Fang Yan1*Arun Sreekumar2*Bharathi Laxman2*Arul M. Chinnaiyan2, 3, 4

David M. Lubman1

Timothy J. Barder5

1Department of Chemistry2Department of Pathology3Department of Urology4Comprehensive Cancer Center,University of Michigan,Ann Arbor, MI, USA,

5Eprogen, Darien, IL, USA

Protein microarrays using liquid phase fractionationof cell lysates

We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves apI-based fractionation using chromatofocusing in the first dimension followed by non-porous reversed-phase high-performance liquid chromatography (HPLC) of each pIfraction in the second dimension. This allows fractionation of cellular proteins in theliquid phase that could then be arrayed on nitrocellulose slides and used to studyhumoral response in cancer. Protein microarrays have been used to identify potentialserum biomarkers for prostate cancer. It is shown that specific fractions are immuno-reactive against prostate cancer serum but not against serum from healthy individuals.These proteins could serve as sero-diagnostic markers for prostate cancer. Impor-tantly, this method allows for use of post-translationally modified proteins as baits fordetection of humoral response. Proteins eliciting an immune response are identifiedusing the molecular mass and peptide sequence data obtained using mass spectro-metric analysis of the liquid fractions. The fractionation of proteins in the liquid phasemake this method amenable to automation.

Keywords: Liquid chromatography / Microarrays / Prostate cancer / Protein PRO 0443

1 Introduction

Cancer is one of the leading causes of death in the mod-ern world [1]. The term cancer is used when cells losetheir ability to regulate growth, become immortal and inmost cases migrate from their primary location into anew unrelated site. A number of genetic and epigeneticfactors contribute to the development, progression, andinvasiveness of cancer. It thus becomes important tostudy these genetic regulations, which might help designtherapeutic interventions to this deadly disease.

The advent of DNA microarrays has resulted in a largewealth of genetic information on development, progres-sion, and the invasive properties of cancer [2–4]. How-ever, there remains a large vacuum in understanding theproteome of cancer cells. This becomes important sinceproteins form the final denominators for the cellularphenotype. Also it is difficult to draw a linear correlationbetween gene expression and protein function since thelatter depends on many factors such as post-translational

modification, compartmentalization, interaction with otherproteins, and environmental factors such as pH, ionicstrength, temperature, etc. Hence it becomes essentialto develop high-throughput platforms for the study ofproteins in cancer.

Microarray or “biochip” technologies have the potential ofrevolutionizing the analysis of human cancer. By simulta-neously measuring the expression of thousands of pro-teins in clinical specimens, a large number of data pointscan be collected to form a molecular fingerprint of a dis-ease process. Tumor markers correlate with malignancyand are detected in solid tumors, lymph nodes, bone mar-row, stool, serum, or urine. Detecting or monitoring thelevels of tumor markers may aid in diagnosis, staging,population screening, and assessment of response totreatment or for identification of metastatic or recurrentdisease. The application of protein microarray technologyin the study of cancer is poised to have a major impact onthe discovery, characterization, and clinical application ofcancer biomarkers.

Protein microarray methodologies that have been ex-plored for high-throughput screening of changes in pro-tein expression include both antigen and antibody arrays[5–19]. Many of these approaches use recombinant pro-teins obtained from cDNA libraries [12, 13, 15] or phagedisplay libraries [17]. These methods have been shown

Correspondence: Dr. David M. Lubman, Department of Chem-istry, University of Michigan, 930 N. University Ave, Ann Arbor,MI 48109-1055, USAE-mail: dmlubman@umich.eduFax: +1-734-615-8108

Abbreviations: CF, chromatofocusing; FCS, fetal calf serum;IgG, immunoglobulin G; NPS, nonporous silica; PCA, prostatecancer

1228 Proteomics 2003, 3, 1228–1235

These authors contributed equally to this work.

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0707–1228 $17.50�.50/0

DOI 10.1002/pmic.200300443

Proteomics 2003, 3, 1228–1235 Protein microarrays 1229

to have excellent sensitivity that is sufficient for measuringmany clinically important proteins in patient blood sam-ples and sera. A limitation of these technologies thoughhas been that in cellular systems, proteins may undergonumerous post-translational modifications such as phos-phorylation, glycosylation, and acetylations that often playan important part in protein function. A deleterious changein the pattern of post-translational modifications couldresult in transformation of a normal protein into an onco-gene, which may lead to cancer development [20–22].More recent work has sought to use proteins as producedin the cells for analysis of protein markers. Such methodshave included the use of tissue microarrays [23] and theextraction of proteins from cells using either 2-D gel elec-trophoresis or liquid phase separation methods [14, 24].

