Hindawi Publishing CorporationDisease MarkersVolume 35 (2013) Issue 6 Pages 847ndash855httpdxdoiorg1011552013923819

Research ArticleEvaluation of Molecular Species of Prostate-Specific AntigenComplexed with Immunoglobulin M in Prostate Cancer andBenign Prostatic Hyperplasia

Sanja GoI and Miroslava JankoviT

Institute for the Application of Nuclear Energy INEP University of Belgrade Banatska 31b 11080 Zemun Serbia

Correspondence should be addressed to Miroslava Jankovic jmiroslavsbbrs

Received 21 August 2013 Revised 4 October 2013 Accepted 5 October 2013

Academic Editor Ferdinando Mannello

Copyright copy 2013 S Goc and M Jankovic This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This study was aimed at defining molecular species of prostate-specific antigen (PSA) in immune complexes with immunoglobulinM (IgM) Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens there is the possibility that it canselectively recognize knownPSAglycoisoforms PSA-IgMcomplexes and free PSA fractionswere separated from the sera of subjectswith prostate cancer (PCa) and benign prostatic hyperplasia (BPH) by gel filtration and subjected to on-chip immunoaffinity andion-exchange chromatography PSA-immunoreactive species were detected using surface-enhanced laser desorptionionizationtime of flight mass spectrometry The obtained spectra were analyzed for protein and glycan composition The general patternof the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar It comprised major peaks at17 kDa and minor peaks at 28 kDa corresponding to the entire mature glycosylated PSA The main difference was the presenceof incompletely glycosylated 268 kDa species having putative paucimannosidic structures observed in PCa PSA-IgM but not inBPH PSA-IgM Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework forimmune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker

1 Introduction

Efforts in the field of biomarker research have pointed to theexistence of circulating immune complexes as a novel class oftumor markers with diagnostic potential comparable with orgreater than that of the corresponding free biomarker Theyinclude biomarker-immunoglobulin M (IgM) complexeswhich have been found in several neoplastic diseases such ascolorectal liver and prostate cancer [1ndash3] Although they aresupposed to be a valuable adjunct in differential diagnosticsthe biological meaning of this kind of complexes has not beenelucidated that is it is not known whether they have anyphysiological role In addition from the biochemical pointof view there is no insight into their composition in terms ofthe structural properties of the molecules recognized by IgM

Prostate-specific antigen (PSA) a well-known tumormarker for prostate cancer (PCa) has been found com-plexed with IgM in both benign prostatic hyperplasia (BPH)and cancer [3] In general PSA comprises heterogeneous

molecules differing in primary structure and in glycan com-position Structural variability exists among PSA forms inserum seminal plasma and hyperplastic or cancerous tissues[4ndash6] Thus using different experimental approaches morethan 30 immunoreactive glycoisoforms ranging in molecularmass from 6 to 35 kD have been separated from serumseminal plasma or prostate tissue [7ndash12] Investigation of thestructural heterogeneity of PSA revealed that some distinctforms are more frequently associated with PCa than withBPH [13ndash15] For example some isoforms of precursor or pro-PSA are cancer specific whereas other internally cleaved ornicked (multichain) PSA isoforms are characteristic for BPH[16 17]

This study was aimed at complementing existing data onPSA-IgM by defining molecular species of PSA in circulatingimmune complexes Having in mind the oligoreactivity andmultivalency of IgM and its preference for carbohydrate anti-gens there is the possibility that it can selectively recognize

848 Disease Markers

known PSA glycoisoforms [18 19] Beyond the clinical rele-vance for cancer detection this might be of basic importancesince tumor-associated antigens are known to be targets ofimmune surveillance [20 21]

Circulating PSA-IgM complexes separated by gel filtra-tion from subjects with BPH and PCa were subjected to on-chip immunoaffinity profiling using monoclonal anti-PSAantibodiesThedetectedmolecular specieswere subsequentlyanalyzed for protein and glycan composition and comparedto established PSA forms

2 Materials and Methods

21 Chemicals and Reagents Monoclonal anti-free PSA anti-body clone 8A6 (recognizing epitope I) was purchased fromHy Test (Turku Finland) Monoclonal anti-PSA antibodyclone 8311 (recognizing free and complexed PSA forms) wasfrom Medix Biochemica (Kauniainen Finland) SephacrylS-300 was obtained from Pharmacia Biotech (UppsalaSweden) Alkaline phosphatase-conjugated anti-human IgMand p-nitrophenyl phosphate (PNPP) were from InstitutVirionSerion GmbH (Wurzburg Germany) Broad rangeSDS-PAGE molecular mass standards and Silver Stain Kitwere purchased from Bio-Rad (Hercules CA USA) Pro-teinChip PS20 (preactivated surface) Q10 (strong anionexchanger) CM10 (weak cation exchanger) sinapinic acidand ProteinChip all-in-one protein standards II were fromBio-Rad (Hercules CA USA) Microwell plates were fromNUNC (Roskilde Denmark) and star-bottom tubes werefrom Spektar (Cacak Serbia) IgM concentration was deter-mined using an IgMRID kit (INEP Serbia) Total PSA (tPSA)and free PSA (fPSA) were quantitated using the appropriatecommercially available IRMA PSA assays (INEP Serbia)according to the manufacturerrsquos instructions

All other chemicals were reagent grade

22 Serum Samples Serum samples from subjects withbenign prostatic hyperplasia (BPH) and prostate cancer(PCa) seen at INEP Zemun for PSA determination as apart of followup were obtained according to local ethicalstandards (document GSP05 and PR03009 approvedby the Institutional Committee of INEP) Diagnoses wereconfirmed using clinically established protocols based onPSA level physical examination ultrasound and biopsySamples of PCa sera were from patients diagnosed withlocally advanced and advanced cancers

Individual serum samples from 18 patients with PCa andBPH were randomly used (to reflect intrinsic heterogeneity)to form three pools (each 119899 = 6) PSA concentration ranges ofthe PCa pools were 156ndash776 120583gL 354ndash9872 120583gL and 207ndash30633 120583gL PSA concentration ranges of the BPHpools were08ndash51120583gL 04ndash25120583gL and 03ndash51 120583gL

23 Separation of PSA-IgM Complexes by Gel FiltrationPSA-IgM complexes were separated as described previouslywith slight modifications [3] Serum samples (400 120583L) wereapplied to a Sephacryl S-300 column (13 times 25 cm) equili-brated with 005MPBS pH 72 and eluted with the same

buffer and 1mL fractionswere collected PSA-IgMorPSAwasdetected in the expected fractions using solid phase assayswith immobilizedmonoclonal anti-PSA antibody Antibody-coated tubes (12 120583g per tube) were prepared by physicaladsorption overnight at 4∘C After washing with 005MPBSpH 72 tubes were blocked with 05 casein for 3 h at 37∘Cand rinsed again

(a) Gel filtration fractions (fractions 1ndash20) were added(100 120583L) to coated tubes and incubated for 4 h at roomtemperature (RT) After washing thrice with 005MPBSpH 72 alkaline phosphatase-conjugated anti-human IgM(100 120583L) was added and incubated for 2 h at RT

Unbound conjugate was washed and 100120583L of pNPPsubstrate solution was added The reaction was quenchedwith 100120583L 1MNaOH after 30min The absorption wasmeasured at 405 nm using a Wallac 1420 multilabel counter(Perkin Elmer Monza Italy) Fractions containing PSA-IgMwere concentrated and used for further analysis

In parallel the total IgM was detected in void volume gelfiltration fractions after direct immobilization on polystyrenemicrowell plates using alkaline phosphatase-conjugated anti-human IgM as described above

(b) PSA was probed by the addition of correspondinggel filtration fractions (100 120583L of fractions 1ndash30) to anti-PSA coated tubes following simultaneous incubation withiodinated anti-PSA antibody (300000 cpmtube) overnightat RT After washing steps bound radioactivity was detectedon aWallac Wizard 1470 automatic 120574-counter (Perkin ElmerWaltham USA) fPSA peak fractions were concentrated andused for further analysis

24 Immunoelectrophoresis Samples of IgM or PSA-IgMpreparations (10 120583L) were subjected to immunoelectrophore-sis on 1 agar gel for 15 h at 4∘C Purified sheep anti-humanIgM (INEP Zemun Serbia) was used for detection

25 Profiling of PSA-IgMComplexes by on-Chip Immunoaffin-ity Chromatography PSA fractions (PSA-IgM or fPSA) sep-arated by gel filtration were profiled using on-chip immobi-lized monoclonal anti-PSA antibodies as described [12]

The corresponding antibody (5120583L) was applied to eachspot of the preactivated surface protein chip array andincubated in a humid chamber overnight at 4∘C The spotswere washed thrice with 5120583L of 005MPBS pH 72 for1min at RT and then blocked with 005MTris-HCl pH 80buffer for 1 h in a humid chamber at RT After washingthree times using the same procedure 5 120583L of examinedsample was added to each spot and incubated for 2 h in ahumid chamber at RT The spots were rinsed again thricewith 005MPBS pH 72 and twice with deionized water Allprocedures included shaking (150 rpm) After drying 1 120583L of50 sinapinic acid in acetonitriledH

2Otrifluoroacetic acid

(5049901) was added to each spot dried and thenreapplied Nonspecific binding was monitored using IgM(Figure 1 fraction 10) as control sample

Protein chips were analyzed by surface-enhanced laserdesorptionionization time of flight mass spectrometry(SELDI-TOFMS) using the ProteinChipReader Series 4000

Disease Markers 849

05000100001500020000250003000035000400004500050000

0

05

1

15

2

(a)

(b)

(c)

(d)

25

7 9 11 13 15 17 19 21 23 25 27

(cpm

)

Fraction (1mL)

Abso

rban

ce at

405

nm

Figure 1 Representative PSA-immunogram of pooled humanprostate cancer sera Sephacryl S-300 gel filtration of pooled humanPCa sera The elution was monitored by measuring IgM-reactivity(---) and PSA-reactivity (mdash) in selected fractions Fraction volumewas 1mL cpm counts per minute (a) IgM (b) PSA-IgM (c) PSA-ACT (alpha-1-antichymotrypsin) (d) fPSA free PSA

Personal Edition (Bio-Rad) All spectra were acquired in25 kV positive ion acquisition mode in mass range 25ndash300 kDa and with 8815 laser shots per spot The laser energywas 6000 nJ ProteinChip all-in-one protein standards IIwere used for calibration All spectra were analyzed usingCiphergen Express Software 30 (Bio-Rad Hercules CAUSA)

26 Profiling of Free PSA Forms by on-Chip Ion-ExchangeChromatography The spots with incorporated quaternizedammonium groups (Q10 strong anion exchanger) or car-boxymethyl groups (CM 10 weak cation exchanger) wereprewetted twice with 5120583L of 005MTris-HCl pH 8 for5min at RT After removing the prewetting buffer 5120583L ofthe examined sample (BPH fPSA or PCa fPSA) was addedto each spot and incubated for 15 h in a humid chamberat RT The spots were rinsed thrice with 005MTris-HClpH 8 and twice with deionized water All steps includedshaking (300 rpm) After drying 1120583L of 50 sinapinic acidin acetonitriledH

2Otrifluoroacetic acid (5049901)

was added to each spot dried and then reapplied Proteinchips were analyzed as described above

3 Results

31 Separation of PSA-IgM Complexes by Gel Filtration Arepresentative PSA-immunogram revealing typical differ-ential elution positions of the main serum fractions isshown in Figure 1 PSA-IgM (b) is eluted at the trailingedge of the total IgM peak (a) As deduced from its elutionposition and immunoelectrophoretic appearance (data notshown) PSA-immunoreactivity of IgM complexes seemedto be partly associated with low molecular mass IgM Gelfiltration profiles of several individual runs of pooled PCa andBPH sera gave no striking difference in PSA-IgM peaks butPSA-ACT (c) and fPSA (d) peaks were considerably lower inBPH as expected

32 On-Chip Immunoaffinity Chromatography of PSA-IgMComplexes PSA-IgM preparations from pooled PCa andBPH sera were further subjected to on-chip immunoaffinitychromatography to determine PSA forms in the immunecomplexes fPSA fractions were analyzed in parallel as areference

Typical mass spectra of PSA-IgM preparations from BPHand PCa sera profiled using anti-fPSA antibody are shownin Figure 2 and Table 1 The general pattern was similarcomprising dominant peaks at 17 kDa and 28 kDa The maindifference was the presence of a 2688927Da species inPCa PSA-IgM absent from BPH PSA-IgM This appeared asdoubly charged ion which might be related to its structuralproperties In comparison to the species at 268 kDa theobservation of minor peaks at 158 kDa or at 160 kDa whichhave close molecular masses was not considered as relevantdifference In addition to anti-fPSA antibody anti-PSA anti-body recognizing both free and complexed forms was alsoused for on-chip chromatography Comparable spectra wereobtained but the intensity and frequency of particular peaksdiffered slightly However this did not additionally contributeto separation and individual characterization of PSA (datanot shown)

PSA-immunoreactive forms complexed with IgM inboth BPH and PCa overlapped distinct molecular speciesobserved in mass spectra of fPSA fraction (Figure 3) Clusterat 28 kDa was common for both PCa PSA-IgM (Figure 2(a))and PCa fPSA (Figure 3(a)) whereas the 158 kDa species wasmore abundant in PCa fPSA compared to PCa PSA-IgM

As for BPH fPSA fraction displayed a more restrictedpattern with the main forms at 2759581Da and 2824495Da(Figure 3(b)) in contrast to BPH PSA-IgM (Figure 2(b))

