinting [28,29]. The protein isdigested with a proteolytic enzyme, such as trypsin, toproduce a set of tryptic fragments unique to eachprotein. The masses of these peptides are thendetermined by mass spectrometry. The most commonsystem for doing this is matrix-assisted laser desorptiontime-of-¯ight mass spectrometry (MALDI-TOF). Thesystem is highly sensitive and can give molecularweight information at the attomole level (10x14 g fora 10 kD molecule) [30]. The tryptic masses are thenevaluated using a peptide-mass ®ngerprinting tool suchas MS-Fit, Mascot, or Peptide Search. These tools tryto `®t' a user's mass spectrometry data to a proteinsequence in an existing database and thus suggest theidentity of the protein. This form of protein identi®ca-tion will only be successful if the protein beinganalysed is represented in the databases. For proteinswhich have incomplete sequence information, it isnecessary to obtain sequence information for the

protein by Edman degradation [31] or by nano-electrospray mass spectrometry [32]. This sequenceinformation can then be used along with the massspectrometry information to interrogate expressedsequence tag (EST) databases.

Techniques for ensuring the analysis ofspeci®c cell types

Accurate proteomic analysis requires the isolation ofspeci®c morphologically or functionally uniform popu-lations of cells, since the presence of contaminatingcells within a sample is a major obstacle to meaningfulmolecular analysis. This is particularly important inthe analysis of pathological conditions. To minimize oreliminate any potential `interference' from other celltypes, cell enrichment is necessary. Several methods,including ¯ow cytometry, antibody puri®cation, andmore recently micro-dissection, have been used forobtaining speci®c cell types. The method chosendepends in part on the starting material and on theavailability of reagents such as antibodies for puri®ca-tion. Flow cytometric methods are ideally suited tohaematological cells. Antibody-linked magnetic beadscan be used to select cells of the desired type frommechanically disaggregated mixed cell populations [33].Protein expression pro®les of normal breast epithelialand myoepithelial cells have been recently established,the two cell types being separated using doubleantibody magnetic af®nity sorting [34]. However, theuse of such antibody puri®cation methods requires theavailability of antibodies speci®c for the cell type ofinterest and so might not always be feasible.

Micro-dissection can be used to isolate speci®c celltypes. Manual micro-dissection methods, which rely onthe removal of desired material by micro-manipulation

Table 1. Useful proteomic resources and databases available on the World Wide Web

Name Resource WWW address

Aberdeen University Proteomics Facility National proteomics research facility http://www.biochem.abdn.ac.uk/yjef/Protein_Lab.html

Aberdeen 2-D PAGE, Aberdeen University 2D-gel databases for Haemophilus in¯uenzae

and Neisseria meningitidis

http://www.abdn.ac.uk/ymmb023/2dhome.htm

EMBL protein and peptide group Useful information on mass spectrometry http://www.mann.embl-heidelberg.de/Default.htmlExPASy Molecular Biology Server Very extensive proteomic site, includes

SWISS-2DPAGE human 2D-gel database

http://www.expasy.ch/

HSC-2DPAGE (Heart Science Centre,

Hare®eld Hospital, UK)

2D-gel databases for heart (ventricle),

endothelial cells

http://www.hare®eld.nthames.nhs.uk/nhli/protein/

Human and mouse 2-D PAGE databases

(Danish Centre for Human Genomic

Research)

2D-gel databases for human keratinocytes,

bladder transitional cell carcinomas, bladder

squamous cell carcinomas, urine, ®broblasts

http://biobase.dk/cgi-bin/celis

Molecuar Anatomy Laboratory, Indiana

University, USA

2D-gel databases for liver, kidney, brain,

plasma, testis

http://iupucbio1.iupui.edu/frankw/molan.htm

Mycobacterium 2-DE databases, Max

Planck Institute for Infection Biology,Berlin, Germany

2D-gel databases for Mycobacterium bovis

and Mycobacterium tuberculosis

http://www.mpiib-berlin.mpg.de/2D-PAGE/

ProteinProspector Extensive web site with tools for protein

database searching including MS-®t

http://prospector.ucsf.edu/

SIENNA-2DPAGE 2D-gel database of human breast ductalcarcinoma cells

http://www.bio-mol.unisi.it/2d/2d.html

YPD yeast protein database 2D-gel databases for yeast Http://www.proteome.com/databases/index/html



