prostate cancer is 
 one of the most common cancers diagnosed in 
 men in the united states : there were 238  590 
 new cases and 29  720 deaths recorded in 2013 .
prostate specific antigen ( psa ) is currently used as a 
 diagnostic biomarker of prostate cancer .
psa is an androgen - regulated serine protease and a member of the 
 tissue kallikrein family .
the concentration 
 of psa is elevated in the blood serum of prostate cancer patients . 

this is due to the loss of basal cells , basement membrane , and lumen 
 architecture , thus permitting propsa to have direct access to the 
 peripheral circulation .
the simplicity 
 of psa testing , which measures the concentration of psa in blood serum , 
 has prompted its wide use to detect and screen prostate cancer .
however , 
 psa testing in the diagnostic gray zone does not clearly distinguish 
 between benign prostate hypertrophy and prostate cancer . to develop new candidate biomarkers for prostate cancer , a 
 deeper 
 understanding of the biochemical properties of psa
glycosylation is one of the most common protein 
 post - translational modifications ( ptms ) , and approximately 8% of psa 
 by weight is composed of n - glycans that occupy a single glycosylation 
 site at asn69 .
several papers have reported 
 a variable degree of sialylation of psa in sera between healthy subjects 
 and those with prostate cancer .
the major isoform , consisting of over 90% of psa , has an 
 isoelectric point ( pi ) of 6.9 , whereas the minor one has a pi of 7.2 . 

the glycosylation pattern at high pi was observed to have a high level 
 of sialylation .
these properties were similarly 
 observed in a study of psa isoenzymes from the serum of patients diagnosed 
 with prostate cancer and the serum of patients diagnosed with benign 
 prostate hyperplasia .
psa n - glycans were 
 also reported to be mostly core fucosylated and to have a minor presence 
 of galnac residues with the increasing pi of the psa fraction .
these different glycosylation patterns at different pi s 
 could be employed to develop new diagnostic biomarker for detecting 
 different prostate diseases . to effectively characterize the 
 n - glycosylation of psa , a widely 
 used method is liquid chromatography interfaced with mass spectrometry 
 ( lc  ms or lc  ms / ms ) .
the applicability 
 of these methods to investigate aberrations in glycosylation associated 
 with several diseases has already been demonstrated .
enrichment techniques , such as lectin affinity chromatography or 
 hydrazide chemistry , and peptide labeling have also been used in conjunction 
 with lc 
ms / ms to characterize and quantify the glycosylation 
 sites of glycoproteins . here , we report the comprehensive 
 qualitative and quantitative 
 glycoproteomic studies of two psa isoforms by lc  ms / ms . the 
 overarching goal is to effectively identify the different glycoforms 
 of psa prior to prostate cancer - related glycoproteomic study . our 
 results were compared with findings of 2012 abrf glycoprotein research 
 group ( gprg ) study , of which we were 
 one of the participating laboratories .
the purpose of the 2012 abrf 
 study was to establish a global overview of the approaches and methodologies 
 in glycoproteomics using a consensus among interlaboratory data .
we 
 found a comparable pattern / level of glycosylation between our results 
 and those of the abrf study .
psa 
 and psa - high pi isoform ( psah ) samples 
 were obtained from lee biosolutions ( st . louis , mo ) .
sodium chloride 
 and disodium phosphate were obtained from mallinckrodt chemicals ( phillipsburg , 
 nj ) .
d , l - dithiothreitol ( dtt ) , iodoacetamide ( iaa ) , 
 and ms - grade formic acid were purchased from sigma - aldrich ( st . louis , 
 mo ) .
hplc - grade solvents , including methanol and isopropanol , were 
 purchased from fisher scientific ( pittsburgh , pa ) .
mass spectrometry 
 grade trypsin gold was obtained from promega ( madison , wi ) .
a 20 g aliquot of psa and 
 5 g aliquot of psah were prepared in 50 mm pbs buffer ( ph 7.5 ) 
 ( phosphate buffered saline containing 50 mm disodium phosphate and 
 150 mm sodium chloride ) . in both cases , denaturation was performed 
 at 65 c for 1 h. the samples were then reduced by adding by 
 adding a 1.25 l aliquot of 200 mm dtt prior to incubation at 
 60 c for 45 min .
those reduced samples were then alkylated with 
 the addition of a 5 l aliquot of 200 mm iaa and incubated at 
 37.5 c for 45 min in the dark .
excess iaa was consumed through 
 the addition of a second 1.25 l aliquot of 200 mm dtt .
the 
 reaction was allowed to proceed at 37.5 c for 30 min in the 
 dark .
trypsin was added into the samples using an enzyme / substrate 
 ratio of 1:25 w / w , and the samples were subjected to overnight incubation 
 at 37.5 c for 18 h. to complete the enzymatic digestion , samples 
 were subjected to microwave digestion at 45 c and 50 w for 30 
 min before adding a 0.5 l aliquot of neat formic acid to the 
 samples .
finally , the samples 
 were dried and suspended in 0.1% formic acid prior to lc 
lc  ms / ms was carried 
 out on dionex 3000 ultimate nano - lc system ( dionex , sunnyvale , ca ) 
 interfaced with an ltq orbitrap velos mass spectrometer ( thermo scientific , 
 san jose , ca ) equipped with a nano - esi source .
the psa and psah digests 
 were initially online - purified using a pepmap 100 c18 cartridge ( 3 
 m , 100  , dionex ) .
a 2 g aliquot of psa and 0.4 
 g aliquot of psah digests were injected into the trapping cartridges . 

