PMC2667357 The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with 2-6-linked sialic acid. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma. Different N- glycosylation patterns of the two isoforms of KLK6 were confirmed by glycosidase digestion followed by gel shift mobility assays. Our main finding is that KLK6 from ovarian cancer ascites (but not CSF) is extensively sialylated. These results suggested presence of terminal sialic acid residues on ascites-derived KLK6 but not on the CSF-derived KLK6. KLK6 was further shown to contain only one site of N-glycosylation at residue Asn-134 by site-directed mutagenesis and transient expression in HEK 293 cells. The presence of 2-6-linked sialic acid on KLK6 glycoisoforms was further confirmed using a lectin-antibody sandwich ELISA method. Only the glycoisoform of KLK6 from ovarian cancer ascites fluid showed a concentration-dependent increase in signal that was above background noise (Fig. 3), further confirming the presence, and absence, of sialic acid on KLK6 from ovarian cancer ascites and CSF, respectively. Consistent with results described above, the glycan structures present on KLK6 derived from ovarian cancer ascites fluid were shown to be highly heterogeneous and almost exclusively sialylated, save for one identified non-sialylated glycopeptide (Fig. 4A). The majority of the identified structures were core-fucosylated bi-, tri-, or tetra-antennary glycans with a varying number of terminal galactose-linked sialic acids. Two exceptions were observed; the ion at m/z 1192.48 lacked a terminal sialic acid residue and the ion at m/z 1400.22 contained a terminal sialic acid linked to an N-acetylglucosamine directly, instead through galactose. Conversely, a single major peak at m/z 1152.14 was observed for KLK6 from CSF, corresponding to a tri-antennary core-fucosylated glycopeptide (Fig. 4B). The minor peaks identified were indicative of tri- and tetra-antennary structures heterogeneous in respect to core fucosylation and terminal fucosylation and galactosylation. Two minor peaks (at m/z 1303.19 and 1370.88) were found to contain terminal sialic residues. Considering the mRNA up-regulation of several sialyltransferases and the general deregulation of sialylation pathways in ovarian cancer (15–22) it was not surprising that KLK6 isolated from ascites fluid of ovarian cancer patients was found to be modified with glycan structures containing 2-6-linked sialic acid. On the other hand, KLK6 from CSF of healthy individuals was, for the most part, lacking in sialic acid groups. The almost exclusive presence of sialic acid moieties on KLK6 derived from ovarian cancer cells could, in the future, serve to the further development and refinement of KLK6 as an improved ovarian cancer biomarker. ***************** PMC2785108 Human Protein C (hPC) is glycosylated at three Asn-X-Ser/Thr and one atypical Asn-X-Cys sequons. The N-glycans of hPC are complex di- and tri-sialylated structures, and we measured 78% site occupancy at Asn-329 (the Asn-X-Cys sequon). The N-glycans of tg-PC are complex sialylated structures, but less branched and partially sialylated. The porcine mammary epithelial cells glycosylate the Asn-X-Cys sequon with a similar efficiency as human hepatocytes even at these high expression levels, and site occupancy at this sequon was not affected by expression level. Interestingly, glycans with GalNAc in the antennae were predominant at the Asn-329 site. The N-glycosylation site at Asn-329 contains the unusual sequon Asn-X-Cys (X is any amino acid except Pro) that is also found in bovine Protein C [5]. HPC has been reported to be 70–80% glycosylated at this sequon by SDS PAGE analysis, and bovine Protein C is reported to be fully glycosylated [5–8]. N-glycosylation of the atypical Asn-X-Cys sequon has been characterized in the literature for only six other proteins: human alpha1T- glycoprotein, human von Willebrand Factor, human CD69, human