In this work, we present a new method for preparinglarge numbers of intact proteins from cell lysates formicrochip array analysis. This method involves separat-ing intact proteins from cell lysates using chromato-focusing in the first dimension [25–29]. Each fraction isthen separated in a second dimension by nonporoussilica RP-HPLC [30–35]. The result is a 2-D liquid phasefractionation of the proteins from the cell lysate, at rela-tively high purity. Using this method, hundreds of iso-lated proteins in the liquid phase can be collected forspotting on the microchip array. The fractionated pro-teins can then be spotted on a nitrocellulose slide andused to study humoral response by exposing them tosera from cancer patients and normal individuals. Thismethod allows for comprehensive analysis of the cancerproteome using very small amounts of analyte obtainedby fractionation.

2 Materials and methods

2.1 Chemicals

Urea, thiourea, iminodiacetic acid, DTT, octyl glucoside,glycerol, bis-tris, trifluoroacetic acid, methanol, acetoni-trile, �-mercaptoethanol, Tween 20, PMSF, and normaldonkey serum were obtained from Sigma (St. Louis, MO,USA), Cy5-conjugated anti-rabbit immunoglobulin G(IgG), biotin-conjugated anti-human IgG and Cy5-conju-gated streptavidin were obtained from Jackson Immu-noResearch Laboratories (West Grove, PA, USA). RPMIand fetal calf serum (FCS) were obtained from Invitrogen(Grand Island, NY, USA). The reagents used were in themost pure form commercially available. Polybuffer 74was purchased from Amersham Pharmacia Biotech (Pis-cataway, NJ, USA). Water was purified using the Milli-Qwater filtration system (Bedford, MA, USA). All the sol-vents were HPLC-grade unless otherwise specified.

2.2 Sample preparation

Cell culture: LnCAP cells were cultured in RPMI mediumsupplemented with 10% FCS at 37�C and 5% CO2.Approximately 1 million cells were plated in 100 mm tis-sue culture dishes (Becton Dickinson, Franklin Lakes,NJ, USA). Log phase cells were harvested, washed inPBS and pelleted. The cell pellets were lysed and usedfor fractionation. Protein extraction: Cell pellets werereconstituted in lysis buffer. The lysis buffer consistedof 7 M urea, 2 M thiourea, 100 mM DTT, 0.5% Biolyte3–10, 2% octyl glucoside, and 1 mM PMSF. The cellpellets were lysed at room temperature for half anhour, followed by centrifugation at 35 000 rpm at 4�Cfor 1 h. The supernatant was stored at �80�C for futureuse. Sample preparation for chromatofocusing (CF):A PD 10 column equilibrated with 25 mM bis-tris in6 M urea and 0.2% octyl glucoside, was used to ex-change the cell lysate from the lysis buffer to the abovebuffer.

2.3 Chromatofocusing on LnCAP cell lysate

In the CF experiment two buffers, a start buffer and anelution buffer, were employed. The start buffer was 25 mM

bis-tris, pH 7.1, and the elution buffer was polybuffer74:water in a ratio of 1:10, pH 4.0. Both buffers were pre-pared in 6 M urea and 0.2% octyl glucoside. Iminodiaceticacid was used to adjust the pH of both buffers. The PS-HPCF 1D column was equilibrated with the start bufferuntil the pH of the effluent was 7.1. Sample was appliedto the column with multiple injections. Once a stablebaseline was achieved, the elution buffer was switchedon to elute the proteins on the column in an isocraticmode. UV detection was performed at 280 nm and thepH of the effluent was monitored using a flow-throughonline pH probe. The pH fractions were collected in0.2 pH intervals and 15 fractions in total were collectedin the range of pH 7–4. The CF separation was stoppedwhen the pH of the effluent reached 4.