with a probe or small-gauge syringe needle, areadequate for obtaining suf®cient tissue for polymerasechain reaction (PCR) analysis [35,36], but cannotprovide suf®cient tissue for proteomic studies andhave several disadvantages, including the necessity fora skilled operator and susceptibility to contamination.New methods based on laser capture techniques havehad a major impact on the micro-dissection of solidtissues and tumours. Laser capture is precise and rapidin comparison with other methods of micro-dissectionand has been used successfully in initial studies toselect speci®c cells for proteomic analysis [37±39].

Current limitations of proteomics

While proteomics offers a new approach to the studyof many pathological conditions, there are still severaltechnological limitations. From the pathologist's per-spective, one of the most important of these is thatformalin-®xed archival tissue cannot be used for 2D-gel electrophoresis, as the cross-linking of proteins byaldehyde ®xatives renders them insoluble. Anotherissue with proteomics is the time-consuming nature ofthe procedures, in particular the time taken to perform2D-gel electrophoresis.

There are also several speci®c limitations of 2D-gelelectrophoresis. It cannot adequately separate hydro-phobic proteins; Wilkins et al. [40] calculated thehydrophobicity values of 427 bacterial and yeastproteins and found that there was a clear hydro-phobicity cut-off point beyond which 2D-gel electro-phoresis was ineffective. Another serious drawback isthe inability of 2D-gel electrophoresis to detect lowabundance proteins; there is no equivalent in proteinbiochemistry of PCR in molecular biology and currentprotein staining techniques do not allow the visualiza-tion of low abundance proteins in 2D-gels. Abundantproteins, especially structural proteins such as b-actin,can also pose problems by obscuring other more minorproteins [41].

While 2D-gel electrophoresis can resolve post-transcriptionally or proteolytically modi®ed proteinsfrom their `parent' molecules, this can complicateprotein identi®cation. It has recently been suggestedthat up to a quarter of the protein spots visualized in a2D-gel are modi®ed proteins, so it is not uncommon toisolate what is effectively the same protein frommultiple spots on a gel [1]. The limitations posed by2D-gel electrophoresis may be overcome in the nearfuture by advances in other methods of proteinseparation, some of which are described later, butthey are still an important consideration when plan-ning research using proteomics.

Applications of proteomics in the study ofdisease

There is a great deal of current interest in theapplication of proteomics to the study of individual

diseases and research in these areas is continuallyexpanding.

Cancer research

Because of the potential of proteomics to identify newtumour markers, there is much current interest inapplying proteomics to the study of tumours, but todate only a limited number of studies have been carriedout.

Extensive 2D-gel electrophoresis of bladder tumourcells and urinary proteins has led to the developmentof a comprehensive 2D database for bladder carci-noma that includes pro®les of both transitional andsquamous cell carcinomas [42]. The same group hasalso developed a database of excreted proteins foundin the urine of bladder cancer patients. The database ismade up of scanned and annotated 2D-gels. Spots onthe gels which have de®nite protein identities foundfrom subsequent proteomic analysis are labelled oneach gel. The clinical results of these programmes are,however, somewhat disappointing, as only one candi-date marker for squamous cell carcinoma has beenproposed. This putative marker, psoriasin, is a lowmolecular weight calcium-binding protein which is alsoexcreted by cultured keratinocytes. It is excreted in theurine of squamous cell carcinoma patients and mayhave the potential to be developed as a diagnosticmarker for squamous cell carcinoma. Considerablework will be needed to evaluate this and there are somepotential problems. For example, there is a possibilitythat reversible benign metaplastic lesions may tem-porarily express psoriasin. Strati®ed squamous epithe-lium in the female trigone may also secrete psoriasin,which can thus be detected in the urine of some healthyfemales. An ELISA test for psoriasin is currently underdevelopment, and its use in large-scale studies may leadto a true evaluation of the usefulness of psoriasin as adiagnostic marker [43].