Taking the mass spectra of PSA-IgM preparationsobtained from different pools of sera together subtle differ-ences (some peaks were occasionally present) were noticeablebut they all revealed PSA separation into several peaks thatis they suggested the presence of different PSA glycoisoformsin immune complexes with IgM

33 Assessment of Glycosylation of PSA Molecular SpeciesComplexed with IgM Taking into account the theoreticalmass of PSA peptides and their carbohydrate moiety theobserved PSA-IgM species were matched to amino acidsequence and glycan composition of established PSA iso-forms (Table 2) The deduced glycan masses of dominantpeaks at 17 kDa in BPH PSA-IgM and PCa PSA-IgM rangedbetween 1121 and 1259Da They could be attributed tooligomannosidic structures (Man4 or Man5)

The distinct PCa PSA species at 2688927 is supposed tocontain trimannosyl corepaucimannosidic structures (MM)based on the deduced glycans mass of 80016Da

PSA species at 28 kDa are assessed to contain fucosylatedor nonfucosylated monosialylated biantennary chain withterminal N-acetylglucosamine (GnNaFNaGnF) or galactose(NaAANa ANaFNaAF) In addition the modificationthrough bisecting glucosaminylation (NaGnFbiGnNaFbi) isalso suggested

850 Disease Markers

2688927 + 2H1465895 + 1H

1602695 + 1H1713681 + 1H

1725485 + 1H1738281 + 1H

2083608 + 1H 2406708 + 1H 2585393 + 1H

2807048 + 1H2823786 + 1H

2890806 + 1H

15000 20000 25000 30000

Inte

nsity

075

05

025

0

mz

1288962 + 1H1259044 + 1H

(a)

1288914 + 1H

1464354 + 1H1584514 + 1H

1712676 + 1H

1724457 + 1H1737732 + 1H

2082911 + 1H 2408237 + 1H 2597036 + 1H

2804952 + 1H2824103 + 1H

2890673 + 1H

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

1263959 + 1H

1289488 + 1H

2081214 + 1H

1464775 + 1H

2416384 + 1H 2588536 + 1H

15000 20000 25000 30000

0

01

02

Inte

nsity

mz

(c)

Figure 2 Representative spectra of PSA-immunoreactive species complexed with IgM On-chip chromatography was performed using anti-fPSA monoclonal antibody array Corresponding samples were incubated for 2 h in a humid chamber at RT The spots were rinsed andafter drying 50 sinapinic acid was added to each spot dried and then reapplied The spectra were obtained by SELDI-TOFMS The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2Hdouble charged ion (a) PCa PSA-IgM (PSA-IgM from PCa sera) (b) BPH PSA-IgM (PSA-IgM from BPH sera) (c) IgM control samplePeaks found on the control chip were considered as nonspecific The region below 12 kDa is not shown since it was obscured by non-specificbinding Highlighted peak at 2688927Da was detected only in PCa samples

289 kDa peaks in both BPH and PCa have estimated gly-canmass of 2817DaThis is higher thanmass of complete PSAglycan (2352Da) and it might be assigned to triantennaryoligosaccharide chains

Due to the limited amounts of particular PSA speciesfor direct analysis the predictions were validated using ion-exchange chromatography of uncomplexed PSA as referenceTaking into account known data on isoelectric point ofPSA glycoisoforms (pI 69ndash77) corresponding samples wereseparated on anion and cation exchangers at pH 80 Theresults obtained indicated that incompletely glycosylated PSA(17 kDa) showed weak binding to both ionic exchangers incontrast to sialylated forms (28 kDa) which bound stronglyto anionic exchanger (Figure 4) Thus the observed chargeheterogeneity of PSA molecular species was in agreementwith their predicted various degrees of glycosylation

4 Discussion

According to the observed molecular masses PSA has beenclassified into two more or less heterogeneous groups ofglycosylated (gp28 gp22 gp18 and gp12) or nonglycosylated(p26-full length nonglycosylated PSA p20 p16 p10 and p6)peptides [8] Lower molecular mass PSA results from proteincleavage at several different sites among which major formscomprise those cuts at either Lys182Ser183 or Lys145Ser146or at both residues [4 16]

Our results indicate that the major molecular speciesof PCa PSA and BPH PSA found complexed with IgMcorresponded to gp18 which is glycosylated PSA amino acidresidues 1ndash145 In additionminor forms corresponding to theentire mature glycosylated PSA species that is gp28 werealso observed

Disease Markers 851

Table 1 Summary of PSA-immunoreactive species complexed with IgM in PCa and BPH serum pools

Sample PSA119898119911 Mean (SD) PeakP I P II P III 119898119911 frequency ()

PCa

1602995 1602178 1615058 1606744 (7212) 1001713681 1714265 1713973 (413) 661725485 1721410 1726133 1724343 (2560) 1001738281 1738147 1737829 1738086 (232) 1002688927 2689418 2689172 (347) 662807048 2805844 2807826 2806906 (999) 1002823786 2821013 2828217 2824339 (634) 1002890806 2887531 2897221 2891853 (4929) 100

BPH

1584514 1605460 33 (33)1712676 1714241 1713695 1713537 (794) 1001724457 1726152 1721650 1724076 (2274) 1001737732 1739620 1738440 1738597 (954) 1002804952 2809572 2807406 2807310 (2311) 1002824103 2828975 2822728 2825269 (3283) 1002890673 2892609 2892316 2891866 (1044) 100

SD standard deviation119898119911 mass to charge ratio and P pool (119899 = 6) Frequency of peaks observed when analyzed individually in each pool (IndashIII)

2587218 + 2H1359620 + 1H

2817639 + 2H1517559 + 1H

1584630 + 1H

1604330 + 1H

1726559 + 1H 1904198 + 1H 2080716 + 1H 2364265 + 1H 2587218 + 1H

2803287 + 1H

2817639 + 1H

2890200 + 1H

15000 20000 25000 30000

0

02

04

Inte

nsity

mz

(a)

2581132 + 2H1355180 + 1H

2759581 + 2H

2824495 + 2H

1641264 + 1H1726191 + 1H

1741661 + 1H

1785821 + 1H 2005999 + 1H

2105973 + 1H2312378 + 1H 2581105 + 1H

2804820 + 1H

2824495 + 1H

2889975 + 1H

15000 20000 25000 300000

05

10

15

Inte

nsity

mz

(b)

Figure 3 Representative spectra of free PSA-immunoreactive species On-chip chromatography of free PSA was performed using anti-fPSAmonoclonal antibody array The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis shows signalintensity of the ions M+ 2H double charged form (a) PCa fPSA (free PSA from PCa sera) (b) BPH fPSA (free PSA from BPH sera)

852 Disease Markers

Table 2 Assessment of glycosylation of PSA molecular species complexed with IgM

PSA amino acidsequence theoreticalmass1

PSA group2PSA PSA glycan (G) Glycan (G)

Observed mass Calculated mass3 Theoreticalmass Putative composition4 Structure

PCa BPH PCa BPH

1ndash145 (1612350) + G gp18

1713681 1712676 101331 100326105496 Man4

1725485 1724457 113135 112107

1738281 1737732 125931 125382 121711 Man5

1ndash237 (2608911) + G gp28

2688927 8001673069

89282 MM

2807048 2804952 198137 196041 189900 GnNaFNaGnF

193370 NaAANa

2823786 2824103 214875 215192 206100 ANaFNaAF

212080 NaGnFbiGnNaFbi

2890806 2890673 281895 281762 286260 Na[NaNa][NaNa]Na

1Theoretical average masses of PSA amino acid sequences were obtained using PeptideMass software (httpwebexpasyorgpeptide masspeptide-mass-dochtml)2According to Isono et al 2002 [8]3PSA glycan mass was calculated as the difference between observed PSA mass and theoretical mass of corresponding PSA protein sequence4Putative glycan compositions were proposed based on established complete PSA glycan chain (NaNaF 235216Da) It is depicted using the nomenclature ofthe Consortium for Functional Glycomics+G glycosylationThe blue square Gn (N-acetylglucosamine) the green circle M or Man (mannose) the yellow circle A (galactose) the red triangle F (fucose) the purplediamond Na (sialic acid) bi (bisected)

PSA is defined as a 28430Da glycoprotein containing 8of carbohydrates mainly as a biantennary N-linked oligosac-charide of N-acetyllactosamine type with sialic acid group atthe end of each of the two branches [22] However availabledata indicate that there is pronounced heterogeneity underboth normal and pathological conditions PSA isoformsdiffer generally in sialic acid content as well as outer chainfeatures [23ndash25] Besides biantennary chains monoanten-nary sugar chains and peripheral structures composed onlyof N-acetylhexosamine residues also occur

The results obtained in this study indicated that PSAspecies complexed with IgM mostly contained incompleteglycan chain lacking sialic acid Thus the main PSA speciescomplexed with IgM are assessed to have oligomannosidicstructure in both BPH and PCa In addition paucimanno-sidic structure is assigned to PCa PSA-IgM

Alterations in PSA glycosylation were reported in bothBPH and PCa [26ndash30] Specifically PSA forms with reducedsialylation fucosylation changes and increased pI werefound in PCa

As for unusual mannosidic structures found in PSA-IgMthey can be correlatedwith glycans of PSA isoformdesignatedas PSA-A1015840 [24] It represented a minor fraction that is only06 of the total PSA isoforms and comprised four PSAspecies ranging from 268089 to 272958Da

It seems that newly found IgM-bound isoforms couldhave diagnostic potential to distinguish PCa and BPHbut with no advantage in comparison to other establishedparameters (total PSA-IgM or fPSAPSA) Due to intrinsicheterogeneity of total PSA-IgMand complexity of themethodapplied their possible relevance should be confirmed by test-ing different types of samples using different experimentaldesigns suitable for clinical use However the finding ofdistinct IgM-bound PSA forms may be of interest in lightof recent data indicating paucimannose as marker of humancancers [31 32]

In contrast to these poorly glycosylated PSA speciesfound in complex with IgM themain fPSA species containedpredominantly mono- or disialylated biantennary chainsThe fPSA fraction was used as the reference because it issupposed to comprise an abundant species that can be recog-nized by IgM PSA complexed with ACT was not consideredrelevant for this interaction since it predominantly containscovalently bound enzymatically active PSA

PSA glycosylation is very important for its biomarkerpotential and this was investigated in a number of studiesunder diverse physiological and pathological conditions [26]Generally an examination of PSA glycosylation especiallynumerous PSA speciesglycoforms is faced with the problemof small amounts preventing isolation in sufficient quantity

Disease Markers 853

2820019 + 2H

1582721 + 1H 1721234 + 1H 2098894 + 1H 2349855 + 1H 2591809 + 1H

2799956 + 1H

2820091 + 1H2881627 + 1H

2806951 + 2H 1522470 + 1H

1584249 + 1H1604169 + 1H

1724538 + 1H1737394 + 1H

2806951 + 1H

0

02

04

(B)

(A)

15000 20000 25000 30000

Inte

nsity

0

02

04

Inte

nsity

mz

15000 20000 25000 30000mz

(a)

1738933 + 1H1752391 + 1H

2102763 + 1H

2763666 + 1H2792866 + 1H

2822404 + 1H2889975 + 1H

2801211 + 2H 1723755 + 1H 2801211 + 1H

15000 20000 25000 300000

02

04

(B)

(A)

Inte

nsity

mz

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

Figure 4 Representative spectra of free PSA isoforms On-chip ion-exchange chromatography of PCa fPSA (a) and BPH fPSA (b) wasperformed on anion exchanger Q10 (A) and cation exchanger CM10 (B) array at pH 8 The 119909-axis represents the distribution of ions bymass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2H double charged ion Peaks corresponding toPSA-immunoreactive species are considered as relevant

for glycan analysis In addition the limitation is related to theknown influence of both glycan and peptide on PSA behaviorduring electrophoretic and chromatographic separations

Using data on the PSA standard preparation obtained bymass spectrometry assessment of PSAglycosylation by calcu-lation of glycan mass has already been proved relevant [24]In this study this was combinedwith on-chip immunoaffinitychromatography and ion-exchange chromatography whichenables simultaneous sensitive detection of various PSA gly-coforms Thus despite the differences in methods and detec-tion systems used for this analysis and those reported in thereference literature generalizations about the composition ofPSA isoforms based on congruence in patterns of protein andglycanmoieties can bemade Differences between calculatedand theoretical masses extended to 01 kDa which may bedue to common ion adducts observed in mass spectrometryHowever for the PSA species at 289Da the difference wassignificant PSA species having masses higher than 284 kDa

are also reported in seminal plasma and serum but they arenot characterized in detail [6 8 11 12] Their relation to PSAforms containing multiantennary chains or to pro-PSA assuggested by some authors cannot be confirmed or denied

It is known that the structural integrity of PSA sig-nificantly influences its biological activity and ability tointeract with different molecules As the first line of defenseIgM has a special position in the immune system andthe discovery of PSA-IgM complexes opens an interestingpoint beyond its biomarker potential Is the binding ofmodified carbohydrates as in the case of PSA a generalprinciple not only of basic but also of clinical interest forinstance in relation to cancer immunotherapy Differentactive and passive vaccines including carbohydrate-basedvaccines to aberrant glycostructures are under clinical trialsbut have had limited success at proof of principle and efficacystages [33] Characteristic PSA glycoforms bound to IgMas emerged from this study direct further investigations to