the purified peptides were then separated using a pepmap 100 c18 capillary 
 column ( 75 m i.d .
the separation was achieved at a 350 nl / min flow rate using the following 
 gradient conditions : 010 min , 5% solvent b ( 98% acn with 0.1% 
 formic acid ) ; 1040 min , ramping of solvent b from 5 to 45% ; 
 4045 min , ramping of solvent b from 45 to 80% ; 4550 
 min , maintaining solvent b at 80% ; 5051 min , decreasing solvent 
 b to 5% ; and 5160 min , sustaining solvent b at 5% .
the first scan event 
 was a full ms scan of 3802000 m / z at a mass resolution of 15  000 .
the collision induced dissociation 
 ( cid ) and higher - energy collision dissociation ( hcd ) ms / ms were performed 
 on the eight most intense ions seen from the first ms scan event . 
 the second scan event was the cid ms / ms of parent ions selected from 
 the first scan event with an isolation width of 3.0 m / z , a normalized collision energy ( ce ) of 35% , and 
 an activation q value of 0.250 .
the third scan event 
 was set to acquire hcd ms / ms of the parent ions selected from the 
 first scan event .
the isolation width of hcd experiment was set to 
 3.0 m / z , and the normalized ce was 
 set to 45% with an activation time of 0.1 ms .
the protein sequence 
 and purity of prostate specific antigen were confirmed using mascot 
 and scaffold .
proteome discoverer , version 1.2 , software ( thermo scientific , 
 san jose , ca ) was used to generate a mascot generic format file ( * .mgf ) 
 which was subsequently employed for database searching using mascot , 
 version 2.3.2 ( matrix science inc . ,
parent ions were 
 selected from a mass range of 30010  000 da with a minimum 
 peak count of 1 .
the parameters from mascot daemon were set to search 
 against uniprotkb ( uniprot release 2014_06 ) .
oxidation of methionine 
 was set as a variable modification , and carbamidomethylation of cysteine 
 was set as a fixed modification .
tandem ms ions were searched within 
 a 0.8 da mass tolerance , and the peptide sequences were identified 
 within 10 ppm .
the results from mascot were imported into scaffold 
 3 ( proteome software , inc . ,
the protein identifications were based on an ion score 
 cutoff higher than 30 and a minimum number of peptides of two .
the identification of psa and psah glycopeptides was achieved by 
 glypid 2.0 and glycoseq with additional manual confirmation .
glypid 
 2.0 used a combination of hcd / cid scoring 
 and precursor mass - based searching to detect and characterize glycopeptides . 
 because trypsin is well - known to produce incomplete cleavage at c - terminal 
 lysine residues , we searched glycopeptides for theoretical psa sequon - containing 
 peptides .
three sequences , nksvillgr , avcggvlvhpqwvltaahcirnk , 
 and avcggvlvhpqwvltaahcirnksvillgr , have valid 
 identifications of glycopeptides in tryptic digest psa samples .
a 
 default glycan library was modified to consider sulfated / phosphorylated 
 versions of glycans .
it contains 413 glycan compositions with combinations 
 of glcnac , man , fuc , and neuac .
glycoseq ( available 
 as open source software at http://sourceforge.net/projects/glycoseq/ ; manuscript in preparation ) was used to determine glycan sequences 
 ( i.e. , topology or cartoon - graph representation ) using a de novo sequencing 
 algorithm . briefly , starting with the y1 ion , the glycan topology 
 is grown using cid fragmentation peaks while adhering to n - linked 
 glycan synthesis rules . all identified 
 glycopeptides and associated glycan structures were manually confirmed . 