Initial protein pro®les have been de®ned in sometumours, but many of these studies are limited in theirusefulness, as no attempt has been made to purify thetumour cells. In the study of breast cancer, comparative2D-gel electrophoresis analysis of whole tissue sampleshas shown that decreased expression of cytokeratins andtropomyosins is associated with malignant progression[44]. In another study [45], 2D-gel electrophoresispro®ling of breast invasive ductal carcinomas found adistinctive pattern of 32 protein spots expressed stronglyby whole samples of breast ductal carcinomas, but notby whole samples of normal breast tissue. Comparisonof this gel to gel images contained in the SWISS-2DPAGE database allowed the identity of some of thespots to be determined.

A recent study of protein expression in renal cellcarcinoma has created a ®rst generation 2D-geldatabase for human kidney proteins. A variety oftechniques were employed including comparison of2D-gel images, amino acid analysis, N-terminal aminoacid sequencing, and immuno-detection, to identify 43



out of 2789 separated polypeptides. Comparison ofprotein expression patterns between whole samples ofnormal and tumour kidney tissues showed that fourpolypeptides synthesized by normal kidney were notpresent in renal carcinoma tissue. The techniquesemployed by the investigators were suf®cient toidentify only two out of the four proteins; one wasidenti®ed as ubiquinol cytochrome c reductase, whilethe other was mitochondrial NADH-ubiquinoneoxido-reductase complex I. Neither protein has a genelocus in a region known to be deleted in renal cellcarcinoma, so the loss of expression of these twoproteins may be due to unknown deletions, or tochanges in gene transcription or translation [46].

A study of lung tumours using 2D-gel electrophor-esis has shown that different histopathological tumourtypes display differences in their protein expressionpatterns and that an unknown protein is signi®cantlyoverexpressed in primary lung adenocarcinomas com-pared with other types of lung carcinoma and normaltissue [47]. Subsequently it has been found that thereare two variants of the protein, which differ slightly inisoelectric point but not molecular weight. A follow-upstudy has shown that both variants are expressed byprimary lung adenocarcinoma and that their expres-sion correlates closely with the degree of cellular atypiaand histological differentiation [48].

Proteomics is also being used in other areas ofcancer research, especially concerning tumour develop-ment and progression. One of the most importantareas is the study of apoptosis, since a failure of theapoptotic mechanism has been proposed as one of themajor factors in tumour growth and development. Aconsiderable body of research has been undertaken totry to understand the mechanism of apoptosis and toexplain how defects in that mechanism can lead to thedevelopment of cancer and the failure to respond toanti-cancer therapy. Studies of animal cell models haveshown two distinct patterns of proteolysis of proteinsassociated with apoptosis, one of which occurs at anearly stage and the other at a later stage. There is alsoan alteration in the phosphorylation pattern of certainproteins (including p53) in cells induced to undergoapoptosis [49,50]. One of the most recent studies ofprotein expression in apoptosis [51] has used proteo-mics to study nuclear protein expression in Burkittlymphoma BL60 cells. The BL60 cell line has beenused as a model for determining apoptosis-associatedproteins, by subtractive analysis of normal andapoptotic cells. The study examined 36 protein spotsfrom a Coomassie blue-stained gel and was able toassign identities to 33 of the proteins by a combinationof MALDI-TOF mass spectrometry and nano-electrospray mass spectrometry. The other threeproteins were not identi®ed in any of the availabledatabases. While some of the identi®ed proteins werecommon to normal cells, others were known to beinvolved in apoptosis. This study provides the basis forfurther work to construct a comprehensive database ofproteins expressed in apoptosis-induced cells.

Neuropathology

A 2D-gel electrophoresis database of normal brainproteins has recently been created from samples oftotal brain tissue from the parietal lobe cortex. In thedatabase, 400 identi®ed spots correspond to 180different brain proteins, mostly of cytoplasmic ormitochondrial origin. This should serve as a usefulreference against which gels prepared from brainextracts of abnormal individuals can be compared [52].

Proteomics has been used to investigate certainneuropathological disorders. In one study of proteinexpression in the brains of patients with Alzheimer'sdisease, comparative 2D-gel electrophoresis was usedto show differences from normal individuals. Fiveprotein spots were increased in the Alzheimer's group,28 were decreased, and nine were uniquely expressed.The study did not determine the protein identities butprovides a useful reference for future studies [53].

