India n .l numal of Biochemi stry & Bioph ys ics Vnl. 39. October 2002. pp. 29 1-302

Minireview

Proteomics-A new player in the post-genomic era

Kapil Maithal

Dr B R Ambcdkar Center for Biomedi ca l Research , Uni versity o f Delhi , Delhi I 10007

Receil 'ed 27 Jllll e 2002; accepleri 9 A lIglIsl 2002

In the post-genom ic era the concept of personal ized mcd icine and molec ular medi cine emph asizes the utility of th .: proteomi cs appro;:lCh . Proteomi cs is the global anal ysis of ce llul ar proteins and complement s the genomi cs approach. Protein s. in principle do all the work of the ce ll and ultimatel y dictate all biological processes and the ce llul ar fat e. Proteo mi cs uses a combination of sophisticated tec hniques including two-d imensional (2D) gel electrophoresis. image anal ys is. mass spectrometry. ami no ac id sequencing and bioinformatics to identify and characteri ze proteins. Thi s rev iew aims at providing the va ri ous approaches and pitfall s assoc iated with thi s technique and gives a brief overview of the utility of thi s approach in the area of biomedica l research .

Introduction Proteomics encompasses the study of expressed

proteins. including identification and elucidation of the structure-function relationship under healthy condi tion s and di sease conditions I. Proteomics along with genomics can provide a holi stic view of the biology underlying cellular alteration processes . The knowledge of proteome is quite essential , as the protein express ion level s are quite necessary to determine the critical changes that occur as part of di sease pathogenesis. Thi s is because proteins are often expressed at concentrations and forms (post­translational modifications etc.) that cannot be predicted from a mere analysis of mRNA. Proteomics also provides a route to understand the protein interactions at the level of biochemical pathways and the effect of various factors on them.

The 1980s saw the rise in the area of genomics with the sequencing of whole genomes of organisms, like Helicobacter pylOli , Mycobacterium tuberculosis' , and chromosome 2 of PlasmodiulI/ Ialcipanllll". Further, the genome of Drosophila lII elallogaster, which constitutes a s taggering length of -180 million bases has been decodeds. But, probably one of the most important events of thi s century was the announcement of the successful completion of the human genome project by two groups- the Human Genome consortium and Celera Genomics , on 26 June 2000. The consortium sugges ted that the total number of gene were only

E. Mail: maithalk @rediffmal.com ; Fax : 91 -11 -7666248; Tel.: 91-11-7666272, 91-11-7667151

around 38 ,000 as compared to the earlier es timates of over 1,00,000. A large proportion of these ge nes are either not annotated or have no t been repo rted before6

.7

.

A parallel area to genomics is proteomics, which refers to the study of all proteins produced by a species , much as the genome is the entire set of genes. The term 'proteomics' indicates PROTEins expressed by a genOME and is the sys temati c analysis of protein profiles of ti ssues8.~. Unlike the genome, the proteome is dynamic and varies with time depending on chemical, physical and biolog ical changes. This makes proteomics an essential too l for not only identifying diagnostic markers but al so developing candidates for drug targeting Ill .

History The term " proteomics" was introduced around

1995 by Marc Wilkins. Wilkins defined proteomics as "the study of proteins, how they're modified , when and where they're expressed, how they're invol ved in the metabolic pathways and how they interact with each other". The two major tools of proteomics are two-dimensional gel electrophoresis, which evolved in its modern form in the early 1970s and modern mass spectrometry including electrospray ioni zation and MALDI-TOF, that was developed only in late 1980S11.1 2.

Various landmarks in the historical evoluti on of proteomics are shown in Table I.

Proteomics and Genomics Unlike the genome, which is a constant feature of

an organism, the proteome varies with nature of

292 INDI AN 1. BIOCHEM. BIOPHYS .. VOL. 39. OCTOBER 2002

Table I - L~ndmark s in lhe hi s l ll ric~1 evolulion of proleomics

Year Landmark

I X60 Friedrich Miescher idenlified acid and basic prolein componenls in cell nuclei which was mi stakenly be lieved 10

carry lhe genelic male ri ~ 1

1940 Fkadle and Talum linked genes 10 unique prolein producls and formu laled lhe one gene - one prolein concepl lhal has now been rev ised as one gene codes more lhan one prolein.