this strategy was also applied for other glycoprotein impurities that 
 are present in the samples .
the findings of these searches are discussed 
 in detail in the results and discussion . although psa possesses a single glycosylation site at asn69 , competitive 
 ionization of co - eluting peptides still presents a challenge that 
 limits the ms / ms identification of glycopeptides because a lower number 
 of glycopeptides would be subjected to tandem ms .
therefore , we created 
 an inclusion list consisting of all theoretical m / z values possibly associated with three peptide 
 backbones , thus ensuring the acquisition of a large number of tandem 
 ms data to facilitate identification of more glycopeptides .
the theoretical m / z values were based on every theoretical 
 peptide backbone sequence using the glycomod tool from the expasy 
 web site .
these m / z values were 
 used to create extracted ion chromatograms ( eics ) using xcalibur qual 
 browser 2.1 ( thermo scientific , san jose , ca ) . a mass accuracy of 
 5 ppm or better
was then applied to confirm ions prior to manual evaluation 
 of tandem ms data . however , only 9% of the detected ions depicted 
 a mass accuracy of 7 ppm .
the mass range was 
 set to full ftms scan with 7-point smoothing enabled and a mass tolerance 
 of 10 ppm allowed .
prostate specific antigen in the samples analyzed 
 here was identified with sequence coverages of 85.6 and 86.5% from 
 psa and psah samples , respectively ( data not shown ) . peptides containing 
 the glycosylation sequon ( n69ks ) were not detected in the 
 proteomics analysis , thus suggesting that the n69 residue 
 is glycosylated .
protein identifications in proteomics analysis were 
 based on an ion score cutoff higher than 30 and a minimum number of 
 peptides of two . according to spectral count data generated by scaffold 

software , spectral counts of psa accounted for 86% of the total spectral 
 count data ( 15 identified peptides ) , whereas the spectral counts of 
 psah accounted for 78% of the total spectral count data ( 17 peptides ) . 