Another recent study of Alzheimer's disease andschizophrenia, also by comparative 2D-gel electrophor-esis, examined protein expression in post-mortemhippocampal tissue. While sample numbers wererelatively small (seven in each abnormal group andseven controls), eight proteins were found to beexpressed in decreased amounts in the schizophrenicpatients and eight in increased amounts. In theAlzheimer's disease patients, 35 proteins displayeddecreased expression and 73 displayed increasedexpression. Decreased expression of one particularprotein, diazepam binding inhibitor, was observed inboth schizophrenic and Alzheimer's patients; althoughthis may be due to the action of drugs prescribed forthese conditions, it is also possible that it represents aunique change [54]. Despite their limitations, bothstudies point the way to a greater understanding of themolecular basis of neuropathological disorders.

A proteomic approach has also been used toinvestigate demyelination in a mouse model. Theexpression of proteins was examined in the opticnerves of transgenic mice with a c-myc-induceddegenerative myelin disorder. There were markeddifferences in the amounts of cytoskeletal proteinsassociated with gliosis in the optic nerves of theabnormal mice, compared with the normal. Theexpression of several major myelin proteins wasmarkedly reduced in the transgenic mice, including®ve isoforms of myelin basic protein, four isoforms ofcyclic nucleotide 3k-phosphodiesterase, and myelin-associated glycoprotein. The study provides the basisfor future more detailed investigation and reveals anumber of candidate molecular markers for demyeli-nation [55].

Cardiovascular disease

In a recent study on cardiac hypertrophy, 2D-gelelectrophoretic patterns were compared from culturednormal rat cardiac myocytes and myocytes withphenylephrine-induced hypertrophy. Eleven proteinsdisplayed quantitative changes in expression in cardiac



hypertrophy, with three showing a decrease and eightan increase. Following peptide mass pro®ling and

database matching, all 11 spots were identi®ed. Five

were found to be isoforms of the myosin light chain, at

least one form of which is known to be associated with

cardiac hypertrophy. The others represented proteins

not previously known to be associated with the

condition [56].Application of proteomics to the study of dilated

cardiomyopathy has resulted in the establishment of a

human myocardial database containing 150 identi®edproteins. This was used as a baseline for comparisons

with protein pro®les from patients with dilated

cardiomyopathy. Signi®cant differential expression

was found for 25 proteins and it was possible to

identify 12 proteins [57].

Proteomics applied to microbiology

Many of the current applications of proteomic tech-

niques have been in microbiology, with much work

being carried out to link the genomes and proteomes of

a variety of bacterial and yeast species. The proteome

of E. coli has been particularly well investigated since

1983 and today has a 2D-gel database containingapproximately 1600 different protein spots. There are

data containing identi®cation information for many of

these proteins and also information on how their

expression patterns change according to different

bacterial growth conditions. Three hundred and ®fty

of the 4000 E. coli genes have now been matched to

approximately 400 protein spots (2D-gel electrophor-

esis will detect isomers as well as unique proteins) [58].

This type of analysis has paved the way for similar

work to be carried out on pathogenic micro-organisms.

Salmonella typhimurium, which is an enteric pathogenresponsible for many cases of gastroenteritis in

humans, is one of these pathogens whose proteome

has been investigated. In an analysis of S. typhimurium

cell envelope proteins, 53 proteins were identi®ed by

2D-gel electrophoresis followed by N-terminal sequen-

cing. Approximately 20% of these proteins had no

match in current sequence databases [59]. It is hoped

that a better understanding of the proteome of S.

typhimurium will identify which proteins are associated

with virulence.The immune response to infection with Helicobacter

pylori has been studied and 20 proteins have been

identi®ed which were reactive with the serum of

infected patients. All the proteins were identi®ed as

H. pylori proteins [60]. A follow-up study investigated

the possibility of using one of these proteins as an

antigen for generating an H. pylori vaccine, due to its

consistently high reactivity. Detailed analysis was

carried out using many proteomic techniques, includ-

ing 2D-gel electrophoresis and mass spectrometry.Endoprotease digestion was carried out, followed by

MALDI-TOF peptide sizing and sequence analysis. As

a result, it was possible to identify the protein as

corresponding to an open reading frame in the H.pylori genome [61].