2.4 RP-HPLC on pI fractions

RP-HPLC was performed using PS-HPCF 2D (4.6�33 mm)columns. Solvent A was 0.1% TFA in water and Solvent B0.1% TFA in acetonitrile. The gradient was run from 5%to 15% in 1 min, 15% to 25% in 2 min, 25% to 31% in2 min, 31% to 41% in 10 min, 41% to 47% in 6 min, 47%to 67% in 4 min, finally up to 100% B in 3 min and heldfor another 1 min, then back to 5% in 1 min at a flow rateof 1 mL/min. The column temperature was 40�C higherthan the ambient temperature. UV absorptions weremonitored at 214 nm. RP fractions were collected using

1230 F. Yan et al. Proteomics 2003, 3, 1228–1235

a fraction collector. The fractions were dried down com-pletely by Speed-Vac at 75�C and stored at �80�C untilfurther use.

2.5 Protein identification

The targeted UV peak in the second-dimensional RP-HPLC chromatogram was collected and aliquoted intothree fractions. One portion was dried down completelyand used for microarray experiments. A second portionwas dried down completely and reconstituted in 50%ACN with 2% formic acid. The resulting solution was di-rectly infused into an ESI-TOF-MS (LCT; Micromass,Manchester, UK) at 10 �L/min to determine intact molec-ular weight values of the proteins in the fraction [20, 38].The other portion of the liquid band was dried down toeliminate ACN and TFA. 1 M NH4HCO3 and 10 mM DTTwere then added to a final concentration of 100 mM and1 mM, respectively. A sequence-specific endopeptidasesuch as trypsin was then mixed with the denatured pro-teins at the ratio of 1:50. The mixture was incubated at37�C for 24 h. MALDI-TOF-MS (TOFSpec2E; Micromass)was subsequently employed to analyze peptide massfingerprints (PMFs), which were then used to search forthe registered peptide masses of proteins in the data-base. The intact molecular weights from LCT and thePMF obtained using MALDI-TOF-MS were together usedto identify the protein in the database. Further confirma-tion of the protein identity was obtained by MS/MSsequence analysis using the MALDI QTOF MS (Micro-mass).

2.6 Printing the fractionated proteins

The fractionated proteins were resuspended in 25 �L buf-fer containing 125 mM Tris-Cl (pH 6.8), 4% SDS and 2%�-mercaptoethanol (Sigma, St. Louis, MO, USA). Thiswas the minimum volume required for spotting in ourinitial work. The samples were denatured by heating at70�C for 2 h followed by a final denaturation at 100�C for10 min in a thermal cycler (Perkin Elmer Life Sciences,Boston, MA, USA). The samples were transferred to a96-well microtiter plate (MJ Research, Waltham, MA,USA) and printed on nitrocellulose slides (Schleicher &Schuell, Keene, NH, USA) using a GMS 417 microarrayprinter (Genetic MicroSystems, Woburn, MA, USA). Inlater work, the spotting was performed using MagnaSpotter microarray printer (Bioautomation). Using thissystem the spotting volume of the protein was reducedto 2.5 �L, allowing the ability to use concentrated frac-tions as baits. Each spot measured approximately 300 �min diameter and was placed 1200 �m apart. The platesused to print are 96-well V bottom plates from Major Sci-

entific. The slides were either used immediately or storedat 4�C. They were dried for 1 h at room temperaturebefore further processing.

2.7 Hybridization of slides

Nitrocelluose slides containing spotted proteins wereblocked in TBS containing 1% non-fat milk and 5% nor-mal donkey serum (Sigma) in 0.1% Tween-20 (Sigma),for 1 h at room temperature (RT). The slides were rinsedwith TBS and incubated with either serum from prostatecancer patients or normal individuals (1:50) in a micro-caster chamber (Schleicher & Schuell) for 2 h at RT. Theywere washed six times with TBS-T (TBS containing 0.1%Tween-20), each for 5 min. The slides were then incu-bated with biotin-conjugated anti-human IgG (1:200;Jackson ImmunoResearch Laboratories) for 30 min atRT. They were then washed with TBS-T and incubatedwith Cy5-conjugated streptavidin (1:250; Jackson Immu-noResearch Laboratories) for 30 min. The slides werefinally washed and dried as described above and ana-lyzed using a microarray scanner (Axon Instruments,Foster City, CA, USA).

2.8 Data analysis

The scanned image was analyzed using Gene Pix 4 soft-ware (Axon Instruments). The average median intensity ofstreptavidin Cy3 spots (n = 20) was calculated and usedto normalize across hybridizations. Briefly, for the pur-pose of normalization, the average median intensities forstreptavidin Cy3 were calculated independently for eachslide. Each of these values were then divided against thehighest value of average median intensity for streptavidinCy3 obtained among the different hybridizations, to ob-tain a factor which would define the variation caused byeither spotting or hybridization. The average medianintensity for each reactive protein in each slide was thendivided by this factor to get a normalized value for eachmeasurement. Arbitrary threshold values were assignedfor each reactive protein based on its ability to classifythe cancer and normal samples.