1953 Idenlifi cali on of the double-slranded slruclure of lhe DNA by Wal son and Crick . 1956 Separalion of pro leins wilh a combinalion of paper and starch gellwo-dimensional eleclrophon~s i s achieved. 196 1 Modern concepl of gene express ion following discovery of messenger RNA. deciphering of gcncl ie code and

descriplion of lheory of genelic regulalion of prolein synlhcs is. 1967 Prolein sequencing defined and aUlomaled by Edman and 8 egg. 11)75 The modern form of I wo-dime n s i o n~1 eleclrophoresis o f proleins by high reso lu lion separa li on. 191\2 T he concepl of mapping lhe human prOleome was pUI forward by A nderson and Anderson. II)X6 Rodcrick coinedlhe word "Genomics" . 11)95 Defin i li on of the proleollle as given by Wilk ins. :WOO Sequencing of lhe h um~n genome compleled.

ti ss ue. the state of development, health or d isease and effec t o f drug treatment 13

. A single gene may generate more than one fini shed protein product, wh ich may I . . f . I I' f'f 1 ~ I '; T I lave mInor or major unctIona ( l erences . -. le protein production pathway is shown in Fig. I .

The mall1 difference between geno l11l cs and proteo l11i cs li es in their approach. Genomi cs starts with the gene and makes inferences about it s products (proteins). Proteomics on other hand beg ins with the functi onally modified protei n and works back to the gene responsible for its producti on. Parallel to genomics. proteomics research can be categori zed as stru ctural and functional. Structural proteomics, or prote in ex press ion. measures the number and types o f prote ins present in normal and diseased cell sl('. Thi s approach is use ful in defining the structure of proteins in a ce ll. Some o f these protei ns may be targets for drug discovery. On other hand function al proteomics is the study of the biologica l acti v iti es associated w ith prote ins and the intr icate inter-relati onship among th em I 7. 1~. Basicall y, proteol11i cs can be di v ided into

ex press ion proteomics, the study o f global changes in prote in ex press ion, and cell-map proteomics, the systemati c study o f protein-protein interactions through the isolati on of protein compl exes l ~ . The minimum proteome size can be ca lcu lated fro m the size and 2-D polyacry lamide ge l electrophores is (2-D PAGE) separated prote ins. Proteomics is based on Icacli ng edge technological capab i I ity for undertak i ng the mass screening of proteins and their post­translational modifications in whole organisms as we ll as in their ti ssues in normal and diseased states. There are three main steps in proteome research: i) Separati on o f individual proteins by 2-D polyacry lamide gel electrophoresis (2-D PAGE); ii )

DNA

Transcri ption l~ RNA

Process ing j ~ mRNA

I Translation

r Protein --..

Transcripti onal control

Post -transcri prion a I contro l (eg. A ltern ative splicing, al ternative polyadenylat ion, RNA editing)

Translati onal and degradation contro ls Translational frameshi fti ng

>200 known pos t­translation modifications (eg. Phosphorylation, glycosylalion. pept ide cleavage)

Fig. I - Pro lein producl ion palh \\'ay

Identi ficat ion by mass spect rometry or N -termi nal sequencing of individual proteins recovered from the gel ; iii ) Storage, manipUlation, and comparison o f the c1ata using bioinformati cs.

The basie practical approach used in proteomics is shown in Flow chart I .

MAITHAL: PROTEOMICS - A NEW PLA YER IN THE POST-GENOMIC ERA 293

Flow C hart-I

Whole ce ll /ti ssue lysa te

~ Protein solubli zati on and separation

1 3 . Two-d imensional (2D) electrophores is

b. Visuali zat ion of proteins

Protein identi fieation and characterization

a. In-gel digestion with trypsin. b. MALO[ analysis of the supernatant after in-gel digestion. c. Database analysis

Protein Target

Protein solublization and separation For the separation of complex protein mixtures,

two-dimensional (20) electrophoresis is currently the only technique that can reveal hundreds or even thousands of pro teins at a time. In this method prote ins are separated on the basi s of charge in the first dimension (isoelectric focusing) and molecular mass in the second dimension (reducing and denaturing PAGE). But, a good 2D-PAGE must adhere to some basic requirements , like: Reproducibilty: Maximum reproducibility of the 2D maps is a prerequis ite to determine any change in the proteome expression.

Resolution : Optimum resolution is required to prevent any cross-contamination of spots in 2D gels.

Maximum loading capacity: Quantity of protein that could be loaded on a PAGE should be optimized to identify low abundant proteins. Automation : Automation is desired for both simplification and high-throughput analysis of prote ins.

Class ical 2D e lectrophoresis with pH gradients generated by a carrier ampholyte (CA) were limited in their resolution, reproducibility and protein-loading capacit/ ,,·21. By contrast, immobilized pH gradients in 20 electrophoresis22.23 (abbreviated to IPG-Dalt -proteins are first separated according to their isoelectric po int, and then by molecular weight, which is measured in Dalton) have proved to be amazingly flexible with respect to the requirements of proteome analys is.