The study of antibiotic resistance in pathogenicmicrobes is of great importance to medical microbiol-ogists and to specialists in infectious disease. A recentstudy has been carried out on erythromycin resistancein the pathogen Streptococcus pneumoniae. This bac-terium has developed widespread penicillin resistanceand the incidence of erthyromycin resistance is alsorising. In an effort to understand the mechanism oferythromycin resistance, proteomic methodologieswere used to examine protein expression in erythro-mycin-susceptible and -resistant strains. 2D-gel electro-phoresis was used to separate the proteins expressed byeach strain. Comparison of electrophoresis patternsshowed that a 38 500 D protein with an iso-electricpoint of 6.27 was expressed by the resistant strain, butnot by the susceptible strain. Following peptide massmapping, this protein was found to be glyceraldehyde-3-phosphate dehydrogenase. The particular resistantstrain was one displaying the M phenotype oferythromycin resistance, in which resistance is asso-ciated with an active ef¯ux process which reducesintracellular levels of erythromycin. Strains displayingan alternative mechanism of erythromycin resistance,where enzymatic methylation of ribosomal RNAblocks erythromycin binding to the ribosome (theMLS phenotype), expressed a protein pro®le nodifferent from that of the susceptible strains [62].

Research has also been carried out on yeast, as thisorganism offers a simple model for the integration ofgenomic and proteomic information in eukaryoticcells. Mass spectrometric identi®cation of yeast pro-teins by nano-electrospray tandem mass spectrometry,followed by database searching using multiplesequence tags, has been used to identify 150 individualyeast proteins as resolved by 2D-gel electrophoresis.More than 30 of these were novel and matchedpreviously uncharacterized open reading frames in theyeast genome [63]. Another study of yeast proteomics[64] has been used to construct a detailed proteomicmethodological outline which could easily be modi®edand employed in studies of other more complexeukaryotic cells. Research has continued in the ®eldof yeast proteomics with the development of a yeastprotein database. Efforts are now being undertaken tocarry out quantitative studies using 2D-gel electro-phoresis. One such recent study has been able to showa good correlation between protein abundance, mRNAabundance, and codon bias for identi®ed spots andrepresents a successful effort to integrate informationfrom proteomics with information from genomicresearch [65].

Future developments

At the moment, much research is being doneto improve the ef®ciency of proteomic techniques,especially with regard to automation, sample through-



put, and sensitivity. Since 2D-gel electrophoresis iscurrently the mainstay of protein separation inproteomic research, current developments have focusedon automated systems for excising individual spotsfrom 2D-gels and for protein digestion, followed bypeptide mass mapping using mass spectrometry withintegrated peptide identi®cation software [66]. Anothervery new and promising method is micro-channel-based separation, which has already been employed inthe rapid resolution of small amounts of nucleic acidmixtures. A methodology for micro-channel-basedSDS capillary gel electrophoresis of proteins has beendemonstrated [67] which offers high speed and resolu-tion as well as economy. The system utilizes a chipwith the micro-channels photo-etched onto it andproteins to be analysed labelled with ¯uorescein.Detection of individual proteins following electrophor-esis is by laser-induced ¯uorescence. The technique wasshown to resolve a mixture of six ¯uorescent-labelledproteins ranging in size from 9 to 116 kD within3.5 min. Such a rapid technique will allow a highthroughput of samples. There is also much researchbeing performed to develop protein `chips', which canbe considered to be analogous to DNA micro-arrays.

Conclusions

The development of proteomics represents an excitingnew way to examine pathological processes at themolecular level and is already leading to improvementsin the understanding of many conditions. It will beparticularly important to perform proteomic experi-ments with speci®c populations of cells, to generatesigni®cant information about cell-speci®c proteinexpression pro®les and how these change in differentdiseases. As the technology of proteomics analysiscontinues to improve, it will be possible in the nearfuture to combine genomic and proteomic informationto obtain a more comprehensive picture of manypathological conditions.

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Proteomics: a new approach to the study of disease

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