3 Results and discussion

In order to develop a microarray of cancer proteins, pros-tate cancer cell line, LnCAP, was fractionated using a pIgradient by chromatofocusing at 0.2 pI intervals. Each ofthese pI fractions were collected and separated in thesecond dimension by nonporous silica (NPS) RP-HPLC.A profile of the total fractionation from pH 4–7 is shownin Fig. 1. Some of the fractions obtained after chromato-

Proteomics 2003, 3, 1228–1235 Protein microarrays 1231

Figure 1. 2-D map of the liquidseparations of the LnCAP cellsusing chromatofocusing over apH range of 4–7 followed byseparation in the second dimen-sion using NPS RP-HPLC.

focusing contained as many as 70–90 proteins. As arepresentative example the different proteins separatedby NPS RP-HPLC from the pI fraction 5 are shown inFig. 2. It is estimated that the sample was fractionatedinto as many as 1400 protein bands. Many of these bandscontain single proteins, but some contain more than oneprotein. The presence of one or more proteins could beconfirmed by MW measurements using ESI-TOF-MS.Nevertheless, this method provides cellular proteins witha relatively high degree of purity, which could then beused for microarray experiments. The relative amount of

Figure 2. NPS RP-HPLC separation of CF fraction #5 atpH 6.0–6.2.

protein collected in each fraction from the CF column andloaded onto the HPLC separation, as determined by theBradford protein assay, is shown in Fig. 3. More proteinsare usually recovered from the mid-pH fractions than to-wards the end of the pH range as expected. In theseexperiments using analytical columns, generally up to5 mg of protein could be loaded on the first dimensionwithout overloading the column and losing significantresolution. The total recovery of protein from the first di-mension has been found to be around 51% over therange of pH 4–7. The latter can be increased by extendingthe pH range to 8.5. In addition, some acidic proteins(with pI below 4) and highly hydrophobic proteins mightbe lost during the fractionation. An additional salt washand isopropanol wash might provide a means to recover

Figure 3. Concentration profile as a function of pI for thechromatofocusing of the LnCAP cell line from pH 7.0 topH 4.0. The most acidic fraction is on the right and themost basic on the left.

1232 F. Yan et al. Proteomics 2003, 3, 1228–1235

Figure 4. Detection of humoral response in serum fromnormal and prostate cancer patients. LnCAP cells werechromatofocused at pI 6.0–6.2 and fractionated usingNPS based RP-HPLC. The fractions were denatured andspotted on nitrocellulose slides. The slides were probedwith serum (A) from PCA patients or (B) normal indivi-duals. Specific fractions showed immunoreactivity withPCA (arrows) that was not seen with serum from normalindividuals.

these proteins. Nevertheless, on average we expectaround 150 �g total protein recovered from each pI frac-tion. The proteins from each pI fraction are further sepa-rated by NPS RP-HPLC, into about 70–80 fractions withhigh degree of purity. The recovery of proteins in eachfraction by NPS RP-HPLC depends on the proteins buton average is around 80% [35]. The result is that on

average around 1.5 �g of protein is collected from eachprotein band. This corresponds to approximately 12 pgof protein or 0.05 fmol for a 30 kDa protein. The sensitivityof the arrays could be increased tenfold by using a moreconcentrated protein solution as baits. Importantly, thismethod could be adapted to preparative scale such thatlarge amounts of material would be available for con-structing many protein arrays. In the present work ap-proximately 100 microarrays were spotted from eachcollection.

As a representative example, a slide-containing proteinsfractionated in the pH range of 4–7 were probed witheither serum from healthy individuals or from sera fromprostate cancer patients. Figure 4 shows one such repre-sentative example of the array containing 96 fractionsseparated in the pH range of 6–6.2. Immunoreactivitywas observed with specific fractions when probed withprostate cancer serum (Fig. 4A, arrow) which was notseen in serum from healthy subjects (Fig. 4B). This indi-cated the presence of auto-antibodies in serum fromprostate cancer patients against specific cancer asso-ciated proteins.