other proteins that were detected in the case of the psa sample were 
 triosephosphate isomerase ( 6% of total spectral counts , three peptides ) , 
 prostaglandin - h2 d - isomerase ( 3% of the total spectral count 
 data , two peptides ) , and growth / differentiation factor 15 ( 5% of total 
 spectral count data , two peptides ) . other proteins detected in the 
 case of the psah sample were triosephosphate isomerase ( 12% of the 
 total spectral count data , eight peptides ) , prosaposin ( 5% of the 
 total spectral count data , two peptides ) , prostaglandin - h2 d - isomerase ( 2% of the total spectral count data , two peptides ) , and 
 growth / differentiation factor 15 ( 3% of the total spectral count data , 
 two peptides ) .
furthermore , glypid analysis using these proteins did 
 not identify any valid glycopeptides associated with above - mentioned 
 proteins , yet they all have the glycosylation sequences .
therefore , 
 all glycopeptides reported here are unique to psa and are not originating 
 from the other proteins observed in the lc  ms / ms analysis . 
 additionally , the y1 ions reported here are unique to psa and are 
 not from any contaminant proteins observed in the lc 
since psa is known to have only one n - linked glycosylation 
 site at asn69 , it is adequate to interpret its glycosylations with 
 cid or hcd tandem ms . if the cid or hcd spectra include glycan fragmentation 
 or glycopeptide diagnostic ions in addition to the expected y1 ion 
 ( peptide + glcnac ) , then the glycopeptides associated with asn69 were 
 confirmed .
for example , a y1 ion associated with asn69 detected at 
 an m / z value of 602 or 601 corresponded 
 to doubly charged nksvillgr + glcnac .
it is observed in low 
 abundance ( < 8% ) when y1 ion intensities are incredibly high .
however , 
 hexose rearrangement is not the case in this study because y1 ion 
 intensities associated with the psa glycosylation site are not that 
 high , as can be seen in cid ms / ms scans ( supporting 
 information figure 1 ) . in cases
when the theoretical m / z values of glycopeptides were used , 
 they were confirmed by matching 
 the retention times of the glycopeptides that were confirmed by tandem 
 ms . using a 5 ppm cutoff mass accuracy allowed glycopeptides that 
 have similar molecular weights
for example , a 
 glycopeptide possessing 2 fuc residues is only 1 da different from 
 that possessing an neuac residue , which can be differentiated using 
 this 5 ppm cutoff . moreover , their retention time differences were 
 also used to distinguish them , since a glycopeptide containing neuac 
 is retained longer by lc than a counterpart glycopeptide containing 
 2 fuc .
overall , the use of the aforementioned criteria results in 
 an overall averaged mass accuracy of 3.05 ppm for all identified glycopeptides . in total ,
tryptic digestion of the samples resulted in the formation 
 of three peptide backbones containing the glycosylation site , namely , 
 nksvillgr ( 1723 min ) , avcggvlvhpqwvltaahcirnk 
 ( 2621 min ) , and avcggvlvhpqwvltaahcirnksvillgr 
 ( 3136 min ) , as shown in figure 1a . 

there are 56 glycopeptides observed for psa , as summarized in table 1 . because 55 of the structures were identified on 
 the nksvillgr backbone , the discussion will be mainly focused on this 
 peptide backbone .
one missing glycoform associated with the nksvillgr 
 backbone is hexnac3hex3dhex1neuac1 , which was detected on the 
 avcggvlvhpqwvltaahcirnk backbone .
figure 1b  d illustrates averaged full ms with observed 
 n - glycans detected on the nksvillgr
there were 11 neutral 
 n - glycans observed at 17.5 min ( figure 1b ) , 
 36 monosialylated and 2 sulfated / phosphorylated n - glycans were observed 
 at 19.5 min ( figure 1c ) , and 6 disialylated 

the m / z values ( charge state 
 = 3 ) of these glycopeptides were listed with confirmed glycan structures 
 if they were supported by tandem ms data . otherwise , the most likely 
 structures were described as previously reported .
there were 32 n - glycans 
 confirmed by tandem ms data , as shown in table 1 .
total ion chromatogram of psa digests with different retention 
 times of three peptide backbones possessing the asn69 glycosylation 
 site ( a ) .
different retention times correspond to the detection 
 of neutral glycoforms ( b ) , sulfated / phosphorylated ( s / p ) neutral glycoforms 
 and monosialylated glycoforms ( c ) , and disialylated glycoforms ( d ) .
the identifications of glycan structures 
 were supported by ms / ms , which are included in supporting information figure 1 . for psah
, similar retention times were detected with 
 different 
 types of n - glycans , as mentioned above ( figure 2a ) . in total , 57 n - glycans
all of the n - glycans 
 were detected on the nksvillgr backbone except hexnac6hex4dhex2neuac1 , 
 which was detected only on the avcggvlvhpqwvltaahcirnk 
 backbone .
figure 2b  d depicts an averaged 
 full ms with identified n - glycans associated with the nksvillgr backbone . 