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

848 Disease Markers

known PSA glycoisoforms [18 19] Beyond the clinical rele-vance for cancer detection this might be of basic importancesince tumor-associated antigens are known to be targets ofimmune surveillance [20 21]

Circulating PSA-IgM complexes separated by gel filtra-tion from subjects with BPH and PCa were subjected to on-chip immunoaffinity profiling using monoclonal anti-PSAantibodiesThedetectedmolecular specieswere subsequentlyanalyzed for protein and glycan composition and comparedto established PSA forms

2 Materials and Methods

21 Chemicals and Reagents Monoclonal anti-free PSA anti-body clone 8A6 (recognizing epitope I) was purchased fromHy Test (Turku Finland) Monoclonal anti-PSA antibodyclone 8311 (recognizing free and complexed PSA forms) wasfrom Medix Biochemica (Kauniainen Finland) SephacrylS-300 was obtained from Pharmacia Biotech (UppsalaSweden) Alkaline phosphatase-conjugated anti-human IgMand p-nitrophenyl phosphate (PNPP) were from InstitutVirionSerion GmbH (Wurzburg Germany) Broad rangeSDS-PAGE molecular mass standards and Silver Stain Kitwere purchased from Bio-Rad (Hercules CA USA) Pro-teinChip PS20 (preactivated surface) Q10 (strong anionexchanger) CM10 (weak cation exchanger) sinapinic acidand ProteinChip all-in-one protein standards II were fromBio-Rad (Hercules CA USA) Microwell plates were fromNUNC (Roskilde Denmark) and star-bottom tubes werefrom Spektar (Cacak Serbia) IgM concentration was deter-mined using an IgMRID kit (INEP Serbia) Total PSA (tPSA)and free PSA (fPSA) were quantitated using the appropriatecommercially available IRMA PSA assays (INEP Serbia)according to the manufacturerrsquos instructions

All other chemicals were reagent grade

22 Serum Samples Serum samples from subjects withbenign prostatic hyperplasia (BPH) and prostate cancer(PCa) seen at INEP Zemun for PSA determination as apart of followup were obtained according to local ethicalstandards (document GSP05 and PR03009 approvedby the Institutional Committee of INEP) Diagnoses wereconfirmed using clinically established protocols based onPSA level physical examination ultrasound and biopsySamples of PCa sera were from patients diagnosed withlocally advanced and advanced cancers

Individual serum samples from 18 patients with PCa andBPH were randomly used (to reflect intrinsic heterogeneity)to form three pools (each 119899 = 6) PSA concentration ranges ofthe PCa pools were 156ndash776 120583gL 354ndash9872 120583gL and 207ndash30633 120583gL PSA concentration ranges of the BPHpools were08ndash51120583gL 04ndash25120583gL and 03ndash51 120583gL

23 Separation of PSA-IgM Complexes by Gel FiltrationPSA-IgM complexes were separated as described previouslywith slight modifications [3] Serum samples (400 120583L) wereapplied to a Sephacryl S-300 column (13 times 25 cm) equili-brated with 005MPBS pH 72 and eluted with the same

buffer and 1mL fractionswere collected PSA-IgMorPSAwasdetected in the expected fractions using solid phase assayswith immobilizedmonoclonal anti-PSA antibody Antibody-coated tubes (12 120583g per tube) were prepared by physicaladsorption overnight at 4∘C After washing with 005MPBSpH 72 tubes were blocked with 05 casein for 3 h at 37∘Cand rinsed again

(a) Gel filtration fractions (fractions 1ndash20) were added(100 120583L) to coated tubes and incubated for 4 h at roomtemperature (RT) After washing thrice with 005MPBSpH 72 alkaline phosphatase-conjugated anti-human IgM(100 120583L) was added and incubated for 2 h at RT

Unbound conjugate was washed and 100120583L of pNPPsubstrate solution was added The reaction was quenchedwith 100120583L 1MNaOH after 30min The absorption wasmeasured at 405 nm using a Wallac 1420 multilabel counter(Perkin Elmer Monza Italy) Fractions containing PSA-IgMwere concentrated and used for further analysis

In parallel the total IgM was detected in void volume gelfiltration fractions after direct immobilization on polystyrenemicrowell plates using alkaline phosphatase-conjugated anti-human IgM as described above

(b) PSA was probed by the addition of correspondinggel filtration fractions (100 120583L of fractions 1ndash30) to anti-PSA coated tubes following simultaneous incubation withiodinated anti-PSA antibody (300000 cpmtube) overnightat RT After washing steps bound radioactivity was detectedon aWallac Wizard 1470 automatic 120574-counter (Perkin ElmerWaltham USA) fPSA peak fractions were concentrated andused for further analysis

24 Immunoelectrophoresis Samples of IgM or PSA-IgMpreparations (10 120583L) were subjected to immunoelectrophore-sis on 1 agar gel for 15 h at 4∘C Purified sheep anti-humanIgM (INEP Zemun Serbia) was used for detection

25 Profiling of PSA-IgMComplexes by on-Chip Immunoaffin-ity Chromatography PSA fractions (PSA-IgM or fPSA) sep-arated by gel filtration were profiled using on-chip immobi-lized monoclonal anti-PSA antibodies as described [12]

The corresponding antibody (5120583L) was applied to eachspot of the preactivated surface protein chip array andincubated in a humid chamber overnight at 4∘C The spotswere washed thrice with 5120583L of 005MPBS pH 72 for1min at RT and then blocked with 005MTris-HCl pH 80buffer for 1 h in a humid chamber at RT After washingthree times using the same procedure 5 120583L of examinedsample was added to each spot and incubated for 2 h in ahumid chamber at RT The spots were rinsed again thricewith 005MPBS pH 72 and twice with deionized water Allprocedures included shaking (150 rpm) After drying 1 120583L of50 sinapinic acid in acetonitriledH

2Otrifluoroacetic acid

(5049901) was added to each spot dried and thenreapplied Nonspecific binding was monitored using IgM(Figure 1 fraction 10) as control sample

Protein chips were analyzed by surface-enhanced laserdesorptionionization time of flight mass spectrometry(SELDI-TOFMS) using the ProteinChipReader Series 4000

Disease Markers 849

05000100001500020000250003000035000400004500050000

0

05

1

15

2

(a)

(b)

(c)

(d)

25

7 9 11 13 15 17 19 21 23 25 27

(cpm

)

Fraction (1mL)

Abso

rban

ce at

405

nm

Figure 1 Representative PSA-immunogram of pooled humanprostate cancer sera Sephacryl S-300 gel filtration of pooled humanPCa sera The elution was monitored by measuring IgM-reactivity(---) and PSA-reactivity (mdash) in selected fractions Fraction volumewas 1mL cpm counts per minute (a) IgM (b) PSA-IgM (c) PSA-ACT (alpha-1-antichymotrypsin) (d) fPSA free PSA

Personal Edition (Bio-Rad) All spectra were acquired in25 kV positive ion acquisition mode in mass range 25ndash300 kDa and with 8815 laser shots per spot The laser energywas 6000 nJ ProteinChip all-in-one protein standards IIwere used for calibration All spectra were analyzed usingCiphergen Express Software 30 (Bio-Rad Hercules CAUSA)

26 Profiling of Free PSA Forms by on-Chip Ion-ExchangeChromatography The spots with incorporated quaternizedammonium groups (Q10 strong anion exchanger) or car-boxymethyl groups (CM 10 weak cation exchanger) wereprewetted twice with 5120583L of 005MTris-HCl pH 8 for5min at RT After removing the prewetting buffer 5120583L ofthe examined sample (BPH fPSA or PCa fPSA) was addedto each spot and incubated for 15 h in a humid chamberat RT The spots were rinsed thrice with 005MTris-HClpH 8 and twice with deionized water All steps includedshaking (300 rpm) After drying 1120583L of 50 sinapinic acidin acetonitriledH

2Otrifluoroacetic acid (5049901)

was added to each spot dried and then reapplied Proteinchips were analyzed as described above

3 Results

31 Separation of PSA-IgM Complexes by Gel Filtration Arepresentative PSA-immunogram revealing typical differ-ential elution positions of the main serum fractions isshown in Figure 1 PSA-IgM (b) is eluted at the trailingedge of the total IgM peak (a) As deduced from its elutionposition and immunoelectrophoretic appearance (data notshown) PSA-immunoreactivity of IgM complexes seemedto be partly associated with low molecular mass IgM Gelfiltration profiles of several individual runs of pooled PCa andBPH sera gave no striking difference in PSA-IgM peaks butPSA-ACT (c) and fPSA (d) peaks were considerably lower inBPH as expected

32 On-Chip Immunoaffinity Chromatography of PSA-IgMComplexes PSA-IgM preparations from pooled PCa andBPH sera were further subjected to on-chip immunoaffinitychromatography to determine PSA forms in the immunecomplexes fPSA fractions were analyzed in parallel as areference

Typical mass spectra of PSA-IgM preparations from BPHand PCa sera profiled using anti-fPSA antibody are shownin Figure 2 and Table 1 The general pattern was similarcomprising dominant peaks at 17 kDa and 28 kDa The maindifference was the presence of a 2688927Da species inPCa PSA-IgM absent from BPH PSA-IgM This appeared asdoubly charged ion which might be related to its structuralproperties In comparison to the species at 268 kDa theobservation of minor peaks at 158 kDa or at 160 kDa whichhave close molecular masses was not considered as relevantdifference In addition to anti-fPSA antibody anti-PSA anti-body recognizing both free and complexed forms was alsoused for on-chip chromatography Comparable spectra wereobtained but the intensity and frequency of particular peaksdiffered slightly However this did not additionally contributeto separation and individual characterization of PSA (datanot shown)

PSA-immunoreactive forms complexed with IgM inboth BPH and PCa overlapped distinct molecular speciesobserved in mass spectra of fPSA fraction (Figure 3) Clusterat 28 kDa was common for both PCa PSA-IgM (Figure 2(a))and PCa fPSA (Figure 3(a)) whereas the 158 kDa species wasmore abundant in PCa fPSA compared to PCa PSA-IgM

As for BPH fPSA fraction displayed a more restrictedpattern with the main forms at 2759581Da and 2824495Da(Figure 3(b)) in contrast to BPH PSA-IgM (Figure 2(b))

Taking the mass spectra of PSA-IgM preparationsobtained from different pools of sera together subtle differ-ences (some peaks were occasionally present) were noticeablebut they all revealed PSA separation into several peaks thatis they suggested the presence of different PSA glycoisoformsin immune complexes with IgM

33 Assessment of Glycosylation of PSA Molecular SpeciesComplexed with IgM Taking into account the theoreticalmass of PSA peptides and their carbohydrate moiety theobserved PSA-IgM species were matched to amino acidsequence and glycan composition of established PSA iso-forms (Table 2) The deduced glycan masses of dominantpeaks at 17 kDa in BPH PSA-IgM and PCa PSA-IgM rangedbetween 1121 and 1259Da They could be attributed tooligomannosidic structures (Man4 or Man5)

The distinct PCa PSA species at 2688927 is supposed tocontain trimannosyl corepaucimannosidic structures (MM)based on the deduced glycans mass of 80016Da

PSA species at 28 kDa are assessed to contain fucosylatedor nonfucosylated monosialylated biantennary chain withterminal N-acetylglucosamine (GnNaFNaGnF) or galactose(NaAANa ANaFNaAF) In addition the modificationthrough bisecting glucosaminylation (NaGnFbiGnNaFbi) isalso suggested

850 Disease Markers

2688927 + 2H1465895 + 1H

1602695 + 1H1713681 + 1H

1725485 + 1H1738281 + 1H

2083608 + 1H 2406708 + 1H 2585393 + 1H

2807048 + 1H2823786 + 1H

2890806 + 1H

15000 20000 25000 30000

Inte

nsity

075

05

025

0

mz

1288962 + 1H1259044 + 1H

(a)

1288914 + 1H

1464354 + 1H1584514 + 1H

1712676 + 1H

1724457 + 1H1737732 + 1H

2082911 + 1H 2408237 + 1H 2597036 + 1H

2804952 + 1H2824103 + 1H

2890673 + 1H

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

1263959 + 1H

1289488 + 1H

2081214 + 1H

1464775 + 1H

2416384 + 1H 2588536 + 1H

15000 20000 25000 30000

0

01

02

Inte

nsity

mz

(c)

Figure 2 Representative spectra of PSA-immunoreactive species complexed with IgM On-chip chromatography was performed using anti-fPSA monoclonal antibody array Corresponding samples were incubated for 2 h in a humid chamber at RT The spots were rinsed andafter drying 50 sinapinic acid was added to each spot dried and then reapplied The spectra were obtained by SELDI-TOFMS The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2Hdouble charged ion (a) PCa PSA-IgM (PSA-IgM from PCa sera) (b) BPH PSA-IgM (PSA-IgM from BPH sera) (c) IgM control samplePeaks found on the control chip were considered as nonspecific The region below 12 kDa is not shown since it was obscured by non-specificbinding Highlighted peak at 2688927Da was detected only in PCa samples

289 kDa peaks in both BPH and PCa have estimated gly-canmass of 2817DaThis is higher thanmass of complete PSAglycan (2352Da) and it might be assigned to triantennaryoligosaccharide chains

Due to the limited amounts of particular PSA speciesfor direct analysis the predictions were validated using ion-exchange chromatography of uncomplexed PSA as referenceTaking into account known data on isoelectric point ofPSA glycoisoforms (pI 69ndash77) corresponding samples wereseparated on anion and cation exchangers at pH 80 Theresults obtained indicated that incompletely glycosylated PSA(17 kDa) showed weak binding to both ionic exchangers incontrast to sialylated forms (28 kDa) which bound stronglyto anionic exchanger (Figure 4) Thus the observed chargeheterogeneity of PSA molecular species was in agreementwith their predicted various degrees of glycosylation