We next investigated the humoral response generatedagainst specific cancer proteins using a larger pool ofserum samples. Figure 5 shows the profile of auto-anti-

Figure 5. Detection of humoralresponse in PCA against frac-tion B6. (A) LnCAP cells werefractionated at pH 6.8–7. Thefractions were spotted on nitro-cellulose-coated slides andprobed with serum from normaland PCA patients. Specificimmunoreactivity was observedwith fraction B6, when probedwith PCA sera but not withserum from normal individuals(A). When a total of 34 serumsamples (15 normals and 19prostate cancer) were used foranalysis, 8 out of the 19 PCAsera showed the presence ofspecific antibody repertoireagainst fraction B6, giving asensitivity of 42% (B). However,only 1 out of the 15 normal seraused showed the presence ofantibodies against fraction B6,giving a specificity of 93% (B).

Proteomics 2003, 3, 1228–1235 Protein microarrays 1233

bodies against fraction B6, fractionated at pH 6.8–7. Aspecific repertoire of antibodies against fraction B6 wasfound in serum from prostate cancer patients while negli-gible immunoreactivity was observed using serum fromnormal individuals (Fig. 5A). Quantitation of the datarevealed 8 out of a total 19 prostate cancer (PCA) serareacted with fraction B6 giving a sensitivity of 42% whileonly 1 out of the 15 normals showed reactivity, giving aspecificity of 93%. Such high degree of specificity makesfraction B6 a useful candidate to identify PCA samples.This is especially so since prostate specific antigen(PSA), the routine marker used in the clinic, shows a spec-ificity of only 30–35% [36]. This implies that there is a highdegree of false positivity (60–65%) associated with PSA.

As a result of such low specificity, PSA is used only as aprimary screen to detect PSA. Thus, in this context, frac-tion B6 seems to be a promising candidate for monitoringthe occurrence of PCA using serum samples to supple-ment PSA levels. The low percentage of sensitivityobserved with fraction B6 becomes less of an issue sincePCA is wide spread in the general population.

The sensitivity and specificity of monitoring PCA usingserum markers could be increased by multiplexing datafrom multiple biomarkers. With this aim, additional spotswere investigated for their immunoreactive profile using24 serum samples, consisting of 12 normals and 12 can-cers (Figs. 6, 7). Proteins in these spots were identified

Figure 6. Detection of humoralresponse in prostate canceragainst creatine kinase fractio-nated at pH 6–6.2. LnCAP cellswere fractionated at pH 6–6.2.The fractions were spotted onnitrocellulose-coated slides andprobed with serum from normaland PCA patients. A total of 24serum samples (12 normals and12 prostate cancer) were usedfor analysis. 10 out of the 12PCA sera showed the presenceof specific antibody repertoireagainst creatine kinase, giving asensitivityof 83%. However, only2 out of the 12 normal sera usedshowed the presence of anti-bodies against creatine kinase.

Figure 7. Detection of humoralresponse in PCA against 54 kDanuclear RNA-DNA binding pro-tein fractionated at pH 6–6.2.LnCAP cells were fractionatedat pH 6–6.2. The fractions werespotted on nitrocellulose-coatedslides and probed with serumfrom normal and PCA patients. Atotal of 24 serum samples (12 nor-mals and 12 PCA) were used foranalysis. 10 out of the 12 PCAsera showed the presence of spe-cific antibody repertoire againstRNA-DNA binding protein, givinga sensitivity of 83%. However,only 5 out of the 12 normal seraused showed the presence ofantibodies against this protein.

1234 F. Yan et al. Proteomics 2003, 3, 1228–1235

using MALDI-TOF and their molecular weights weredetermined using LCT-MS. Figure 6 shows the profile ofauto-antibodies against mitochondrial creatine kinasewhich was fractionated from LnCAP cells at pH 6–6.2.Based on the profile of immunoreactivity shown by PCAand normal samples, a threshold value of 900 normalizedintensity units was used above which the serum was con-sidered to show specific immune response. When such acriteria was used, 10 out the 12 PCA samples displayed aspecific repertoire of auto-antibodies against mitochon-drial creatine kinase. In contrast, only 2 out of 10 normalsshowed any reactivity. Thus, the sensitivity and specificityfor mitochondrial creatine kinase was 83%. Interestingly,mitochondrial creatine kinase has been associated withadenocarcinoma and its detection in sera has been corre-lated to poor outcome of the cancer [39]. In this regard ourresults showing the presence of auto-antibody repertoirecould be due to overexpression of this protein in PCA.Another interesting protein which showed presence ofauto-antibody repertoire was the 54 kDa non-POU do-main containing nuclear RNA-DNA binding protein. Thiswas also fractionated from LnCAP cells at pH 6–6.2(Fig. 7). Among the 12 PCA sera tested 10 of the serumshowed specific immune response. Thus, using a novelmethod of fractionating proteins from cancer cells andprotein microarrays, this study identified three novel pro-teins that seem to have elicited a specific antibodyresponse in PCA. Interestingly, the specificity of immuneresponse to each of these three proteins showed a highdegree of specificity (58–93%). This was much higherthan the specificity exhibited by PSA (30–35%).