there were 15 neutral n - glycans observed at 17.5 min ( figure 2b ) , 29 monosialylated and 2 sulfated / phosphorylated 
 n - glycans observed at 19.5 min ( figure 2c ) , 
 and 10 disialylated n - glycans detected at 22 min ( figure 2d ) .
manual annotations of the cid ms / ms of 37 glycopeptides 
 are depicted in supporting information figure 
 1 .
total ion chromatogram of psah digests with different retention 
 times of three peptide backbones possessing the asn69 glycosylation 
 site ( a ) .
different retention times correspond to the detection 
 of neutral glycoforms ( b ) , sulfated / phosphorylated ( s / p ) neutral glycoforms 
 and monosialylated glycoforms ( c ) , and disialylated glycoforms ( d ) .
the identifications of glycan structures 
 were supported by ms / ms , which are included in supporting information figure 1 .
assigning glycan structure from cid ms / ms is achievable 
 because 
 the mass difference between the glycan residues of glycopeptides , 
 such as hex , hexnac , fuc , or neuac , is rather large .
also , sequencing 
 n - glycan structures follows some rules because specific glycosyltransferases / exoglycosidases 
 are involved in trimming or attachment of each glycan residue during 
 the biosynthesis of n - glycans .
the precursor m / z values of glycopeptides were confirmed using a 5 ppm cutoff 
 mass accuracy because the detection of precursor ions was achieved 
 by an orbitrap ftms analyzer .
the intriguing features of n - glycans 
 associated with psa are the 
 presence of an galnac residue instead of gal followed by glcnac and 
 sulfation / phosphorylation . in this study , many n - glycans 
 with these characteristics were detected , some of which were confirmed 
 by tandem ms , as depicted in figure 3 .
the 
 glycan fragments of a glycopeptide with m / z 1034.1406 were assigned with diagnostic ions originating 
 from glycan residues ( figure 3a ) .
the annotation 
 of this tandem ms demonstrates that this glycopeptide is core fucosylated 
 and monosialylated followed by either a gal or glcnac residue .
the 
 core fucosylation was confirmed on the basis of the existence of a 
 y1 ion ( peptide backbone + glcnac ) with fucose , which 
 has an m / z value of 675.3 .
one of 
 the fragment peaks with an m / z value 
 of 1303.95 represents the loss of a galnac residue . also , we confirmed 
 that there are two co - eluting isomeric glycopeptides .
in the low m / z region , diagnostic ions with m / z values of 495.16 and 698.35 confirmed 
 galnac + neuac and glcnac + galnac + neuac glycan structures , respectively . 

in addition , the presence of a 657.19 m / z value affirms the presence of a glcnac + gal + neuac glycan structure . 