4 Discussion

According to the observed molecular masses PSA has beenclassified into two more or less heterogeneous groups ofglycosylated (gp28 gp22 gp18 and gp12) or nonglycosylated(p26-full length nonglycosylated PSA p20 p16 p10 and p6)peptides [8] Lower molecular mass PSA results from proteincleavage at several different sites among which major formscomprise those cuts at either Lys182Ser183 or Lys145Ser146or at both residues [4 16]

Our results indicate that the major molecular speciesof PCa PSA and BPH PSA found complexed with IgMcorresponded to gp18 which is glycosylated PSA amino acidresidues 1ndash145 In additionminor forms corresponding to theentire mature glycosylated PSA species that is gp28 werealso observed

Disease Markers 851

Table 1 Summary of PSA-immunoreactive species complexed with IgM in PCa and BPH serum pools

Sample PSA119898119911 Mean (SD) PeakP I P II P III 119898119911 frequency ()

PCa

1602995 1602178 1615058 1606744 (7212) 1001713681 1714265 1713973 (413) 661725485 1721410 1726133 1724343 (2560) 1001738281 1738147 1737829 1738086 (232) 1002688927 2689418 2689172 (347) 662807048 2805844 2807826 2806906 (999) 1002823786 2821013 2828217 2824339 (634) 1002890806 2887531 2897221 2891853 (4929) 100

BPH

1584514 1605460 33 (33)1712676 1714241 1713695 1713537 (794) 1001724457 1726152 1721650 1724076 (2274) 1001737732 1739620 1738440 1738597 (954) 1002804952 2809572 2807406 2807310 (2311) 1002824103 2828975 2822728 2825269 (3283) 1002890673 2892609 2892316 2891866 (1044) 100

SD standard deviation119898119911 mass to charge ratio and P pool (119899 = 6) Frequency of peaks observed when analyzed individually in each pool (IndashIII)

2587218 + 2H1359620 + 1H

2817639 + 2H1517559 + 1H

1584630 + 1H

1604330 + 1H

1726559 + 1H 1904198 + 1H 2080716 + 1H 2364265 + 1H 2587218 + 1H

2803287 + 1H

2817639 + 1H

2890200 + 1H

15000 20000 25000 30000

0

02

04

Inte

nsity

mz

(a)

2581132 + 2H1355180 + 1H

2759581 + 2H

2824495 + 2H

1641264 + 1H1726191 + 1H

1741661 + 1H

1785821 + 1H 2005999 + 1H

2105973 + 1H2312378 + 1H 2581105 + 1H

2804820 + 1H

2824495 + 1H

2889975 + 1H

15000 20000 25000 300000

05

10

15

Inte

nsity

mz

(b)

Figure 3 Representative spectra of free PSA-immunoreactive species On-chip chromatography of free PSA was performed using anti-fPSAmonoclonal antibody array The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis shows signalintensity of the ions M+ 2H double charged form (a) PCa fPSA (free PSA from PCa sera) (b) BPH fPSA (free PSA from BPH sera)

852 Disease Markers

Table 2 Assessment of glycosylation of PSA molecular species complexed with IgM

PSA amino acidsequence theoreticalmass1

PSA group2PSA PSA glycan (G) Glycan (G)

Observed mass Calculated mass3 Theoreticalmass Putative composition4 Structure

PCa BPH PCa BPH

1ndash145 (1612350) + G gp18

1713681 1712676 101331 100326105496 Man4

1725485 1724457 113135 112107

1738281 1737732 125931 125382 121711 Man5

1ndash237 (2608911) + G gp28

2688927 8001673069

89282 MM

2807048 2804952 198137 196041 189900 GnNaFNaGnF

193370 NaAANa

2823786 2824103 214875 215192 206100 ANaFNaAF

212080 NaGnFbiGnNaFbi

2890806 2890673 281895 281762 286260 Na[NaNa][NaNa]Na

1Theoretical average masses of PSA amino acid sequences were obtained using PeptideMass software (httpwebexpasyorgpeptide masspeptide-mass-dochtml)2According to Isono et al 2002 [8]3PSA glycan mass was calculated as the difference between observed PSA mass and theoretical mass of corresponding PSA protein sequence4Putative glycan compositions were proposed based on established complete PSA glycan chain (NaNaF 235216Da) It is depicted using the nomenclature ofthe Consortium for Functional Glycomics+G glycosylationThe blue square Gn (N-acetylglucosamine) the green circle M or Man (mannose) the yellow circle A (galactose) the red triangle F (fucose) the purplediamond Na (sialic acid) bi (bisected)

PSA is defined as a 28430Da glycoprotein containing 8of carbohydrates mainly as a biantennary N-linked oligosac-charide of N-acetyllactosamine type with sialic acid group atthe end of each of the two branches [22] However availabledata indicate that there is pronounced heterogeneity underboth normal and pathological conditions PSA isoformsdiffer generally in sialic acid content as well as outer chainfeatures [23ndash25] Besides biantennary chains monoanten-nary sugar chains and peripheral structures composed onlyof N-acetylhexosamine residues also occur

The results obtained in this study indicated that PSAspecies complexed with IgM mostly contained incompleteglycan chain lacking sialic acid Thus the main PSA speciescomplexed with IgM are assessed to have oligomannosidicstructure in both BPH and PCa In addition paucimanno-sidic structure is assigned to PCa PSA-IgM

Alterations in PSA glycosylation were reported in bothBPH and PCa [26ndash30] Specifically PSA forms with reducedsialylation fucosylation changes and increased pI werefound in PCa

As for unusual mannosidic structures found in PSA-IgMthey can be correlatedwith glycans of PSA isoformdesignatedas PSA-A1015840 [24] It represented a minor fraction that is only06 of the total PSA isoforms and comprised four PSAspecies ranging from 268089 to 272958Da

It seems that newly found IgM-bound isoforms couldhave diagnostic potential to distinguish PCa and BPHbut with no advantage in comparison to other establishedparameters (total PSA-IgM or fPSAPSA) Due to intrinsicheterogeneity of total PSA-IgMand complexity of themethodapplied their possible relevance should be confirmed by test-ing different types of samples using different experimentaldesigns suitable for clinical use However the finding ofdistinct IgM-bound PSA forms may be of interest in lightof recent data indicating paucimannose as marker of humancancers [31 32]

In contrast to these poorly glycosylated PSA speciesfound in complex with IgM themain fPSA species containedpredominantly mono- or disialylated biantennary chainsThe fPSA fraction was used as the reference because it issupposed to comprise an abundant species that can be recog-nized by IgM PSA complexed with ACT was not consideredrelevant for this interaction since it predominantly containscovalently bound enzymatically active PSA

PSA glycosylation is very important for its biomarkerpotential and this was investigated in a number of studiesunder diverse physiological and pathological conditions [26]Generally an examination of PSA glycosylation especiallynumerous PSA speciesglycoforms is faced with the problemof small amounts preventing isolation in sufficient quantity

Disease Markers 853

2820019 + 2H

1582721 + 1H 1721234 + 1H 2098894 + 1H 2349855 + 1H 2591809 + 1H

2799956 + 1H

2820091 + 1H2881627 + 1H

2806951 + 2H 1522470 + 1H

1584249 + 1H1604169 + 1H

1724538 + 1H1737394 + 1H

2806951 + 1H

0

02

04

(B)

(A)

15000 20000 25000 30000

Inte

nsity

0

02

04

Inte

nsity

mz

15000 20000 25000 30000mz

(a)

1738933 + 1H1752391 + 1H

2102763 + 1H

2763666 + 1H2792866 + 1H

2822404 + 1H2889975 + 1H

2801211 + 2H 1723755 + 1H 2801211 + 1H

15000 20000 25000 300000

02

04

(B)

(A)

Inte

nsity

mz

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

Figure 4 Representative spectra of free PSA isoforms On-chip ion-exchange chromatography of PCa fPSA (a) and BPH fPSA (b) wasperformed on anion exchanger Q10 (A) and cation exchanger CM10 (B) array at pH 8 The 119909-axis represents the distribution of ions bymass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2H double charged ion Peaks corresponding toPSA-immunoreactive species are considered as relevant

for glycan analysis In addition the limitation is related to theknown influence of both glycan and peptide on PSA behaviorduring electrophoretic and chromatographic separations

Using data on the PSA standard preparation obtained bymass spectrometry assessment of PSAglycosylation by calcu-lation of glycan mass has already been proved relevant [24]In this study this was combinedwith on-chip immunoaffinitychromatography and ion-exchange chromatography whichenables simultaneous sensitive detection of various PSA gly-coforms Thus despite the differences in methods and detec-tion systems used for this analysis and those reported in thereference literature generalizations about the composition ofPSA isoforms based on congruence in patterns of protein andglycanmoieties can bemade Differences between calculatedand theoretical masses extended to 01 kDa which may bedue to common ion adducts observed in mass spectrometryHowever for the PSA species at 289Da the difference wassignificant PSA species having masses higher than 284 kDa

are also reported in seminal plasma and serum but they arenot characterized in detail [6 8 11 12] Their relation to PSAforms containing multiantennary chains or to pro-PSA assuggested by some authors cannot be confirmed or denied

It is known that the structural integrity of PSA sig-nificantly influences its biological activity and ability tointeract with different molecules As the first line of defenseIgM has a special position in the immune system andthe discovery of PSA-IgM complexes opens an interestingpoint beyond its biomarker potential Is the binding ofmodified carbohydrates as in the case of PSA a generalprinciple not only of basic but also of clinical interest forinstance in relation to cancer immunotherapy Differentactive and passive vaccines including carbohydrate-basedvaccines to aberrant glycostructures are under clinical trialsbut have had limited success at proof of principle and efficacystages [33] Characteristic PSA glycoforms bound to IgMas emerged from this study direct further investigations to

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 849

05000100001500020000250003000035000400004500050000

0

05

1

15

2

(a)

(b)

(c)

(d)

25

7 9 11 13 15 17 19 21 23 25 27

(cpm

)

Fraction (1mL)

Abso

rban

ce at

405

nm

Figure 1 Representative PSA-immunogram of pooled humanprostate cancer sera Sephacryl S-300 gel filtration of pooled humanPCa sera The elution was monitored by measuring IgM-reactivity(---) and PSA-reactivity (mdash) in selected fractions Fraction volumewas 1mL cpm counts per minute (a) IgM (b) PSA-IgM (c) PSA-ACT (alpha-1-antichymotrypsin) (d) fPSA free PSA

Personal Edition (Bio-Rad) All spectra were acquired in25 kV positive ion acquisition mode in mass range 25ndash300 kDa and with 8815 laser shots per spot The laser energywas 6000 nJ ProteinChip all-in-one protein standards IIwere used for calibration All spectra were analyzed usingCiphergen Express Software 30 (Bio-Rad Hercules CAUSA)

26 Profiling of Free PSA Forms by on-Chip Ion-ExchangeChromatography The spots with incorporated quaternizedammonium groups (Q10 strong anion exchanger) or car-boxymethyl groups (CM 10 weak cation exchanger) wereprewetted twice with 5120583L of 005MTris-HCl pH 8 for5min at RT After removing the prewetting buffer 5120583L ofthe examined sample (BPH fPSA or PCa fPSA) was addedto each spot and incubated for 15 h in a humid chamberat RT The spots were rinsed thrice with 005MTris-HClpH 8 and twice with deionized water All steps includedshaking (300 rpm) After drying 1120583L of 50 sinapinic acidin acetonitriledH

2Otrifluoroacetic acid (5049901)

was added to each spot dried and then reapplied Proteinchips were analyzed as described above

3 Results

31 Separation of PSA-IgM Complexes by Gel Filtration Arepresentative PSA-immunogram revealing typical differ-ential elution positions of the main serum fractions isshown in Figure 1 PSA-IgM (b) is eluted at the trailingedge of the total IgM peak (a) As deduced from its elutionposition and immunoelectrophoretic appearance (data notshown) PSA-immunoreactivity of IgM complexes seemedto be partly associated with low molecular mass IgM Gelfiltration profiles of several individual runs of pooled PCa andBPH sera gave no striking difference in PSA-IgM peaks butPSA-ACT (c) and fPSA (d) peaks were considerably lower inBPH as expected

32 On-Chip Immunoaffinity Chromatography of PSA-IgMComplexes PSA-IgM preparations from pooled PCa andBPH sera were further subjected to on-chip immunoaffinitychromatography to determine PSA forms in the immunecomplexes fPSA fractions were analyzed in parallel as areference

Typical mass spectra of PSA-IgM preparations from BPHand PCa sera profiled using anti-fPSA antibody are shownin Figure 2 and Table 1 The general pattern was similarcomprising dominant peaks at 17 kDa and 28 kDa The maindifference was the presence of a 2688927Da species inPCa PSA-IgM absent from BPH PSA-IgM This appeared asdoubly charged ion which might be related to its structuralproperties In comparison to the species at 268 kDa theobservation of minor peaks at 158 kDa or at 160 kDa whichhave close molecular masses was not considered as relevantdifference In addition to anti-fPSA antibody anti-PSA anti-body recognizing both free and complexed forms was alsoused for on-chip chromatography Comparable spectra wereobtained but the intensity and frequency of particular peaksdiffered slightly However this did not additionally contributeto separation and individual characterization of PSA (datanot shown)