The sample (eg, ti ssue, serum) is solubili sed. and the proteins are separated into their po lypeptide subunits. Thi s mi xture is then separated by isoelec tri c focusing (IEF). The most co mmon means of protein solubi li zation employed in proteomic studi es is to lyse the ti ssue either chemically or mechanicall y. Sub­cellu lar fractions may also be prepared to permit the ana lysis of specific cellular compartments such as the nuclear or mitochondrial fraction s. The ex tract can be incubated with nucleases to remove DNA or RN A molecules, wh ich can interfere with electrophores is. Sodium dodecyl sulphate (SDS) is the detergent of cho ice for one-dimensi onal po lyacrylamide ge l electrophoresis (PAGE), but being a negative ly charged detergent a high concentration of SDS is not recommended for IEF. Therefore, zwitterionic detergents are used, like 3-(3-chloramidopropyl) dimethyl ammonio-l-propanesulphonate (CHAPS) or non-ionic detergents such as Nonidet P-40 (N P40). A high concentration of urea is used to denature the proteins.

The two main shortcomings of protein separation and solubilization are:

(a) Solubilization of hydrophobic proteins; but in recent past new reagents have been developed, which permit the solubilization of hydrophobic proteins. These reagents include chaotropes such as thiourea; improved surfactants, which have greater compatibility with chaotropes, such as sulphobetaines; and reducing agents such as tributyl phosphine.

(b) The second shortcoming is presence of large number of proteins with simi lar charges and masses, which will yield overlapping results. To solve thi s problem, sequential extraction method has been developed24. The first step in this process is an initial protein extraction using Tris buffer. The insoluble fraction is then extracted using a solution containing 8 M urea, 4% CHAPS, and dithiothreitol. The remaining insoluble pellet is very rich in membrane proteins and is then extracted with 5 M urea. 2 M thiourea, 2% CHAPS, 2% SB 3-10 (sulphobetaine), and 2 mM tributyl phosphine25

.

Visualization of proteins following 20-gel electrophoresis is usually done by staining with either Coomassie brilliant blue stain (sensitivity;::: 100 ng) or si lver staining (2-5 ngl6

. However. cross-linking reagents such as glutaraldehyde should be avoided27

The recent development of new fluorescent dyes such as the SYPRO protein dyes have further improved the sensit ivity of protein detecti on28

.2() .

294 INDIA N J. BIOCHEM. BI OP HYS .. VOL. ]9. OCTOB ER 2002

Other protei n separation methods have also becn deve lopcd particularly two-dimensional high­perrurlllance liquid chromatography (HPLC). In thi s mcthod the proteins arc separated by size-exc lusion chromatography in first dimension, and by reversc­phase HPLC in the second dimcnsion. Thc method potentially oilers higher throughput and faster sample re!--olution than 2D-gcl e l cctrophoresis ·~IJ.

Protein identification and ch:u"acterization Once a wcll resol ved t \,io-di mensional PAG E IS

ohtained. the indi vidua l protein spots arc identiried by pcptide-mass ringerprinting.1U2. In-gel digestion or the protein is carried ou t with proteascs, such as trypsin. chymotrypsin, V8 protease or by chemical s like cyanogen bromide to produce a set or proteolytic rragments unique to each protein (Tabie 2). Briefly. the protocol is given below'.1:

L Excisioll of proteiil ballds (spots) ./i·OIl/ polyacl)'lalllide gels a. Rinse the gc l with water. Excise bands or interest with clean scalpel cutting as close to thc edge of the hand ~IS pos~ihle. It is important to reduce the volume or" hackground " gel.

b. Chop thc excised bands into cubes (ca. I x Iml11). Tran~rer gel particles into a microcentriruge tubc (0.5 mlor 1.5 ml Eppendorf). c. Do not touch ge l with ringers! Always \vear g loves .

Rin se g loves wi th water to wash out talcum powder and traces o f dust.

2. Reductioll alld alkylatioll a. Wash thc gel part ic les wi th l Oa-Iso pi or watcr (S

min). Spin clown and remove the liquid.

b. Add ace toni tri le (ca . 3-4 times equa l the volume or gel picces) and wai t ror 10- 15 min until the gel piecc ~

are shrunk - they becollle whitc and st ick together.

c. Spin the ge l particles down anc! remove all liquid. Dry down gel particles in a vacuulll cent riruge.

d. Swell th c gel pieces in I () mM dithinthreitol/O.I !II NH~HCO.1 (add the liquid enough to cover gel) and incubate 1'01' 30 min at S(l '"C to reduce the protein. In­gel reductioll is recommended evcn II' proteins \\ 'cre reduced prior to an electrophoresis.

e. Spin down the gel particles and relll()\'C C\CL'~~ liquid. Shrink the gel pieces w ith acetoil i trile .

f. Rep lan: acetollitrile with 55 IllM iodoace talllidclO . M H~HC03 Incubate for 20 min at room lemperaturc in the clark.

g. Remove iocloacetamicle solution. W<I~h thc gel particles with 150-200 pi of 0.1 M HJHCO, I'm is Ill! n.

h. Spin down the ge l particles and remo\'e all liqui d. Shrink the gel pieces with acetonitrile. i. Spin clown the ge l particles and rem ove ~tli liquid . Dry down ge l partic les in a vaCUUIll cClllrirugc.