alpha-lactalbumin, murine and human fetal antigen 1, and recombinant human epidermal growth factor receptor [9–15]. In these proteins, the reported site occupancy at this sequon ranged from sparse (1% in alpha-lactalbumin) to full (von Willebrand Factor, and human fetal antigen 1) [15]. Where glycan structures are reported for these sequons, they are complex bi- and tri-antennary with and without both sialylation and fucosylation. As summarized in Table 2, only N-acetylneuraminic acid (Neu5Ac) was detected in both hPC and tg-PC. Total Neu5Ac content of hPC was 50–80% higher than tg-PC, which may mean that the N-glycans of hPC are more highly branched and/or have more complete sialylation than tg-PC. Figures 1B–D show that N-glycans of hPC are di- or tri-sialylated structures, while tg-PC contains primarily mono- and di-sialylated oligosaccharides. Sialylated N-glycans of both hPC and tg-PC are more than 98% of total the N-glycan population, based on relative peak area integration from the chromatograms (Table 2). The ions at m/z 1110 [M-2H]2−, m/z 1183 [M-2H]2−, and m/z 1204 [M-2H]2− found on hPC were assigned as di-sialylated biantennary structures without and with core fucosylation, respectively. Peaks consistent with tri-sialylated triantennary structures were also found at m/z 1438 [M-2H]2− and 1511 [M-2H]2− (MS/MS data not shown). The m/z 1971 [M-H]− peak is identified as a monosialylated biantennary with Gal and GalNAc residues on each antenna (Figure 3A). The ion m/z 424 indicates Hex+HexNAc+59 as described in Harvey 2005 [29] and can be 1,3A4αY6 or 1,3A5Y5. The fragment ion B3α (m/z 655) indicates Neu5Ac+Hex+HexNAc sequence, and B3β (m/z 696) indicates Neu5Ac+HexNAc+HexNAc, reflecting there are possible isomers according to the location of Neu5Ac termination. The same structure with a fucosylation is obtained from the m/z 2117 [M-H]− precursor ion (Figure 3B). Figure 3C shows the MS/MS spectrum of a monosialylated biantennary glycan with two fucosylations. The fragment ions, 2,4A7 (m/z 1915) and 0,2A7 (m/z 2121) were 146 Da higher than the corresponding ions, 2,4A7 (m/z 1769) and 0,2A7 (m/z 1975) found in the MS/MS spectrum of a monosialylated biantennary glycan with a core fucosylation (data not shown), indicating that the N-glycan structure in Figure 3C contains a core fucosylation and a fucosylation on the antennae. As summarized in Figure 4, the N-glycans of both hPC and tg-PC are bi- or tri-antennary complex structures. The N-glycans of hPC are less heterogeneous than those of tg-PC, but more highly branched and more fully sialylated, which is consistent with sialic acid content analysis and NP-HPLC profiles. Analysis by SDS PAGE and western blot techniques estimated a 70–80% occupancy of Asn-329 in hPC [6–8]. We were able to detect only the peptides containing 18O- labeled Asp residues in the peptides containing Asn-97, 248, and 313 for both hPC and tg- PC (data not shown). These data indicate that these N-glycan sites of hPC and tg-PC are fully occupied. However, the peptides containing Asn-329 from both hPC and tg-PC had peptides bearing 18O-labeled Asp or Asn, indicating the N-glycan site is partially occupied, and consistent with previous reports [6–8]. As summarized in Table 3, the site occupancy at Asn-329 for hPC is 78%, which is within the range of the previous reports showing 70–80% occupancy [6–8]. There was no significant difference of the site occupancy between hPC (78%) and tg-PC (74–77%), indicating that the Asn-X-Cys sequon is recognized by the oligosaccharyl transferase complex of the porcine mammary epithelial cells with the same efficiency as that observed in human hepatocytes. The presence of N-glycan was confirmed by oxonium ions observed at m/z 204 (HexNAc), 292 (Neu5Ac), 366 (Hex+HexNAc), 454 (Neu5Ac+Hex), and 657 (Neu5Ac+Hex+HexNAc) in the spectra of glycopeptides. Asn-97 contained a more even distribution of glycan structures than other sites, with the predominant being the