PSA-immunoreactive forms complexed with IgM inboth BPH and PCa overlapped distinct molecular speciesobserved in mass spectra of fPSA fraction (Figure 3) Clusterat 28 kDa was common for both PCa PSA-IgM (Figure 2(a))and PCa fPSA (Figure 3(a)) whereas the 158 kDa species wasmore abundant in PCa fPSA compared to PCa PSA-IgM

As for BPH fPSA fraction displayed a more restrictedpattern with the main forms at 2759581Da and 2824495Da(Figure 3(b)) in contrast to BPH PSA-IgM (Figure 2(b))

Taking the mass spectra of PSA-IgM preparationsobtained from different pools of sera together subtle differ-ences (some peaks were occasionally present) were noticeablebut they all revealed PSA separation into several peaks thatis they suggested the presence of different PSA glycoisoformsin immune complexes with IgM

33 Assessment of Glycosylation of PSA Molecular SpeciesComplexed with IgM Taking into account the theoreticalmass of PSA peptides and their carbohydrate moiety theobserved PSA-IgM species were matched to amino acidsequence and glycan composition of established PSA iso-forms (Table 2) The deduced glycan masses of dominantpeaks at 17 kDa in BPH PSA-IgM and PCa PSA-IgM rangedbetween 1121 and 1259Da They could be attributed tooligomannosidic structures (Man4 or Man5)

The distinct PCa PSA species at 2688927 is supposed tocontain trimannosyl corepaucimannosidic structures (MM)based on the deduced glycans mass of 80016Da

PSA species at 28 kDa are assessed to contain fucosylatedor nonfucosylated monosialylated biantennary chain withterminal N-acetylglucosamine (GnNaFNaGnF) or galactose(NaAANa ANaFNaAF) In addition the modificationthrough bisecting glucosaminylation (NaGnFbiGnNaFbi) isalso suggested

850 Disease Markers

2688927 + 2H1465895 + 1H

1602695 + 1H1713681 + 1H

1725485 + 1H1738281 + 1H

2083608 + 1H 2406708 + 1H 2585393 + 1H

2807048 + 1H2823786 + 1H

2890806 + 1H

15000 20000 25000 30000

Inte

nsity

075

05

025

0

mz

1288962 + 1H1259044 + 1H

(a)

1288914 + 1H

1464354 + 1H1584514 + 1H

1712676 + 1H

1724457 + 1H1737732 + 1H

2082911 + 1H 2408237 + 1H 2597036 + 1H

2804952 + 1H2824103 + 1H

2890673 + 1H

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

1263959 + 1H

1289488 + 1H

2081214 + 1H

1464775 + 1H

2416384 + 1H 2588536 + 1H

15000 20000 25000 30000

0

01

02

Inte

nsity

mz

(c)

Figure 2 Representative spectra of PSA-immunoreactive species complexed with IgM On-chip chromatography was performed using anti-fPSA monoclonal antibody array Corresponding samples were incubated for 2 h in a humid chamber at RT The spots were rinsed andafter drying 50 sinapinic acid was added to each spot dried and then reapplied The spectra were obtained by SELDI-TOFMS The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2Hdouble charged ion (a) PCa PSA-IgM (PSA-IgM from PCa sera) (b) BPH PSA-IgM (PSA-IgM from BPH sera) (c) IgM control samplePeaks found on the control chip were considered as nonspecific The region below 12 kDa is not shown since it was obscured by non-specificbinding Highlighted peak at 2688927Da was detected only in PCa samples

289 kDa peaks in both BPH and PCa have estimated gly-canmass of 2817DaThis is higher thanmass of complete PSAglycan (2352Da) and it might be assigned to triantennaryoligosaccharide chains

Due to the limited amounts of particular PSA speciesfor direct analysis the predictions were validated using ion-exchange chromatography of uncomplexed PSA as referenceTaking into account known data on isoelectric point ofPSA glycoisoforms (pI 69ndash77) corresponding samples wereseparated on anion and cation exchangers at pH 80 Theresults obtained indicated that incompletely glycosylated PSA(17 kDa) showed weak binding to both ionic exchangers incontrast to sialylated forms (28 kDa) which bound stronglyto anionic exchanger (Figure 4) Thus the observed chargeheterogeneity of PSA molecular species was in agreementwith their predicted various degrees of glycosylation

4 Discussion

According to the observed molecular masses PSA has beenclassified into two more or less heterogeneous groups ofglycosylated (gp28 gp22 gp18 and gp12) or nonglycosylated(p26-full length nonglycosylated PSA p20 p16 p10 and p6)peptides [8] Lower molecular mass PSA results from proteincleavage at several different sites among which major formscomprise those cuts at either Lys182Ser183 or Lys145Ser146or at both residues [4 16]

Our results indicate that the major molecular speciesof PCa PSA and BPH PSA found complexed with IgMcorresponded to gp18 which is glycosylated PSA amino acidresidues 1ndash145 In additionminor forms corresponding to theentire mature glycosylated PSA species that is gp28 werealso observed

Disease Markers 851

Table 1 Summary of PSA-immunoreactive species complexed with IgM in PCa and BPH serum pools

Sample PSA119898119911 Mean (SD) PeakP I P II P III 119898119911 frequency ()

PCa

1602995 1602178 1615058 1606744 (7212) 1001713681 1714265 1713973 (413) 661725485 1721410 1726133 1724343 (2560) 1001738281 1738147 1737829 1738086 (232) 1002688927 2689418 2689172 (347) 662807048 2805844 2807826 2806906 (999) 1002823786 2821013 2828217 2824339 (634) 1002890806 2887531 2897221 2891853 (4929) 100

BPH

1584514 1605460 33 (33)1712676 1714241 1713695 1713537 (794) 1001724457 1726152 1721650 1724076 (2274) 1001737732 1739620 1738440 1738597 (954) 1002804952 2809572 2807406 2807310 (2311) 1002824103 2828975 2822728 2825269 (3283) 1002890673 2892609 2892316 2891866 (1044) 100

SD standard deviation119898119911 mass to charge ratio and P pool (119899 = 6) Frequency of peaks observed when analyzed individually in each pool (IndashIII)

2587218 + 2H1359620 + 1H

2817639 + 2H1517559 + 1H

1584630 + 1H

1604330 + 1H

1726559 + 1H 1904198 + 1H 2080716 + 1H 2364265 + 1H 2587218 + 1H

2803287 + 1H

2817639 + 1H

2890200 + 1H

15000 20000 25000 30000

0

02

04

Inte

nsity

mz

(a)

2581132 + 2H1355180 + 1H

2759581 + 2H

2824495 + 2H

1641264 + 1H1726191 + 1H

1741661 + 1H

1785821 + 1H 2005999 + 1H

2105973 + 1H2312378 + 1H 2581105 + 1H

2804820 + 1H

2824495 + 1H

2889975 + 1H

15000 20000 25000 300000

05

10

15

Inte

nsity

mz

(b)

Figure 3 Representative spectra of free PSA-immunoreactive species On-chip chromatography of free PSA was performed using anti-fPSAmonoclonal antibody array The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis shows signalintensity of the ions M+ 2H double charged form (a) PCa fPSA (free PSA from PCa sera) (b) BPH fPSA (free PSA from BPH sera)

852 Disease Markers

Table 2 Assessment of glycosylation of PSA molecular species complexed with IgM

PSA amino acidsequence theoreticalmass1

PSA group2PSA PSA glycan (G) Glycan (G)

Observed mass Calculated mass3 Theoreticalmass Putative composition4 Structure

PCa BPH PCa BPH

1ndash145 (1612350) + G gp18

1713681 1712676 101331 100326105496 Man4

1725485 1724457 113135 112107

1738281 1737732 125931 125382 121711 Man5

1ndash237 (2608911) + G gp28

2688927 8001673069

89282 MM

2807048 2804952 198137 196041 189900 GnNaFNaGnF

193370 NaAANa

2823786 2824103 214875 215192 206100 ANaFNaAF

212080 NaGnFbiGnNaFbi

2890806 2890673 281895 281762 286260 Na[NaNa][NaNa]Na

1Theoretical average masses of PSA amino acid sequences were obtained using PeptideMass software (httpwebexpasyorgpeptide masspeptide-mass-dochtml)2According to Isono et al 2002 [8]3PSA glycan mass was calculated as the difference between observed PSA mass and theoretical mass of corresponding PSA protein sequence4Putative glycan compositions were proposed based on established complete PSA glycan chain (NaNaF 235216Da) It is depicted using the nomenclature ofthe Consortium for Functional Glycomics+G glycosylationThe blue square Gn (N-acetylglucosamine) the green circle M or Man (mannose) the yellow circle A (galactose) the red triangle F (fucose) the purplediamond Na (sialic acid) bi (bisected)

PSA is defined as a 28430Da glycoprotein containing 8of carbohydrates mainly as a biantennary N-linked oligosac-charide of N-acetyllactosamine type with sialic acid group atthe end of each of the two branches [22] However availabledata indicate that there is pronounced heterogeneity underboth normal and pathological conditions PSA isoformsdiffer generally in sialic acid content as well as outer chainfeatures [23ndash25] Besides biantennary chains monoanten-nary sugar chains and peripheral structures composed onlyof N-acetylhexosamine residues also occur

The results obtained in this study indicated that PSAspecies complexed with IgM mostly contained incompleteglycan chain lacking sialic acid Thus the main PSA speciescomplexed with IgM are assessed to have oligomannosidicstructure in both BPH and PCa In addition paucimanno-sidic structure is assigned to PCa PSA-IgM

Alterations in PSA glycosylation were reported in bothBPH and PCa [26ndash30] Specifically PSA forms with reducedsialylation fucosylation changes and increased pI werefound in PCa

As for unusual mannosidic structures found in PSA-IgMthey can be correlatedwith glycans of PSA isoformdesignatedas PSA-A1015840 [24] It represented a minor fraction that is only06 of the total PSA isoforms and comprised four PSAspecies ranging from 268089 to 272958Da

It seems that newly found IgM-bound isoforms couldhave diagnostic potential to distinguish PCa and BPHbut with no advantage in comparison to other establishedparameters (total PSA-IgM or fPSAPSA) Due to intrinsicheterogeneity of total PSA-IgMand complexity of themethodapplied their possible relevance should be confirmed by test-ing different types of samples using different experimentaldesigns suitable for clinical use However the finding ofdistinct IgM-bound PSA forms may be of interest in lightof recent data indicating paucimannose as marker of humancancers [31 32]

In contrast to these poorly glycosylated PSA speciesfound in complex with IgM themain fPSA species containedpredominantly mono- or disialylated biantennary chainsThe fPSA fraction was used as the reference because it issupposed to comprise an abundant species that can be recog-nized by IgM PSA complexed with ACT was not consideredrelevant for this interaction since it predominantly containscovalently bound enzymatically active PSA

PSA glycosylation is very important for its biomarkerpotential and this was investigated in a number of studiesunder diverse physiological and pathological conditions [26]Generally an examination of PSA glycosylation especiallynumerous PSA speciesglycoforms is faced with the problemof small amounts preventing isolation in sufficient quantity

Disease Markers 853

2820019 + 2H

1582721 + 1H 1721234 + 1H 2098894 + 1H 2349855 + 1H 2591809 + 1H

2799956 + 1H

2820091 + 1H2881627 + 1H

2806951 + 2H 1522470 + 1H

1584249 + 1H1604169 + 1H

1724538 + 1H1737394 + 1H

2806951 + 1H

0

02

04

(B)

(A)

15000 20000 25000 30000

Inte

nsity

0

02

04

Inte

nsity

mz

15000 20000 25000 30000mz

(a)

1738933 + 1H1752391 + 1H

2102763 + 1H

2763666 + 1H2792866 + 1H

2822404 + 1H2889975 + 1H

2801211 + 2H 1723755 + 1H 2801211 + 1H

15000 20000 25000 300000

02

04

(B)

(A)

Inte

nsity

mz

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

Figure 4 Representative spectra of free PSA isoforms On-chip ion-exchange chromatography of PCa fPSA (a) and BPH fPSA (b) wasperformed on anion exchanger Q10 (A) and cation exchanger CM10 (B) array at pH 8 The 119909-axis represents the distribution of ions bymass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2H double charged ion Peaks corresponding toPSA-immunoreactive species are considered as relevant

for glycan analysis In addition the limitation is related to theknown influence of both glycan and peptide on PSA behaviorduring electrophoretic and chromatographic separations

Using data on the PSA standard preparation obtained bymass spectrometry assessment of PSAglycosylation by calcu-lation of glycan mass has already been proved relevant [24]In this study this was combinedwith on-chip immunoaffinitychromatography and ion-exchange chromatography whichenables simultaneous sensitive detection of various PSA gly-coforms Thus despite the differences in methods and detec-tion systems used for this analysis and those reported in thereference literature generalizations about the composition ofPSA isoforms based on congruence in patterns of protein andglycanmoieties can bemade Differences between calculatedand theoretical masses extended to 01 kDa which may bedue to common ion adducts observed in mass spectrometryHowever for the PSA species at 289Da the difference wassignificant PSA species having masses higher than 284 kDa

are also reported in seminal plasma and serum but they arenot characterized in detail [6 8 11 12] Their relation to PSAforms containing multiantennary chains or to pro-PSA assuggested by some authors cannot be confirmed or denied