Tahlc 2 - Su~ccptiblc: ckavage ~ite~ or so me of Ihe protcases ~Ind chemical reagent s u~ed in in-ge l di gc~lion (X denotc ~ amino ac id)

Prolea~e/ chcmi ca l rcagc nl

Try psi n

EIiLioproleasc Lys-C

Encioprolcase Arg-C

ElidoprOlcase Asp-N Endoprolease Glu-C

Chymolryps in

SWphviococclIS Ollrells VR Cyanogen bromide

2-N il ro-.'i- lhi ocyanobc ll zoic acid

BNPS-sblOk

Cleavage sile: Typical reaclion co nd ili on~

K-X. R-X where: X;t .'i()mM ammoniulll bicarbo nate. p ll 7.R at 37"e 1'01' -I Prol i nc 10 IRhr K-X whcre X;t Proline 2.'iIllM Tris-HC I. p ll 7.7 wilh ImM EDT.t\ al 37"C

1'0 1' 4 10 18hr H.-X whe re: X;t Pro line IOmM Tri s- II C!' " II 7 . .'i wilh 2 . .'imM D1T ami

.'iOIllM CaCl~ al 37"C ror -11 0 18hr X-I) .'iOIllM phosphale: burrel'. p ll 7.0 al 37"C ror -I lu I Shr E-X whe re X;t Prolinc SOmM ammonium bicarbonal e. " II 7.8 al 37"C ror-l

to I Shr L-X. F-X. V-X. W-X SOIllM ammonium bicarbollale. p H 7.8 al 37"C 1'0 1' -1

10 IShr D-X. E-X 2S IllM Tri s- HC!' pH 8 al ]7"C 1'0 1' 4 10 I Rhr X-M O. t M CN Br ill 70";(J (v/v) HCOO H. al room

teillperaillre 1'0 1' I Rhr X-C (i) Modificati oll ill 6M guallidine- HC!. O.2M Tri s­

aceta te. p H 8. at room temperature. l.'i Illill . (ii ) Cleavage in oM guanidinc- II C!. 0. 1 M sod ium borale. pH 9 at ]7"C 1'0 1' I 2hr

W-X 50lllM I3NPS-skatole in glacial aeelic ac id al room lemperature 1'01' 48hr

MAITHAL: PROTEOMICS - A NEW PLAYER IN THE POST-GENOMIC ERA 295

3. Washing of gel pieces (jor Coomassie-stailled gels ollly) a. If the gel pi eces excised from the gel are still blue rehydrate them in I 00-150 ~ I of 0 . 1 M NH4HCOJ and after 10-15 min add an equal volume of acetonitrile (to get 0.1 M NH4HCO.,!acetonitrile I: I v/v).

b. Vortex the tube for 15-20 min. Then spin down the ge l particles and remove all liquid. Shrink the gel pi eces with acetonitrile. Remove all liquid. Dry down gel particles in a vacuum centrifuge.

c. Repeat step 3 if necessary.

4. Ill-gel digestioll with trypsill a. Rehydrate gel particles in the digestion buffer containing 50 mM NH.jHCOJ, 5 mM CaCh and 12.5

ng/~ I of trypsin at 4°C (ice bucket) for 30-45 min . (A fter 15-20 min check out the samples and add more buffer if all liquid is absorbed by the ge l pieces).

b. Remove the remaining supernatant. Add 5-25 ~I of the same buffer but without trypsin to cover gel pieces and keep them wet during enzyme cleavage.

c. Leave the sa mpl es at 37°C overnight.

5. MALDI a1lalysis of the supernatallt after in-gel digestion a. After overnight incubation spin down the water droplets condensed on the tube lid. Take up small (1-2 ~ I) aliquot of the supernatant for MALDl analysis.

6. Extractio1l of peptides a. If Nano ES MS/MS analysis is necessary tryptic peptides should be extracted from the gel particles. Add 10-15 ~I of 25 mM NH4 HCOJ . Incubate at 37°C for 15 min with shaking. Spin down the gel particles and add acetonitrile (1-2 times equal the volume of gel particles). b. Incubate at 3]DC for 15 min with shaking. Spin down the liquid and collect the supernatant.

c. Add 40-50 iJl of 5% formi c acid . Vortex for 15 min at 37°C.

d . Spin down the gel particles and add acetonitrile (1-2 times equal the volume of gel particles). Incubate at 3]DC for 15 min with shaking. Spin down the gel particles and pull the extracts together.

e. Dry down the extracts in a vacuum centrifuge.

The masses of these peptides are then determined by mass spectrometry, usually using matrix-assisted laser desorption time-of flight mass spectrometry (MALDI-TOF). The system is highly sensitive and

can give information even at picomolar peptide concentration J4

.