therefore , the sialylation followed by gal and galnac residues co - eluted , 
 as confirmed in this single tandem ms .
also , on the basis of the assignment 
 of fragment ions of glycans , we distinguished between the attachment 
 of galnac followed by antenna glcnac and bisecting structures . when 
 a series of hexnac fragmentation were detected as associated with 
 antenna glcnac without assignment of a glc residue ,
regarding bisection structures , 
 the presence of 2glcnac + man + glcnac , 2glcnac + 2man + glcnac , and 
 2glcnac + 3man + glcnac was detected , which correspond to m / z values of 886 , 967 , and 1048 , respectively .
two examples 
 of identified glycoforms whose structures were confirmed 
 by cid ms / ms .
the glycan fragments of hexnac5hex4dhex1neuac1 
 glycopeptide with m / z 1034.1406 
 ( a ) support the presence of a galnac residue followed by a glcnac 
 residue on its antenna .
the annotation of a sulfated / phosphorylated 
 ( s / p ) glycopeptide , determined as hexnac5hex4dhex1s / p1 glycopeptide 
 with m / z 963.7589 ( b ) , suggests 
 the occurrence of sulfation / phosphorylation on the glcnac residue 
 before the galnac residue .
figure 3b illustrates annotated tandem 
 ms 
 of a sulfated / phosphorylated glycopeptide corresponding to m / z 963.7589 .
the sulfation / phosphorylation 
 on a glcnac residue was determined on the basis of the detection of 
 fragment ions at m / z 1303.87 and 
 1344.45 , the difference of which corresponds to a doubly charged sulfate 
 or phosphate ion . because the peak at m / z 1344.45 originated from the loss of a galnac residue , this suggests 
 that the occurrence of sulfation / phosphorylation is on a glcnac residue . 

also , the presence of m / z values 
 of 1221.05 and 1262.04 supports the occurrence of sulfation / phosphorylation 
 on a glcnac residue .
the difference between these two assignments 
 of sulfation / phosphorylation was 0.5 da , which fits well within the 
 mass accuracy of cid ms / ms .
on the other hand , no diagnostic ions 
 representing a sulfated / phosphorylated glycan residue were observed 
 from hcd ms / ms , as shown in supporting information 
 figure 2 .
overall , more of core - fucosylated and/or disialylated 
 glycoforms were identified in psah than in normal psa . moreover , highly 
 branched glycan structures such as tri- or tetra - antennary structures 
 were more abundant in psah than in normal psa
there 
 was a difference in the number of identified n - glycans from 
 the three peptide backbones , as shown in tables 1 and 2 .
this might be due to the fact that 
 the ionization efficiency of peptides becomes substantially higher 
 as the mobile phase s organic content increases . as a result , 

some of the glycopeptides with minor intensities would not be subjected 
 to tandem ms experiments .
quantitative results of all identified glycopeptides associated 
 with the three peptide backbones are illustrated in supporting information figure 4a , b for psa and psah , respectively . 

glycopeptides with relatively low intensities associated with the 
 nksvillgr backbone were not detected in the other two peptides backbones , 
 which is believed to be mainly due to competitive ionization .
the quantitation of all identified glycopeptides 
 associated with the three detected peptide backbones is summarized 
 in tables 1 and 2 .
average 
 peak areas and standard deviation values ( std ) were measured from 
 technical triplicates .
figure 4 shows 
 the quantitation results of n - glycans on the nksvillgr backbone .
there 
 are 66 common n - glycans with 68 total identified glycoforms that were 
 quantitatively compared between psa and psah samples .
the abundances 
 of two glycopeptides associated with the peptides avcggvlvhpqwvltaahcirnk 
 and avcggvlvhpqwvltaahcirnksvillgr were excluded 
 because their ionization efficiencies might be different from those 
 of other glycopeptides associated with the peptide nksvillgr .
figure 4b depicts a separate comparison of two glycoforms 
 that demonstrated a higher abundance for psah relative to that in 
 all other structures .
the most abundant glycoform for psa was determined 
 to be hexnac3hex4dhex1neuac1 ( 13.3% ) .
four glycopeptides possessing 
 different glycan structures , namely , hexnac3hex6neuac1 ( 9.3% ) , hexnac4hex4neuac1 
 ( 7.9% ) , hexnac4hex4dhex1neuac1 ( 6.8% ) , and hexnac5hex5neuac1 
 ( 5.7% ) were detected as the next most abundant ions .
on the other 
 hand , glycopeptides with hexnac4hex5dhex1neuac1 ( 28.9% ) and 
 hexnac4hex5dhex1neuac2 ( 27% ) were observed at higher intensities 
 in the psah sample .
glycopeptides possessing hexnac5hex5neuac1 
 ( 6.8% ) , hexnac5hex4dhex1neuac2 ( 6.6% ) , hexnac5hex5neuac2 
 ( 5.3% ) , and hexnac5hex4dhex1neuac1 ( 5.2% ) glycans were observed 
 as the next most abundant ions .
the glycoform of hexnac5hex4dhex1s / p 
 was identified in both psa and psah with different abundances ( 0.52 
 and 0.28% , respectively ) .
an interesting observation related to this 
 structure is that the summation of the relative abundances of hexnac5hex4dhex1 
 and hexnac5hex4dhex1s / p from psah was determined to be comparable 
 with the intensity of hexnac5hex4dhex1s / p from psa . on the other hand , 