It is known that the structural integrity of PSA sig-nificantly influences its biological activity and ability tointeract with different molecules As the first line of defenseIgM has a special position in the immune system andthe discovery of PSA-IgM complexes opens an interestingpoint beyond its biomarker potential Is the binding ofmodified carbohydrates as in the case of PSA a generalprinciple not only of basic but also of clinical interest forinstance in relation to cancer immunotherapy Differentactive and passive vaccines including carbohydrate-basedvaccines to aberrant glycostructures are under clinical trialsbut have had limited success at proof of principle and efficacystages [33] Characteristic PSA glycoforms bound to IgMas emerged from this study direct further investigations to

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

850 Disease Markers

2688927 + 2H1465895 + 1H

1602695 + 1H1713681 + 1H

1725485 + 1H1738281 + 1H

2083608 + 1H 2406708 + 1H 2585393 + 1H

2807048 + 1H2823786 + 1H

2890806 + 1H

15000 20000 25000 30000

Inte

nsity

075

05

025

0

mz

1288962 + 1H1259044 + 1H

(a)

1288914 + 1H

1464354 + 1H1584514 + 1H

1712676 + 1H

1724457 + 1H1737732 + 1H

2082911 + 1H 2408237 + 1H 2597036 + 1H

2804952 + 1H2824103 + 1H

2890673 + 1H

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

1263959 + 1H

1289488 + 1H

2081214 + 1H

1464775 + 1H

2416384 + 1H 2588536 + 1H

15000 20000 25000 30000

0

01

02

Inte

nsity

mz

(c)

Figure 2 Representative spectra of PSA-immunoreactive species complexed with IgM On-chip chromatography was performed using anti-fPSA monoclonal antibody array Corresponding samples were incubated for 2 h in a humid chamber at RT The spots were rinsed andafter drying 50 sinapinic acid was added to each spot dried and then reapplied The spectra were obtained by SELDI-TOFMS The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2Hdouble charged ion (a) PCa PSA-IgM (PSA-IgM from PCa sera) (b) BPH PSA-IgM (PSA-IgM from BPH sera) (c) IgM control samplePeaks found on the control chip were considered as nonspecific The region below 12 kDa is not shown since it was obscured by non-specificbinding Highlighted peak at 2688927Da was detected only in PCa samples

289 kDa peaks in both BPH and PCa have estimated gly-canmass of 2817DaThis is higher thanmass of complete PSAglycan (2352Da) and it might be assigned to triantennaryoligosaccharide chains

Due to the limited amounts of particular PSA speciesfor direct analysis the predictions were validated using ion-exchange chromatography of uncomplexed PSA as referenceTaking into account known data on isoelectric point ofPSA glycoisoforms (pI 69ndash77) corresponding samples wereseparated on anion and cation exchangers at pH 80 Theresults obtained indicated that incompletely glycosylated PSA(17 kDa) showed weak binding to both ionic exchangers incontrast to sialylated forms (28 kDa) which bound stronglyto anionic exchanger (Figure 4) Thus the observed chargeheterogeneity of PSA molecular species was in agreementwith their predicted various degrees of glycosylation

4 Discussion

According to the observed molecular masses PSA has beenclassified into two more or less heterogeneous groups ofglycosylated (gp28 gp22 gp18 and gp12) or nonglycosylated(p26-full length nonglycosylated PSA p20 p16 p10 and p6)peptides [8] Lower molecular mass PSA results from proteincleavage at several different sites among which major formscomprise those cuts at either Lys182Ser183 or Lys145Ser146or at both residues [4 16]

Our results indicate that the major molecular speciesof PCa PSA and BPH PSA found complexed with IgMcorresponded to gp18 which is glycosylated PSA amino acidresidues 1ndash145 In additionminor forms corresponding to theentire mature glycosylated PSA species that is gp28 werealso observed

Disease Markers 851

Table 1 Summary of PSA-immunoreactive species complexed with IgM in PCa and BPH serum pools

Sample PSA119898119911 Mean (SD) PeakP I P II P III 119898119911 frequency ()

PCa

1602995 1602178 1615058 1606744 (7212) 1001713681 1714265 1713973 (413) 661725485 1721410 1726133 1724343 (2560) 1001738281 1738147 1737829 1738086 (232) 1002688927 2689418 2689172 (347) 662807048 2805844 2807826 2806906 (999) 1002823786 2821013 2828217 2824339 (634) 1002890806 2887531 2897221 2891853 (4929) 100

BPH

1584514 1605460 33 (33)1712676 1714241 1713695 1713537 (794) 1001724457 1726152 1721650 1724076 (2274) 1001737732 1739620 1738440 1738597 (954) 1002804952 2809572 2807406 2807310 (2311) 1002824103 2828975 2822728 2825269 (3283) 1002890673 2892609 2892316 2891866 (1044) 100

SD standard deviation119898119911 mass to charge ratio and P pool (119899 = 6) Frequency of peaks observed when analyzed individually in each pool (IndashIII)

2587218 + 2H1359620 + 1H

2817639 + 2H1517559 + 1H

1584630 + 1H

1604330 + 1H

1726559 + 1H 1904198 + 1H 2080716 + 1H 2364265 + 1H 2587218 + 1H

2803287 + 1H

2817639 + 1H

2890200 + 1H

15000 20000 25000 30000

0

02

04

Inte

nsity

mz

(a)

2581132 + 2H1355180 + 1H

2759581 + 2H

2824495 + 2H

1641264 + 1H1726191 + 1H

1741661 + 1H

1785821 + 1H 2005999 + 1H

2105973 + 1H2312378 + 1H 2581105 + 1H

2804820 + 1H

2824495 + 1H

2889975 + 1H

15000 20000 25000 300000

05

10

15

Inte

nsity

mz

(b)

Figure 3 Representative spectra of free PSA-immunoreactive species On-chip chromatography of free PSA was performed using anti-fPSAmonoclonal antibody array The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis shows signalintensity of the ions M+ 2H double charged form (a) PCa fPSA (free PSA from PCa sera) (b) BPH fPSA (free PSA from BPH sera)

852 Disease Markers

Table 2 Assessment of glycosylation of PSA molecular species complexed with IgM

PSA amino acidsequence theoreticalmass1

PSA group2PSA PSA glycan (G) Glycan (G)

Observed mass Calculated mass3 Theoreticalmass Putative composition4 Structure

PCa BPH PCa BPH

1ndash145 (1612350) + G gp18

1713681 1712676 101331 100326105496 Man4

1725485 1724457 113135 112107

1738281 1737732 125931 125382 121711 Man5

1ndash237 (2608911) + G gp28

2688927 8001673069

89282 MM

2807048 2804952 198137 196041 189900 GnNaFNaGnF

193370 NaAANa

2823786 2824103 214875 215192 206100 ANaFNaAF

212080 NaGnFbiGnNaFbi

2890806 2890673 281895 281762 286260 Na[NaNa][NaNa]Na

1Theoretical average masses of PSA amino acid sequences were obtained using PeptideMass software (httpwebexpasyorgpeptide masspeptide-mass-dochtml)2According to Isono et al 2002 [8]3PSA glycan mass was calculated as the difference between observed PSA mass and theoretical mass of corresponding PSA protein sequence4Putative glycan compositions were proposed based on established complete PSA glycan chain (NaNaF 235216Da) It is depicted using the nomenclature ofthe Consortium for Functional Glycomics+G glycosylationThe blue square Gn (N-acetylglucosamine) the green circle M or Man (mannose) the yellow circle A (galactose) the red triangle F (fucose) the purplediamond Na (sialic acid) bi (bisected)

PSA is defined as a 28430Da glycoprotein containing 8of carbohydrates mainly as a biantennary N-linked oligosac-charide of N-acetyllactosamine type with sialic acid group atthe end of each of the two branches [22] However availabledata indicate that there is pronounced heterogeneity underboth normal and pathological conditions PSA isoformsdiffer generally in sialic acid content as well as outer chainfeatures [23ndash25] Besides biantennary chains monoanten-nary sugar chains and peripheral structures composed onlyof N-acetylhexosamine residues also occur

The results obtained in this study indicated that PSAspecies complexed with IgM mostly contained incompleteglycan chain lacking sialic acid Thus the main PSA speciescomplexed with IgM are assessed to have oligomannosidicstructure in both BPH and PCa In addition paucimanno-sidic structure is assigned to PCa PSA-IgM

Alterations in PSA glycosylation were reported in bothBPH and PCa [26ndash30] Specifically PSA forms with reducedsialylation fucosylation changes and increased pI werefound in PCa

As for unusual mannosidic structures found in PSA-IgMthey can be correlatedwith glycans of PSA isoformdesignatedas PSA-A1015840 [24] It represented a minor fraction that is only06 of the total PSA isoforms and comprised four PSAspecies ranging from 268089 to 272958Da

It seems that newly found IgM-bound isoforms couldhave diagnostic potential to distinguish PCa and BPHbut with no advantage in comparison to other establishedparameters (total PSA-IgM or fPSAPSA) Due to intrinsicheterogeneity of total PSA-IgMand complexity of themethodapplied their possible relevance should be confirmed by test-ing different types of samples using different experimentaldesigns suitable for clinical use However the finding ofdistinct IgM-bound PSA forms may be of interest in lightof recent data indicating paucimannose as marker of humancancers [31 32]

In contrast to these poorly glycosylated PSA speciesfound in complex with IgM themain fPSA species containedpredominantly mono- or disialylated biantennary chainsThe fPSA fraction was used as the reference because it issupposed to comprise an abundant species that can be recog-nized by IgM PSA complexed with ACT was not consideredrelevant for this interaction since it predominantly containscovalently bound enzymatically active PSA

PSA glycosylation is very important for its biomarkerpotential and this was investigated in a number of studiesunder diverse physiological and pathological conditions [26]Generally an examination of PSA glycosylation especiallynumerous PSA speciesglycoforms is faced with the problemof small amounts preventing isolation in sufficient quantity

Disease Markers 853

2820019 + 2H

1582721 + 1H 1721234 + 1H 2098894 + 1H 2349855 + 1H 2591809 + 1H

2799956 + 1H

2820091 + 1H2881627 + 1H

2806951 + 2H 1522470 + 1H

1584249 + 1H1604169 + 1H

1724538 + 1H1737394 + 1H

2806951 + 1H

0

02

04

(B)

(A)

15000 20000 25000 30000

Inte

nsity

0

02

04

Inte

nsity

mz

15000 20000 25000 30000mz

(a)

1738933 + 1H1752391 + 1H

2102763 + 1H

2763666 + 1H2792866 + 1H

2822404 + 1H2889975 + 1H

2801211 + 2H 1723755 + 1H 2801211 + 1H

15000 20000 25000 300000

02

04

(B)

(A)

Inte

nsity

mz

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

Figure 4 Representative spectra of free PSA isoforms On-chip ion-exchange chromatography of PCa fPSA (a) and BPH fPSA (b) wasperformed on anion exchanger Q10 (A) and cation exchanger CM10 (B) array at pH 8 The 119909-axis represents the distribution of ions bymass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2H double charged ion Peaks corresponding toPSA-immunoreactive species are considered as relevant

for glycan analysis In addition the limitation is related to theknown influence of both glycan and peptide on PSA behaviorduring electrophoretic and chromatographic separations

Using data on the PSA standard preparation obtained bymass spectrometry assessment of PSAglycosylation by calcu-lation of glycan mass has already been proved relevant [24]In this study this was combinedwith on-chip immunoaffinitychromatography and ion-exchange chromatography whichenables simultaneous sensitive detection of various PSA gly-coforms Thus despite the differences in methods and detec-tion systems used for this analysis and those reported in thereference literature generalizations about the composition ofPSA isoforms based on congruence in patterns of protein andglycanmoieties can bemade Differences between calculatedand theoretical masses extended to 01 kDa which may bedue to common ion adducts observed in mass spectrometryHowever for the PSA species at 289Da the difference wassignificant PSA species having masses higher than 284 kDa

are also reported in seminal plasma and serum but they arenot characterized in detail [6 8 11 12] Their relation to PSAforms containing multiantennary chains or to pro-PSA assuggested by some authors cannot be confirmed or denied

It is known that the structural integrity of PSA sig-nificantly influences its biological activity and ability tointeract with different molecules As the first line of defenseIgM has a special position in the immune system andthe discovery of PSA-IgM complexes opens an interestingpoint beyond its biomarker potential Is the binding ofmodified carbohydrates as in the case of PSA a generalprinciple not only of basic but also of clinical interest forinstance in relation to cancer immunotherapy Differentactive and passive vaccines including carbohydrate-basedvaccines to aberrant glycostructures are under clinical trialsbut have had limited success at proof of principle and efficacystages [33] Characteristic PSA glycoforms bound to IgMas emerged from this study direct further investigations to

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 851

Table 1 Summary of PSA-immunoreactive species complexed with IgM in PCa and BPH serum pools

Sample PSA119898119911 Mean (SD) PeakP I P II P III 119898119911 frequency ()

PCa

1602995 1602178 1615058 1606744 (7212) 1001713681 1714265 1713973 (413) 661725485 1721410 1726133 1724343 (2560) 1001738281 1738147 1737829 1738086 (232) 1002688927 2689418 2689172 (347) 662807048 2805844 2807826 2806906 (999) 1002823786 2821013 2828217 2824339 (634) 1002890806 2887531 2897221 2891853 (4929) 100

BPH

1584514 1605460 33 (33)1712676 1714241 1713695 1713537 (794) 1001724457 1726152 1721650 1724076 (2274) 1001737732 1739620 1738440 1738597 (954) 1002804952 2809572 2807406 2807310 (2311) 1002824103 2828975 2822728 2825269 (3283) 1002890673 2892609 2892316 2891866 (1044) 100