The extract obtained by in-ge l di gestion is resu spended in mlllimum volume o f 50% acetonitrile/5 % formic acid and analyzed l L ing MALDI-TOF mass spec trometer. Preliminary MALDI results will determine whether or not a cl ean­up using C 18 ZipTips (Millipore) or HPLC separati o n of the peptides is required. Generally , the MALDI matrices used for unseparated digests are alpha­cyano-4-hydroxycinammic acid in 50% acetonitril e I 1% TFA (10 mg/ml) and 2,5-dihydroxybenzoic ac id (DHB) , in 20% acetonitrilell % TFA (10 mg/ml ). Some of the matrices that are generally used in MALDI-TOF are li sted in Table 3.

The proteolyti c masses obtained are then evaluated using a peptide-mass fingerprinting tool such as MS­Fit, Mascot or Peptide Search. These tools try to determine protein from existing database. It is important to note that thi s exercise will only be successful if the protein being analysed is represented in the databases. For proteins, which have inco mpl ete sequence information , it is necessary to obtain sequence information for the protein by Edman degradation 35 or by tandem mass spectrometryJ6. Thi sequence information can then be used along with the mass spectrometry information to search the expressed sequence tag (EST) databases. Some of the URLs associated with database searching are li s ted in Table 4.

Applications of Proteomics in Biomedical Research Proteomics offer great potenti al in unrave ling

complex biological proble ms such as the nature of particular molecular complexes or pathways in disease pathogenesis or alteration due to dietary deficiency or drug treatment. This has led to tremendous progress in the area of bi omedical research.

Callcer Cancer proteomics comprises of identi fi cation and

quantitati ve analysis of di fferentially expressed proteins in malignant cells, at di fferent stages of di sease, from preneoplasia to neoplasia re lati ve to normal , healthy tissue. Proteomics is important in identification of biomarkers because the proteome reflects both the intrinsic genetic programs of the ce ll and the impact of its immediate environment.

At the protein level , di stinct changes occur during the transformati on of a healthy ce ll into a neoplasti c

296 INDI AN 1. BIOCHEM . BIOPHYS., VOL. 39, OCTOBER 2002

Table 3-Commonmatri ces used MALDI-TOF analysis

Matri x

2-A mino-4-methyl-5-nitropyridine 2-Amino-5-nitropyridine 6-Aza-2-thiothymine Caffe ic ac id ex -Cyano-4-hydroxy cinnami c ac id 2.5-Dihydroxybenzo ic acid

Analytes

Protei ns. oligonucleotides Oligonucleotides Proteins

Laser wave length

355 nm 355 nm 266, 337 nm, 2.94111 m 337, 10600 nm 337 nm 337,355, 2940 nm

2.5-Dihydrox, benzoic ac id and fucose ( I: I) Feruli c ac id

Protei ns, oligonucleotides Proteins. oligosaccharides Proteins. oli gosaccharides, oligonuc leotides. sulfonic acids Protei ns Protei ns. 01 igonucleot ides Proteins

337 nm 337.355.488 nm 532 nm Glycero l wi th rhodamine 6G (0. 1 M)

2-(4-H ydroxyphenylazo) benzoic ac id

3-Hyd roxypicolin ic ac id Nicotinic acid

3-Nitrobenzy l alcohol

Proteins. gangli osides, industri al polymers Oligonucleotides, glycoproteins Proteins. glycopro teins, oli gonucleotides Pro teins

266. 377 nm

266. 308. 355 nm 266 nl11

3-Nit robenzy l alcohol with rhodamine 6G 3-Nit robenzy l alcohol with I ,4-diphenyl-I ,3-butadiene (O. IM)

Proteins Proteins

266 nm 532 nm 337 nm

2-Pyrazinecarboxylic acid Sinapin ic ac id Succ inic acid

Proteins Proteins. industri al polymers Pro teins

266 nm 337.355 nm 2940, 10600 nm

Table 4 - URLs assoc iated with database-search algorith ms

Database Search Algorithm

ProFound Mascot PepSea

MS-Fit MOWSE Peptldent Multildent SEQUEST Mascot PepFrag MS-Tag

URL

http:// 129.85. 19. 192/pro found_binlWebProFound .exe ht tp://www. matri xsc ience.com/cgi/search_form.p I? SEA R C H=PM F http ://pepsea. protana.com/PA_PepSeaForm.html http ://pepsea.protana.co mIPA_PeptidePat tern Form. h t ml http ://prospector.ucsf.edu/ucsfh tml4. O/ms II t. ht m http ://srs. hgm p.mrc. ac. u k/c g i -bi n/mowse http ://www.ex pasy.ch/ tools/peptident.html http: //www.ex pasy.ch/tools/mu Iti identl http://fi elds.sc ri pps.edu/sequestlindex. html http://www. mat ri xsc ience.com/cgi/searc h_form.pl?SEA RCH=M IS http://prowl. roc kefe ller.edu/PROWLlpepfragch. html http://prospector.ucsf.edu/ucs lll! m14. O/mstag f d. h tm

cell , rang ing from altered express ion, different ial prote in modification, and changes in specifi c acti vity, to aberrant locali zati on, all of which may affect cellul ar function . Identi fying and understanding these changes are the underlying areas in cancer

squamous ce ll carcino ma, which could be used as a bi o marker fo r nonin vas ive fo llow up)!) .