quantitative values ( relative 
 abundance ) of n - glycans on the nksvillgr 
 backbone .
( b ) relative 
 abundances of two glycoforms , which shows low - abundance glycoforms 
 for psah by separating the extremely high abundances of hexnac4hex5dhex1neuac1 
 and hexnac4hex5dhex1neuac2 in psah .
an image representing a 
 glycoform was added to the side for which higher quantities were observed .
the abundances of the 46 common 
 n - glycans were compared between 
 the psa and psah samples .
for example , the glycopeptides possessing 
 hexnac3hex6neuac1 ( > 21.7-fold ) , hexnac4hex4neuac1 ( > 13.8-fold ) , 
 hexnac4hex3dhex1neuac1 
 ( > 8-fold ) , hexnac3hex4dhex1neuac1 ( > 6.5-fold ) , and hexnac5hex4neuac1 
 ( > 5-fold ) were detected with higher intensities in the psa sample 
 compared with that of the psah sample .
on the other hand , the glycopeptides 
 with hexnac5hex4dhex1neuac2 ( > 9.2-fold ) , hexnac4hex5dhex1neuac2 
 ( > 4.8-fold ) , and hexnac4hex5dhex1neuac1 ( > 3.2-fold ) were 
 observed 
 with higher abundances in psah than that in psa . as 
 one of the 26 global laboratories that participated in the 2012 abrf 
 glycoprotein research group ( gprg ) study ( the 2012 abrf study ) ,
the goal of the 2012 
 abrf study was to provide a global overview of glycoproteomics methods 
 using a consensus among interlaboratory data . as a result , 57
twenty six data sets collected from the participating 
 laboratories were classified into four clusters from a to d based 
 on data similarity .
the major cluster is c , where the data were summarized 
 from 21 laboratories , including our laboratory . according to abundances , 
 n - glycans were grouped into three categories , including major , intermediate , 
 and minor abundance structures .
they were also an abrf - participating 
 laboratory that identified 42 glycoforms of intact psa / psah glycoprotein 
 using hr 
esi / tof  ms . as shown in supporting information figure 5a , the three studies reported 
 85 total glycoforms associated with psa and psah , with 29 common glycoforms . 

seventeen additional glycoforms were commonly identified between the 
 2012 abrf study and this study .
three glycoforms were reported by behnken et al . and were observed in our study but were not described in 
 the abrf study . our study and
ten glycoforms were described 
 in behnken et al.s study but were not observed in our study , 
 of which four were described in the abrf study .
this discrepancy may 
 be due to the use of different methods : bottom - up gycoproteomics in 
 this study and top - down glycoproteomics in the behnken et al . study . as shown in supporting 
 information figure 5b
, the relative abundances of 18 major / intermediate 
 n - glycans appeared to be comparable with that in the 2012 abrf study . 