SD standard deviation119898119911 mass to charge ratio and P pool (119899 = 6) Frequency of peaks observed when analyzed individually in each pool (IndashIII)

2587218 + 2H1359620 + 1H

2817639 + 2H1517559 + 1H

1584630 + 1H

1604330 + 1H

1726559 + 1H 1904198 + 1H 2080716 + 1H 2364265 + 1H 2587218 + 1H

2803287 + 1H

2817639 + 1H

2890200 + 1H

15000 20000 25000 30000

0

02

04

Inte

nsity

mz

(a)

2581132 + 2H1355180 + 1H

2759581 + 2H

2824495 + 2H

1641264 + 1H1726191 + 1H

1741661 + 1H

1785821 + 1H 2005999 + 1H

2105973 + 1H2312378 + 1H 2581105 + 1H

2804820 + 1H

2824495 + 1H

2889975 + 1H

15000 20000 25000 300000

05

10

15

Inte

nsity

mz

(b)

Figure 3 Representative spectra of free PSA-immunoreactive species On-chip chromatography of free PSA was performed using anti-fPSAmonoclonal antibody array The 119909-axis represents the distribution of ions by mass (mass to charge ratio 119898119911) and the 119910-axis shows signalintensity of the ions M+ 2H double charged form (a) PCa fPSA (free PSA from PCa sera) (b) BPH fPSA (free PSA from BPH sera)

852 Disease Markers

Table 2 Assessment of glycosylation of PSA molecular species complexed with IgM

PSA amino acidsequence theoreticalmass1

PSA group2PSA PSA glycan (G) Glycan (G)

Observed mass Calculated mass3 Theoreticalmass Putative composition4 Structure

PCa BPH PCa BPH

1ndash145 (1612350) + G gp18

1713681 1712676 101331 100326105496 Man4

1725485 1724457 113135 112107

1738281 1737732 125931 125382 121711 Man5

1ndash237 (2608911) + G gp28

2688927 8001673069

89282 MM

2807048 2804952 198137 196041 189900 GnNaFNaGnF

193370 NaAANa

2823786 2824103 214875 215192 206100 ANaFNaAF

212080 NaGnFbiGnNaFbi

2890806 2890673 281895 281762 286260 Na[NaNa][NaNa]Na

1Theoretical average masses of PSA amino acid sequences were obtained using PeptideMass software (httpwebexpasyorgpeptide masspeptide-mass-dochtml)2According to Isono et al 2002 [8]3PSA glycan mass was calculated as the difference between observed PSA mass and theoretical mass of corresponding PSA protein sequence4Putative glycan compositions were proposed based on established complete PSA glycan chain (NaNaF 235216Da) It is depicted using the nomenclature ofthe Consortium for Functional Glycomics+G glycosylationThe blue square Gn (N-acetylglucosamine) the green circle M or Man (mannose) the yellow circle A (galactose) the red triangle F (fucose) the purplediamond Na (sialic acid) bi (bisected)

PSA is defined as a 28430Da glycoprotein containing 8of carbohydrates mainly as a biantennary N-linked oligosac-charide of N-acetyllactosamine type with sialic acid group atthe end of each of the two branches [22] However availabledata indicate that there is pronounced heterogeneity underboth normal and pathological conditions PSA isoformsdiffer generally in sialic acid content as well as outer chainfeatures [23ndash25] Besides biantennary chains monoanten-nary sugar chains and peripheral structures composed onlyof N-acetylhexosamine residues also occur

The results obtained in this study indicated that PSAspecies complexed with IgM mostly contained incompleteglycan chain lacking sialic acid Thus the main PSA speciescomplexed with IgM are assessed to have oligomannosidicstructure in both BPH and PCa In addition paucimanno-sidic structure is assigned to PCa PSA-IgM

Alterations in PSA glycosylation were reported in bothBPH and PCa [26ndash30] Specifically PSA forms with reducedsialylation fucosylation changes and increased pI werefound in PCa

As for unusual mannosidic structures found in PSA-IgMthey can be correlatedwith glycans of PSA isoformdesignatedas PSA-A1015840 [24] It represented a minor fraction that is only06 of the total PSA isoforms and comprised four PSAspecies ranging from 268089 to 272958Da

It seems that newly found IgM-bound isoforms couldhave diagnostic potential to distinguish PCa and BPHbut with no advantage in comparison to other establishedparameters (total PSA-IgM or fPSAPSA) Due to intrinsicheterogeneity of total PSA-IgMand complexity of themethodapplied their possible relevance should be confirmed by test-ing different types of samples using different experimentaldesigns suitable for clinical use However the finding ofdistinct IgM-bound PSA forms may be of interest in lightof recent data indicating paucimannose as marker of humancancers [31 32]

In contrast to these poorly glycosylated PSA speciesfound in complex with IgM themain fPSA species containedpredominantly mono- or disialylated biantennary chainsThe fPSA fraction was used as the reference because it issupposed to comprise an abundant species that can be recog-nized by IgM PSA complexed with ACT was not consideredrelevant for this interaction since it predominantly containscovalently bound enzymatically active PSA

PSA glycosylation is very important for its biomarkerpotential and this was investigated in a number of studiesunder diverse physiological and pathological conditions [26]Generally an examination of PSA glycosylation especiallynumerous PSA speciesglycoforms is faced with the problemof small amounts preventing isolation in sufficient quantity

Disease Markers 853

2820019 + 2H

1582721 + 1H 1721234 + 1H 2098894 + 1H 2349855 + 1H 2591809 + 1H

2799956 + 1H

2820091 + 1H2881627 + 1H

2806951 + 2H 1522470 + 1H

1584249 + 1H1604169 + 1H

1724538 + 1H1737394 + 1H

2806951 + 1H

0

02

04

(B)

(A)

15000 20000 25000 30000

Inte

nsity

0

02

04

Inte

nsity

mz

15000 20000 25000 30000mz

(a)

1738933 + 1H1752391 + 1H

2102763 + 1H

2763666 + 1H2792866 + 1H

2822404 + 1H2889975 + 1H

2801211 + 2H 1723755 + 1H 2801211 + 1H

15000 20000 25000 300000

02

04

(B)

(A)

Inte

nsity

mz

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

Figure 4 Representative spectra of free PSA isoforms On-chip ion-exchange chromatography of PCa fPSA (a) and BPH fPSA (b) wasperformed on anion exchanger Q10 (A) and cation exchanger CM10 (B) array at pH 8 The 119909-axis represents the distribution of ions bymass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2H double charged ion Peaks corresponding toPSA-immunoreactive species are considered as relevant

for glycan analysis In addition the limitation is related to theknown influence of both glycan and peptide on PSA behaviorduring electrophoretic and chromatographic separations

Using data on the PSA standard preparation obtained bymass spectrometry assessment of PSAglycosylation by calcu-lation of glycan mass has already been proved relevant [24]In this study this was combinedwith on-chip immunoaffinitychromatography and ion-exchange chromatography whichenables simultaneous sensitive detection of various PSA gly-coforms Thus despite the differences in methods and detec-tion systems used for this analysis and those reported in thereference literature generalizations about the composition ofPSA isoforms based on congruence in patterns of protein andglycanmoieties can bemade Differences between calculatedand theoretical masses extended to 01 kDa which may bedue to common ion adducts observed in mass spectrometryHowever for the PSA species at 289Da the difference wassignificant PSA species having masses higher than 284 kDa

are also reported in seminal plasma and serum but they arenot characterized in detail [6 8 11 12] Their relation to PSAforms containing multiantennary chains or to pro-PSA assuggested by some authors cannot be confirmed or denied

It is known that the structural integrity of PSA sig-nificantly influences its biological activity and ability tointeract with different molecules As the first line of defenseIgM has a special position in the immune system andthe discovery of PSA-IgM complexes opens an interestingpoint beyond its biomarker potential Is the binding ofmodified carbohydrates as in the case of PSA a generalprinciple not only of basic but also of clinical interest forinstance in relation to cancer immunotherapy Differentactive and passive vaccines including carbohydrate-basedvaccines to aberrant glycostructures are under clinical trialsbut have had limited success at proof of principle and efficacystages [33] Characteristic PSA glycoforms bound to IgMas emerged from this study direct further investigations to

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

852 Disease Markers

Table 2 Assessment of glycosylation of PSA molecular species complexed with IgM

PSA amino acidsequence theoreticalmass1

PSA group2PSA PSA glycan (G) Glycan (G)

Observed mass Calculated mass3 Theoreticalmass Putative composition4 Structure

PCa BPH PCa BPH

1ndash145 (1612350) + G gp18

1713681 1712676 101331 100326105496 Man4

1725485 1724457 113135 112107

1738281 1737732 125931 125382 121711 Man5

1ndash237 (2608911) + G gp28

2688927 8001673069

89282 MM

2807048 2804952 198137 196041 189900 GnNaFNaGnF

193370 NaAANa

2823786 2824103 214875 215192 206100 ANaFNaAF

212080 NaGnFbiGnNaFbi

2890806 2890673 281895 281762 286260 Na[NaNa][NaNa]Na

1Theoretical average masses of PSA amino acid sequences were obtained using PeptideMass software (httpwebexpasyorgpeptide masspeptide-mass-dochtml)2According to Isono et al 2002 [8]3PSA glycan mass was calculated as the difference between observed PSA mass and theoretical mass of corresponding PSA protein sequence4Putative glycan compositions were proposed based on established complete PSA glycan chain (NaNaF 235216Da) It is depicted using the nomenclature ofthe Consortium for Functional Glycomics+G glycosylationThe blue square Gn (N-acetylglucosamine) the green circle M or Man (mannose) the yellow circle A (galactose) the red triangle F (fucose) the purplediamond Na (sialic acid) bi (bisected)

PSA is defined as a 28430Da glycoprotein containing 8of carbohydrates mainly as a biantennary N-linked oligosac-charide of N-acetyllactosamine type with sialic acid group atthe end of each of the two branches [22] However availabledata indicate that there is pronounced heterogeneity underboth normal and pathological conditions PSA isoformsdiffer generally in sialic acid content as well as outer chainfeatures [23ndash25] Besides biantennary chains monoanten-nary sugar chains and peripheral structures composed onlyof N-acetylhexosamine residues also occur

The results obtained in this study indicated that PSAspecies complexed with IgM mostly contained incompleteglycan chain lacking sialic acid Thus the main PSA speciescomplexed with IgM are assessed to have oligomannosidicstructure in both BPH and PCa In addition paucimanno-sidic structure is assigned to PCa PSA-IgM

Alterations in PSA glycosylation were reported in bothBPH and PCa [26ndash30] Specifically PSA forms with reducedsialylation fucosylation changes and increased pI werefound in PCa

As for unusual mannosidic structures found in PSA-IgMthey can be correlatedwith glycans of PSA isoformdesignatedas PSA-A1015840 [24] It represented a minor fraction that is only06 of the total PSA isoforms and comprised four PSAspecies ranging from 268089 to 272958Da

It seems that newly found IgM-bound isoforms couldhave diagnostic potential to distinguish PCa and BPHbut with no advantage in comparison to other establishedparameters (total PSA-IgM or fPSAPSA) Due to intrinsicheterogeneity of total PSA-IgMand complexity of themethodapplied their possible relevance should be confirmed by test-ing different types of samples using different experimentaldesigns suitable for clinical use However the finding ofdistinct IgM-bound PSA forms may be of interest in lightof recent data indicating paucimannose as marker of humancancers [31 32]

In contrast to these poorly glycosylated PSA speciesfound in complex with IgM themain fPSA species containedpredominantly mono- or disialylated biantennary chainsThe fPSA fraction was used as the reference because it issupposed to comprise an abundant species that can be recog-nized by IgM PSA complexed with ACT was not consideredrelevant for this interaction since it predominantly containscovalently bound enzymatically active PSA

PSA glycosylation is very important for its biomarkerpotential and this was investigated in a number of studiesunder diverse physiological and pathological conditions [26]Generally an examination of PSA glycosylation especiallynumerous PSA speciesglycoforms is faced with the problemof small amounts preventing isolation in sufficient quantity

Disease Markers 853

2820019 + 2H

1582721 + 1H 1721234 + 1H 2098894 + 1H 2349855 + 1H 2591809 + 1H

2799956 + 1H

2820091 + 1H2881627 + 1H

2806951 + 2H 1522470 + 1H

1584249 + 1H1604169 + 1H

1724538 + 1H1737394 + 1H

2806951 + 1H

0

02

04

(B)

(A)

15000 20000 25000 30000

Inte

nsity

0

02

04

Inte

nsity

mz

15000 20000 25000 30000mz

(a)

1738933 + 1H1752391 + 1H

2102763 + 1H

2763666 + 1H2792866 + 1H

2822404 + 1H2889975 + 1H

2801211 + 2H 1723755 + 1H 2801211 + 1H

15000 20000 25000 300000

02

04

(B)

(A)

Inte

nsity

mz

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

Figure 4 Representative spectra of free PSA isoforms On-chip ion-exchange chromatography of PCa fPSA (a) and BPH fPSA (b) wasperformed on anion exchanger Q10 (A) and cation exchanger CM10 (B) array at pH 8 The 119909-axis represents the distribution of ions bymass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2H double charged ion Peaks corresponding toPSA-immunoreactive species are considered as relevant

for glycan analysis In addition the limitation is related to theknown influence of both glycan and peptide on PSA behaviorduring electrophoretic and chromatographic separations