• Through 2-DE analy is of co lo rectal carcino mas and healthy co lo nic epithelial ce ll s, Jungblut ef O/. ClO

di scovered a prote in that was expressed exclusive ly in the tumo r ti ssues. Thi s low-molecul ar-we ight prote in . Calgranulin B, was expressed in dyspl asti c po lyps fro m patients with colon carcino ma and ulcerat ive colitis4u. Altho ugh thi s prote in seems to be hi ghl y specifi c for preneopl asti c and neopl as tic ti ssues, its role in carcinogenesis is unclear.

. ,7 proteomt cs' .

Some of the examples where proteomics based approach is used in cancer research is g iven below:

• Ostergaard ef af.38 analyzed the proteome o f 150 bl adder tumors and observed a decline in the express ion o f specific cy tokeratins, psoriasin , ga lect in 7, and strati fin in tumors with a low degree of diffe renti atio n. Furthermore, 2-DE analys is o f urine ex hi bited the presence of psoriasin in patients with

• Sarto ef a f.4 1 constructed a map o f heal thy and renal ce ll carcino ma (RCC) protein s th rough 2-DE analysis o f healthy and RCC kidney ti ssue, which led to the

•

MAITHAL: PROTEOMICS - A NEW PLAYER IN THE POST-GENOMIC ERA 297

identification of ubiquinol cytochrome c reductase as a potential biomarker.

• In an earl y study of tumor progression and metastases in a rat model of mammary adenocarcinoma, using 2-DE, Welch et al. 42 found a protein , P50.9, that correlated in versely with the metastatic phenotype, suggesting quantitative and qualitative protein differences between tumor types.

• In other studies, profiles from various grades of breast tumors have been obtained, which exhibited increase in proliferating cell nuclear anti gen in i nvasi ve carci nomas43

-45

. Di sti nct differences were observed in the protein expression profil es of fibroadenomas and invasive carcinomas.

• Two polypeptide markers, TAO I and T A02 (naps in ), were found preferenti ally ex pressed in 90% of primary lung adenocarcinomas through the use of 2_ DE-l6 --l S.

• Applying 2-DE, Alaiya el al. 49 attempted to proteomically define ovarian tumors into beni gn, borderline and mali gnant types. Malignant tumors ex hibited increases in proliferating cell nuclear antigen. OPI8, pHSP60, HSP90, and calreticulin and decreases in tropomyosin-I and -2 when compared wi th beni gn tumors. The authors were ab le to discriminate between malignant and benign tumors, using a panel of nine proteins. With the use of 2-DE maps to di stin gui sh between prostate carcinoma and benign prostatic hyperp las ia, a similar pattern of

. . b b d50 I proteJll ex press ion as a ove was 0 serve . n addition , malignant tumors displayed increased expression of oncoprotein l8(v), elongati on factor-2, glutathione S-transferase, superox ide dismutase, and tri osephosphate isomerase and decreased expression of cytokerat in 18.

• 2- DE maps of neuroblas toma indicated th at the protein pI9/nm23-H I was expressed at increased concentrati ons compared with limited-stage di sease51

.

The expression of thi s protein was observed to be pos iti ve ly associated with metastases and large tumor mass.

• Us ing MALDI-TOF-MS of 2-DE separated proteins, Sarto et 0/. 5'2 identi fied multimeric isoforms of manganese superox ide di s mutase expressed exc lusively in RCCs. Interestingly, they also observed ex press ion of isomeric forms of glutathione perox idase only in healthy human kidney samples. Modified expression of these proteins renders them as poten tial markers in RCC because they also map to

chromosome loc i 5q2l and 6q21-6q27 , which have been implicated in the oncogenesis of RCC52

.

• Screening for the multipl e forms of the molecu lar chaperone 14-3-3 protein in healthy breast epithel ial cells and breast carcinomas yielded a potenti al marker for the noncancerous cells53

. The 14-3-3s form was observed to be strongly down-regulated in primary breast carcinomas and breast cancer cell lines relat ive to healthy breast epithelial cells. This finding , in ligh t of the evidence that the gene fo r 14-3-3s was found silenced in breast cancer cell s54

, implicates th is protein as a tumor suppressor.