in particular , the n - glycans hexnac3hex4dhex1neuac1 , hexnac4hex5dhex1neuac2 , 
 hexnac5hex3dhex1neuac1 , and hexnac5hex4dhex1neuac1 were 
 observed with a very high overlap in intensities . from minor n - glycans
, 
 we noticed that 6 of the 13 missing structures were sulfated / phosphorylated 
 n - glycans with very low intensities .
one sulfated / phosphorylated glycoform , 
 hexnac5hex4dhex1s / p , was reported in the 2012 abrf study as being 
 less than 1% , but it is present in psa at approximately twice the 
 amount as that in psah .
this particular glycoform was observed in 
 our analysis and with a similar intensity profile variation .
behnken et al . reported a single analysis 
 of quantitation ; thus , no variation was inserted ( supporting information figure 5c ) . among the 32 common glycoforms 

that were observed in our study , most of them showed a comparable 
 trend of intensities between psa and psah . with regard to the 18 major / intermediate 
 glycoforms defined by the 2012 abrf study ,
16 glycoforms were observed 
 by behnken et al . , whereas 2 major / intermediate 
 glycoforms were not reported , including hexnac2hex5 and hexnac4hex4dhex1 . 

most of the major / intermediate glycoforms show comparable abundance 
 profiles except for hexnac4hex5dhex1neuac2 , in which its abundance 
 in psa appeared to be 4.60-fold lower when comparing this study and 
 the 2012 abrf study .
the comprehensive qualitative 
 and quantitative glycoproteomic studies 
 of two psa isoforms were successfully achieved by lc 
, there were 56 n - glycans detected for 
 the psa sample , whereas 57 n - glycans were observed for psah . in total , 
 three sulfated / phosphorylated neutral n - glycans were identified , which 
 were supported by tandem ms .
moreover , several galnac residues that 
 were monitored were followed by glcnac residues on the antenna . from 
 the quantitative results , the levels of the glycosylation patterns 

the glycopeptide possessing hexnac3hex4dhex1neuac1 
 ( 13.3% ) was observed with the highest intensity from psa , whereas 
 the ones with hexnac4hex5dhex1neuac1 ( 28.9% ) and hexnac4hex5dhex1neuac2 
 ( 27% ) were predominantly present from psah . a comparable pattern / level 
 of glycosylation was observed between the cumulative results from 
 the 2012 abrf study and those from a specific participating laboratory . 

these results can be further investigated with the aim of distinguishing 
 glycosylation patterns between healthy and prostate cancer patients .
 prostate 
 specific antigen ( psa ) is currently used as a biomarker 
 to diagnose prostate cancer . 
 psa testing has been widely used to detect 
 and screen prostate cancer . however , in the diagnostic gray zone , 
 the psa test does not clearly distinguish between benign prostate 
 hypertrophy and prostate cancer due to their overlap . to develop more 
 specific and sensitive candidate biomarkers for prostate cancer , an 
 in - depth understanding of the biochemical characteristics of psa ( such 
 as glycosylation ) is needed . 
 psa has a single glycosylation site at 
 asn69 , with glycans constituting approximately 8% of the protein by 
 weight . here , we report the comprehensive identification and quantitation 
 of n - glycans from two psa isoforms using lc  
 ms / ms . 
 there were 
 56 n - glycans associated with psa , whereas 57 n - glycans were observed 
 in the case of the psa - high isoelectric point ( pi ) isoform ( psah ) . 
 
 three sulfated / phosphorylated glycopeptides were detected , the identification 
 of which was supported by tandem ms data . 
 one of these sulfated / phosphorylated 
 n - glycans , hexnac5hex4dhex1s / p1 was identified in both psa and psah 
 at relative intensities of 0.52 and 0.28% , respectively . 
 quantitatively , 
 the variations were monitored between these two isoforms . because 
 we were one of the laboratories participating in the 2012 abrf glycoprotein 
 research group ( gprg ) study , 
 those results were compared to that presented 
 in this study . 
 our qualitative and quantitative results summarized 
 here were comparable to those that were summarized in the interlaboratory 
 study . 
Introduction
Experimental Section
Results and Discussion
Conclusions