Using data on the PSA standard preparation obtained bymass spectrometry assessment of PSAglycosylation by calcu-lation of glycan mass has already been proved relevant [24]In this study this was combinedwith on-chip immunoaffinitychromatography and ion-exchange chromatography whichenables simultaneous sensitive detection of various PSA gly-coforms Thus despite the differences in methods and detec-tion systems used for this analysis and those reported in thereference literature generalizations about the composition ofPSA isoforms based on congruence in patterns of protein andglycanmoieties can bemade Differences between calculatedand theoretical masses extended to 01 kDa which may bedue to common ion adducts observed in mass spectrometryHowever for the PSA species at 289Da the difference wassignificant PSA species having masses higher than 284 kDa

are also reported in seminal plasma and serum but they arenot characterized in detail [6 8 11 12] Their relation to PSAforms containing multiantennary chains or to pro-PSA assuggested by some authors cannot be confirmed or denied

It is known that the structural integrity of PSA sig-nificantly influences its biological activity and ability tointeract with different molecules As the first line of defenseIgM has a special position in the immune system andthe discovery of PSA-IgM complexes opens an interestingpoint beyond its biomarker potential Is the binding ofmodified carbohydrates as in the case of PSA a generalprinciple not only of basic but also of clinical interest forinstance in relation to cancer immunotherapy Differentactive and passive vaccines including carbohydrate-basedvaccines to aberrant glycostructures are under clinical trialsbut have had limited success at proof of principle and efficacystages [33] Characteristic PSA glycoforms bound to IgMas emerged from this study direct further investigations to

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 853

2820019 + 2H

1582721 + 1H 1721234 + 1H 2098894 + 1H 2349855 + 1H 2591809 + 1H

2799956 + 1H

2820091 + 1H2881627 + 1H

2806951 + 2H 1522470 + 1H

1584249 + 1H1604169 + 1H

1724538 + 1H1737394 + 1H

2806951 + 1H

0

02

04

(B)

(A)

15000 20000 25000 30000

Inte

nsity

0

02

04

Inte

nsity

mz

15000 20000 25000 30000mz

(a)

1738933 + 1H1752391 + 1H

2102763 + 1H

2763666 + 1H2792866 + 1H

2822404 + 1H2889975 + 1H

2801211 + 2H 1723755 + 1H 2801211 + 1H

15000 20000 25000 300000

02

04

(B)

(A)

Inte

nsity

mz

15000 20000 25000 300000

02

04

Inte

nsity

mz

(b)

Figure 4 Representative spectra of free PSA isoforms On-chip ion-exchange chromatography of PCa fPSA (a) and BPH fPSA (b) wasperformed on anion exchanger Q10 (A) and cation exchanger CM10 (B) array at pH 8 The 119909-axis represents the distribution of ions bymass (mass to charge ratio 119898119911) and the 119910-axis gives the signal intensity of the ions M+ 2H double charged ion Peaks corresponding toPSA-immunoreactive species are considered as relevant

for glycan analysis In addition the limitation is related to theknown influence of both glycan and peptide on PSA behaviorduring electrophoretic and chromatographic separations

Using data on the PSA standard preparation obtained bymass spectrometry assessment of PSAglycosylation by calcu-lation of glycan mass has already been proved relevant [24]In this study this was combinedwith on-chip immunoaffinitychromatography and ion-exchange chromatography whichenables simultaneous sensitive detection of various PSA gly-coforms Thus despite the differences in methods and detec-tion systems used for this analysis and those reported in thereference literature generalizations about the composition ofPSA isoforms based on congruence in patterns of protein andglycanmoieties can bemade Differences between calculatedand theoretical masses extended to 01 kDa which may bedue to common ion adducts observed in mass spectrometryHowever for the PSA species at 289Da the difference wassignificant PSA species having masses higher than 284 kDa

are also reported in seminal plasma and serum but they arenot characterized in detail [6 8 11 12] Their relation to PSAforms containing multiantennary chains or to pro-PSA assuggested by some authors cannot be confirmed or denied

It is known that the structural integrity of PSA sig-nificantly influences its biological activity and ability tointeract with different molecules As the first line of defenseIgM has a special position in the immune system andthe discovery of PSA-IgM complexes opens an interestingpoint beyond its biomarker potential Is the binding ofmodified carbohydrates as in the case of PSA a generalprinciple not only of basic but also of clinical interest forinstance in relation to cancer immunotherapy Differentactive and passive vaccines including carbohydrate-basedvaccines to aberrant glycostructures are under clinical trialsbut have had limited success at proof of principle and efficacystages [33] Characteristic PSA glycoforms bound to IgMas emerged from this study direct further investigations to

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

854 Disease Markers

structural comparison of different tumor markers found inimmune complexes and their free forms and hold promisefor advancement of their use as targets for cancer detectionand therapy

5 Conclusions

The results obtained indicated distinct differences in im-muno-complexed PCa PSA compared to BPH PSA althoughboth were predominantly composed of nonsialylated incom-pletely glycosylated molecular species It poses the questionof the possible role of glycosylation as a framework forimmunoreactivity and immunoediting representing amech-anism for early elimination or inactivation of aberrant cancermolecules and cells

Acknowledgment

This work is supported by the Ministry of Education Scienceand Technological Development of Serbia project code173010

References

[1] L Beneduce F CastaldiMMarino et al ldquoImprovement of livercancer detection with simultaneous assessment of circulatinglevels of free alpha-fetoprotein (AFP) andAFP-IgM complexesrdquoInternational Journal of BiologicalMarkers vol 19 no 2 pp 155ndash159 2004

[2] L Beneduce F Castaldi M Marino et al ldquoSquamous cellcarcinoma antigen-immunoglobulin M complexes as novelbiomarkers for hepatocellular carcinomardquo Cancer vol 103 no12 pp 2558ndash2565 2005

[3] L Beneduce T Prayer-Galetti A M G Giustinian et alldquoDetection of prostate-specific antigen coupled to immuno-globulin M in prostate cancer patientsrdquo Cancer Detection andPrevention vol 31 no 5 pp 402ndash407 2007

[4] W-M Zhang J Leinonen N Kalkkinen B Dowell andU-H Stenman ldquoPurification and characterization of differentmolecular forms of prostate-specific antigen in human seminalfluidrdquo Clinical Chemistry vol 41 no 11 pp 1567ndash1573 1995

[5] A M Ward J W F Catto and F C Hamdy ldquoProstate spe-cific antigen biology biochemistry and available commercialassaysrdquo Annals of Clinical Biochemistry vol 38 no 6 pp 633ndash651 2001

[6] A Vegvari M Rezeli C Sihlbom et al ldquoMolecular micro-heterogeneity of prostate specific antigen in seminal fluid bymass spectrometryrdquo Clinical Biochemistry vol 45 no 4-5 pp331ndash338 2012

[7] K Jung B Brux M Lein et al ldquoMolecular forms of prostate-specific antigen in malignant and benign prostatic tissuebiochemical and diagnostic implicationsrdquo Clinical Chemistryvol 46 no 1 pp 47ndash54 2000

[8] T Isono T Tanaka S Kageyama and T Yoshiki ldquoStructuraldiversity of cancer-related and non-cancer-related prostate-specific antigenrdquo Clinical Chemistry vol 48 no 12 pp 2187ndash2194 2002

[9] K Jung J Reiche A Boehme et al ldquoAnalysis of subformsof free prostate-specific antigen in serum by two-dimensional

gel electrophoresis potential to improve diagnosis of prostatecancerrdquo Clinical Chemistry vol 50 no 12 pp 2292ndash2301 2004

[10] A Sarrats R Saldova J Comet et al ldquoGlycan characterizationof PSA 2-DE subforms from serum and seminal plasmardquoOMICS vol 14 no 4 pp 465ndash474 2010

[11] A Vegvari M Rezeli C Welinder et al ldquoIdentification ofprostate-specific antigen (PSA) isoforms in complex biologicalsamples utilizing complementary platformsrdquo Journal of Pro-teomics vol 73 no 6 pp 1137ndash1147 2010

[12] M M Kosanovic S R Goc G S Potpara and M M JankovicldquoOn chip immuno-affinity profiling of cancer- and benignhyperplasia-associated free prostate specific antigenrdquo DiseaseMarkers vol 31 no 2 pp 111ndash118 2011

[13] P R Huber H-P Schmid G Mattarelli B Strittmatter G JVan Steenbrugge and A Maurer ldquoSerum free prostate specificantigen isoenzymes in benign hyperplasia and cancer of theprostaterdquo Prostate vol 27 no 4 pp 212ndash219 1995

[14] H Hilz J Noldus P Hammerer F Buck M Luck and HHuland ldquoMolecular heterogeneity of free PSA in sera of patientswith benign andmalignant prostate tumorsrdquo EuropeanUrologyvol 36 no 4 pp 286ndash292 1999

[15] J P Charrier C Tournel S Michel S Comby C Jolivet-Reynaud J Passagot et al ldquoDifferential diagnosis of prostatecancer and benign prostate hyperplasia using two-dimensionalelectrophoresisrdquo Electrophoresis vol 22 no 9 pp 1861ndash18662001

[16] S D Mikolajczyk K M Marker L S Millar et al ldquoA truncatedprecursor form of prostate-specific antigen is a more specificserum marker of prostate cancerrdquo Cancer Research vol 61 no18 pp 6958ndash6963 2001

[17] S D Mikolajczyk L S Millar T J Wang et al ldquolsquoBPSArsquo aspecific molecular form of free prostate-specific antigen isfound predominantly in the transition zone of patients withnodular benign prostatic hyperplasiardquoUrology vol 55 no 1 pp41ndash45 2000

[18] H P Vollmers and S Brandlein ldquoThe ldquoearly birdsrdquo natu-ral IgM antibodies and immune surveillancerdquo Histology andHistopathology vol 20 no 3 pp 927ndash937 2005

[19] H P Vollmers and S Brandlein ldquoNatural IgM antibodies fromparias to parvenusrdquoHistology andHistopathology vol 21 no 12pp 1355ndash1366 2006

[20] G P Dunn A T Bruce H Ikeda L J Old and R D SchreiberldquoCancer immunoediting from immunosurveillance to tumorescaperdquo Nature Immunology vol 3 no 11 pp 991ndash998 2002

[21] G P Dunn L J Old and R D Schreiber ldquoThe three Es ofcancer immunoeditingrdquoAnnual Review of Immunology vol 22pp 329ndash360 2004

[22] A Belanger H Van Halbeek H C B Graves et al ldquoMolecularmass and carbohydrate structure of prostate specific antigenstudies for establishment of an international PSA standardrdquoProstate vol 27 no 4 pp 187ndash197 1995

[23] T Okada Y Sato N Kobayashi et al ldquoStructural characteristicsof the N-glycans of two isoforms of prostate-specific antigenspurified from human seminal fluidrdquo Biochimica et BiophysicaActa vol 1525 no 1-2 pp 149ndash160 2001

[24] J M Mattsson L Valmu P Laakkonen U-H Stenman andH Koistinen ldquoStructural characterization and anti-angiogenicproperties of prostate-specific antigen isoforms in seminalfluidrdquo Prostate vol 68 no 9 pp 945ndash954 2008

[25] K Y White L Rodemich J O Nyalwidhe et al ldquoGlycomiccharacterization of prostate-specific antigen and prostatic acid

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 855

phosphatase in prostate cancer and benign disease seminalplasma fluidsrdquo Journal of Proteome Research vol 8 no 2 pp620ndash630 2009

[26] T Vermassen M M Speeckaert N Lumen S Rottey and JR Delanghe ldquoGlycosylation of prostate specific antigen ant itspotential diagnostic applicationsrdquo Clin Chim Acta vol 413 no19-20 pp 1500ndash1505 2012

[27] S Prakash and P W Robbins ldquoGlycotyping of prostate specificantigenrdquo Glycobiology vol 10 no 2 pp 173ndash176 2000

[28] R Peracaula G Tabares L Royle et al ldquoAltered glycosylationpattern allows the distinction between prostate-specific antigen(PSA) fromnormal and tumor originsrdquoGlycobiology vol 13 no6 pp 457ndash470 2003

[29] MM Jankovic andMM Kosanovic ldquoGlycosylation of urinaryprostate-specific antigen in benign hyperplasia and cancerassessment by lectin-binding patternsrdquo Clinical Biochemistryvol 38 no 1 pp 58ndash65 2005

[30] M M Kosanovic and M M Jankovic ldquoSialylation and fucosy-lation of cancer-associated prostate specific antigenrdquo Journal ofBalkan Union of Oncology vol 10 no 2 pp 247ndash250 2005

[31] C I Balog K Stavenhagen W L Fung C A KoelemanL A McDonnell A Verhoeven et al ldquoN-glycosylation ofcolorectal cancer tissues a liquid chromatography and massspectrometry-based investigationrdquo Molecular amp Cellular Pro-teomics vol 11 pp 571ndash585 2012

[32] B Zipser D Bello-DeOcampo S Diestel M H Tai and BSchmitz ldquoMannitou monoclonal antibody uniquely recognizespaucimannose a marker for human cancer stemness andinflammationrdquo Journal of Carbohydrate Chemistry vol 31 no4-6 pp 504ndash518 2012

[33] H H Wandall and M A Tarp ldquoTherapeutic cancer vaccinesClinical trials and applicationsrdquo inCarbohydrate-BasedVaccinesand Immunotherapies Z Guo and G J Boons Eds pp 333ndash366 John Wiley amp Sons Hoboken NJ USA 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

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Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

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Gastroenterology Research and Practice

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

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