• Using MALDJ-MS system, Bergman ef a l55

detected increases in the ex pressions of nuclear matrix , redox, and cytoskeletal proteins in breast carcinoma rel ative to benign tumors_ Fibroadenoma exhibited an increase in the oncogene product DJ-I. Retinoic ac id-bi ndi ng protein , carbohydrate-bi nding protein , and certain lipoproteins were increased in ovarian carcinoma, whereas Catheps in D was increased in lung adenocarcinoma.

• Liquid chromatography-MS and tandem MS (MSMS) were used to identify thymosin b.4, a 4964-Da protein found only in the outer proliferating zone of the tumor56

.

Currentl y many tumor types are being stud ied using proteomics approach and the use of complementary techniques such as laser cap lLI re microdi ssection57

.58 to isolate mali gnant ce lls for

electrophoretic analys is, have facilitated research in thi s area .

Cardiovascular diseases One of the major causes of mortality in the

developed countries has been attri buted to cardiovascular diseases. The pathogenes is of the cardiac dysfuncti on is still largely unknown, but an overview of the overall changes in protein express ion in heart di sease and heart failure using protcomics based approaches have provided new insigh ts in to the cellul ar mechani sms in volved in cardi ac dysfunct ion. together with new diagnostic markers and therapeuti c opportuniti es.

Application of proteomics to the study of dilated cardiomyopathy has resulted in the es tab li shment of a human myocardial database containi ng 150 identi fied proteins. Compari son of thi s database with protein profiles from patients with dilated card iCJmyopathy showed significant differential expression for 25

298 INDI AN J. BIOCHEM. BIO PHYS .. VOL. 39. OCTO BER 2002

prote ins out of whi ch 12 proteins were identified4o.59

.

Bes ides thi s cardi ac anti gens that e licit specifi c antibody responses ill vivo are being identified. Many of these anti gens associated with acute60 and chronic6 1

rejec ti on after cardi ac transplantati on are being investi gated as potenti al non-invasive markers.

Nellrological disorders Prion di seases like Creutzfeld-Jakob disease (CJD),

charac teri zed by fatal degenerati ve encephalopathy, have been implicated to post-translational modi fications at protein levels. It is important to note that post-translati onal modifications of proteins are de tectable onl y at protein level; hence proteomi cs based approach beco mes vital fo r such anal ys is. Ana lys is of cerebrospinal fluid by Harrington et ai. revealed two proteins, viz. p 130 and p 13 1, which could be used to di scriminate between CJD and other types of dementi a wi th a sensiti vity of 88% and spec ificity of 99%(1~. These proteins are also present in some pati ents with other neurological di sorders without dementia, poss ibly refl ecting neuronal damage rather than disease pathogenesis.

Alzheimer's disease is one of the major causes of clementia. Prote i n express ion pro fi les of the brai n ti ss ues of patients suffe ring from Alzheimer's di sease showed that five protein spots were increased in the Alzhei mer's group, twenty-eight were decreased, and nine were uniquely expressed63

. ]n another study a decreased express ion of one particul ar protein , di azepam binding inhibitor, was observed in both schi zophreni c and Alzheimer's pati ents: although thi s may be due to the action of drugs but can also

. h 6-1 represent a unique c ange .

Infectiolls diseases Genome sequencing of many microorganisms has

been co mpleted65 (Table 5). The main aim of most

studi es has been the search fo r new di aanos ti c ~

markers, candidate antigens fo r vacc ines. and determinants of virulence. Currentl y. studies are underway to unders tand the proteomes of many pathogeni c organi sms like: (a) An analys is of Sa/lIloll ella typliilllllrillJII (an en teri c pathogen responsible for many cases of gas troenteriti s in humans) cell envelope proteins led to identification of 53 proteins by 2D-ge l electrophores is followed by N-terminal sequencing66

. A better unders tandin g of the proteome of S. typliilllllril.1I1/ will be useful in identifying pro teins that are associated with virulence. (b) The immune response to in fec ti on with Helicobacter pylori has been studi ed and 20 prote ins have been identifi ed whi ch were react ive with the serum of infected patients67

•

(c) Eryth romycin res istance in StreptocoCClls pl/eulIloniae has been probed and protein ex press ion in erythromycin-susceptibl e and -res istan t strain s was compared and it was seen that glyceral dehyde-3-phos phate dehydrogenase was expressed by the resistant strain , but not by the susceptib le strai n6x .

(d) Tubercul osis causing bac teri a. Mycobacterilllll tllberclllosis is probably one of the most studi ed mi croorgani sms. Several research groups have been looking at the mycobacteri al proteomes, parti cul arl y h d . 6'J 70 S ' . t e secrete prote lI1s· . everal o t the antigens

identified by these studi es have now been incorporated into tri al vacci nes 7 1.