An important issue in these experiments is the ability toidentify the protein spots that show a humoral response.Identification is performed using a peptide mapping andMS/MS and Mr measurements. ESI-TOF-MS is used tomeasure the Mr of the intact protein directly from the vialfrom which the protein was spotted. The sample canthen be digested by trypsin and analyzed by MALDI-MSand MALDI-MS/MS for peptide mapping and databasesearching. The combination of intact Mr and peptidemapping and MS/MS generally provided a reliable matchagainst the database. The use of peptide mapping aloneoften provided an unreliable identification with a relativelylow MOWSE score. The MS/MS of several peptides usingMALDI-QTOF-MS usually provided the additional infor-mation needed to unambiguously identify the protein.

The use of CF fractionation provides a number of distinctadvantages in these protein array experiments. Themethod results in separation of large numbers of purifiedproteins in the liquid phase. In this initial work over a pHrange of 4–7 nearly 1400 protein bands were observed byUV detection and collected. One could extend the range

by changing the buffers so that additional proteins couldbe collected over a pH range up to 9.0. Further, the CFmethod has provided fractionation in intervals of 0.2 pI,where there is little overlap of proteins between adjacentpI fractions. This contrasts to previous work using theRotofor [38] as a means of liquid phase pI fractionation,where pI lanes are �0.5 pI and there is often overlap ofproteins between adjacent pI fractions. The CF methodallows fractionation of up to 0.05 pI units, thus enablingadditional separation of overlapping proteins. The use ofESI-TOF-MS has shown that some of the protein bandsmay consist of more than one protein so that an additionalstage of fractionation or a narrower pI range could beused to achieve further isolation of proteins if required.

An advantage of the CF method is that using analyticalcolumns it can provide a substantial amount of materialfor each protein band where between 3–5 mg of samplecan be loaded on the column. This may result in as muchas 1 �g of protein on average in each band so that signif-icant amounts of protein may be spotted in each arrayspot. This will be important in the sensitivity for detection,especially for proteins expressed at lower levels wheresensitivity will be at a premium. Also, the high reproduci-bility of the method allows for additional experimentsto be run where the same protein can be recollected forfurther analysis of the spot by MS and a variety of tradi-tional methods. Alternatively, the CF experiment could berun using preparative columns, however, this is at theexpense of resolution, where we have found that it is pre-ferable to run the analytical experiment several times tocollect additional material.

In addition, the use of 2-D liquid phase separation of pro-teins has several other distinct advantages for thesemicroarray experiments. The CF fractionation can be runin 1 h while each of the 15 fractions can each be sepa-rated by NPS RP-HPLC in around 20 min. The entire frac-tionation can be performed in �6 h. In addition, the entireprocess can be automated so that large numbers ofdifferent cell lines can be fractionated for spotting on anarray. In addition, the CF fractionation provides proteinsas expressed by the cells. Analysis of these proteins byMS [38], shows that many of the proteins do not haveMr values that correspond to that of the database andare modified. These modifications as expressed by thecell are often important in the cell cycle and the onsetof cancer [36, 37]. Modified isoforms of protein havebeen shown to have a very specific humoral response topatient plasma using 2-D gel-based studies [40].

We would like to thank Dr. Maureen Kachman and Dr. PhilAndrews for use of the MALDI-QTOF-MS/MS for proteinidentification. This work was supported in part by grantsRO1 GM49500 (DML), from the National Institutes of

Proteomics 2003, 3, 1228–1235 Protein microarrays 1235

Health and by Eprogen, Inc, American Cancer SeriesGrant RSG-02-179-01-M60 (A. M. C.), NCI P50 CA69568(A. M. C.) and DOD PC020558 (A. S.). A. M. C. is a fellow ofthe PEW scholar program.

Received December 20, 2002

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