Limitations of Proteomics There are several technological li mitati ons present

in the current protcomi cs approach. One of these is that formalin-fi xed archival ti ssue cannot be used for 2D-gel electrophores is, as the cross-linking of proteins by aldehyde fi xatives renders them insolu ble. Proteomics is believed to be time-consuming, In

Table 5 - A compari son of some of the genome sizes and the num ber o f genes present in d ifferenl o rgani sms

Organi sm Genome size No. o f genes (MB)

Haellloph illis il//lliel/ ::.ae 1.83 1.740 Helicobac/er priori 1.66 1.590 Me/hal/ococclis jOl/ aschi i 1.66 1.680 E. coli 4.6 4,288 Yeas t (Saccharom rces ceril'iseae) 13 6.000 Bacilllls .llIb/ilis 4.2 4. 100 Drosophila 139 13.60 1 Caellorhablii/is elegalls 97 19,099 Arabiliopsis //wliall a 100 27,000 Human 3200 38.000

.~

MAITHAL: PROTEOM ICS - A NEW PLA YER IN THE POST-GENOMIC ERA 299

particular the time taken to perform 2D-gel electrophores is . Further hydrophobic proteins cannot be resolved properly by 2D-gel electrophores is72

.

Another limitation of 2D-gel e lectrophoresis is in detection of low abundance proteins and it is important to note that the presence of abundant proteins can also mask minor prote ins.

The limitations posed by 2 D-gel electrophoresis may be overcome in the near future by advances in othe r methods or prote in separation, but they are still

an important cons ideration when planning

researc h us ing proteomics.

It is important to note that although MALDI-MS is a workhorse of proteomics, still the re are some limitati ons assoc iated with it. Some of these are mentioned below:

a. Ioni zati on of analyte molecules is a se lec tive process and not quantitati ve. Thi s leads to difficulty in determining the differentia l expression of prote ins, although recent advances in quanti tati ve proteomics using stable isotope labe ling has shown g reat promise.

b. In case of low abundant prote ins the number of peptides observed are usuall y low; thi s has been attributed to poor so lubility of prote in mi xture, se lec tive adsorption , suppress io n of ionization , se lec ti ve io ni zati on and very short peptide lengths. This leads to ambiguity in identification of unknown protein s.

c. MALD1-TOF-MS has a limited ability to deal with prote in mixtures. Thi s re nders a great difficulty in correct identificatio n of the prote in from masses of peptides , wh ich wi ll inc lude fragments from more then, a sing le protein.

But inspite of these shortco mings MALDI-TOF­MS is a popular cho ice primarily because it provides a method for hi gh throughput analysis. Further it is a method o f choice for hi gh abundant proteins for organi sms whose compl ete geno me has been sequenced.

Future Prospects Since there is no equivalent of PCR for

amplifi cati on of low-abundance proteins, a constant need for improvement in the efficiency of proteomic techniques, especia lly with regard to automati on, sample th roughput and sensiti vity is highly desired73

•

Many new complementary technolog ies are be ing deve loped I ike protein arrays 74, the yeast two-hybrid sys tem75, phage-di splay antibody librari es 76, surface-

enhanced laser desorption and ioni sation (SELDI)77.78 etc. which will be a major step forward in express io n profiling or molecular inte raction screelllng. Development in the area of modern mass spectro metry will also he lp in better characteri zati on of proteins7,). Further, a parallel development in the area of bioinformatics will also playa key ro le in database analysis , protein identificati on and development of protein interactio n maps.

Conclusion Proteomi cs attempts to catalog and characteri ze the

proteins, compare variations in the ir expression levels in health and di sease, study the ir inte ractio ns. and identify the ir functional ro les. Proteo illics is a composite study of a set of protei ns and requires new technologies for high throughput analysis. Proteomi cs wi ll contribute greatly to our understanding of gene function in the post-geno mic e ra. Since it is oft en difficult to predic t the function o f a prote in based on homology to other prote ins or even the ir three­dimensional structure, de te rminatio n o f components o f a pro te in complex or of a cellular structure is central in functional analysis. Thi s aspect of proteomic studies is pe rhaps the area of greatest promise 13

. With the ease in clonin g proteins by modern molecul ar biology techniques it will enhance the understanding of the bi oche mi stry of prote ins. processes and pathways. Furthe r, proteomics is the link between genes, prote ins and disease and many drugs either act by targeting prote ins or are prote ins. so it can be expected that proteomics wi ll pl ay an important role for drug di scovery and development I J

In addition, many molecular markers of disease, the basis of diagnostics, are proteins so prote in express ion maps can be used as a guide to drug desig n.

The development of proteomics renders us with a powerful tool to examine patho logica l processes at the molecular level and identify sets of prote ins (pathways or cluste rs) th at are responsible for causing it. Thi s will help in develo ping di agnostic and therapeutic preparations. Further, as the techno logy of proteomics analys is w ill further improve, it wi ll be possible in the near future to combine genomi c and proteomic informati o n to obtain a more co mprehensive picture of many patho log ical conditions and a lso develo pmenta l processes.

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