fucosylated triantennary sialylated species H5. Asn-248 was glycosylated predominantly with the biantennary sialylated species H1 (not fucosylated), and contained lower amounts of fucosylated glycans H2, H3, and H5. Asn-313 was predominantly glycosylated with the tri- antennary sialylated species H4 (non-fucosylated), and also had low levels of fucosylation at this site. Asn-329 was largely glycosylated with the fucosylated biantennary sialylated species H3, which contains GalNAc in one of the antennae. The microheterogeneity of tg-PC from the two expression levels is summarized in Figure 8 B–C. At Asn-97 and Asn-248, the biantennary monosialylated T1 and T3 (fucosylated) species were predominant, with no clear preference for fucosylation. At Asn-313, both animals had a significant amount of species T2, which has GalNAc in one antennae, in addition to the T1 and T3 species. At Asn-329, the majority of the MRM signal was due to glycans which has a GalNAc in one antenna (T4, T9, and T11), although there was also a presence (~15–25% of peak areas) from the T1 species. One interesting feature of the microheterogeneity analysis is that for both hPC and tg-PC, glycans with GalNAc in the antennae (H3 for hPC and T4, T9, T11 for tg-PC) were more abundant at Asn-329 than at Asn-97, 248, and 313. The N-glycans of hPC are complex di- and tri-sialylated structures, and MRM analysis resulted in a measured 78% site occupancy at Asn-329, which agrees with previous reports that used SDS-PAGE methods. We analyzed the microheterogeneity of hPC N- glycosylation, and found that there was a distinct bias for particular structures being present at each of the four sites, and that glycans with GalNAc in the antennae were predominant at the Asn-329 site. The N-glycans of tg-PC were complex sialylated structures (with Neu5Ac only), but were less branched and partially sialylated. Similar to hPC, we found that glycans with GalNAc in the antennae were predominant at Asn-329. ******************* PMC3322569 The CID- MS3 spectra of the Y0-ions in Figs. 2A–2C (m/z 828) were all matched to the tryptic D93VSTPPTVLPDNFPR107 peptide of Insulin-like growth factor II (IGF-II, UniProt/KB accession P01344) with ion scores of 67 (p 0.05 threshold; 26), 39 (p 0.05 threshold; 21) and 63 (p 0.05 threshold; 21) for the Hex2HexNAc2 (Fig. 2D), HexHexNAc2 (not shown), and HexHexNAc (Fig. 2F) glycoform, respectively (Table I). We also acquired ECD-MS2 spectra of the triply charged D93VSTPPTVLPDNFPR107 glycopeptides from IGF-II, with Hex2HexNAc2 (Fig. 2G), HexHexNAc2 (Fig. 2H) and HexHex- NAc (Fig. 2I) glycans. A glycosylated c6 fragment was indeed observed at m/z 979.44 (Fig. 2G), which showed that Thr96 harbored a single HexHexNAc. The c7 fragment was observed at m/z 1445.62, which mapped the second HexHex-NAc to Thr99. The glycan sequence, determined as two sep- arate HexHexNAc-O-Ser/Thr structures by CID-MS2 (Fig. 2A), was thus mapped by ECD-MS2 (Fig. 2G) to two individual amino acids, i.e. Thr96 and Thr99 of IGF-II. The ECD-MS2 spectrum of the triply charged HexHexNAc2 glycoform (Fig. 2H) allowed us to verify the peptide sequence and the pres- ence of a HexHexNAc2 moiety within the Asp93-Val94-Ser95- Thr96-Pro97-Pro98-Thr99 region. In contrast, the c7-ion was detected with the additional mass of HexHexNAc (365.13 Da) at m/z 1080.49, thereby pinpointing the glycosylation site to Thr99 of IGF-II as previously described (52). Taken together, these experiments also revealed the site occupancy (macro- heterogeneity) within the D93VSTPPTVLPDNFPR107 tryptic glycopeptide, i.e. the initial HexHexNAc glycosylation occurs at Thr99 whereas the second HexHexNAc is attached to Thr96. For the C-terminal tryptic peptide A342VAVTLQSH350 from protein YIPF3 a single HexNAc, in accordance with the Tn-antigen, (GalNAc -O-Ser/Thr, Fig. 3A) was identified. The ECD-MS2 346 spectrum showed that the HexNAc was attached to the Thr residue (Fig. 3B). The HexHexNAc glycoform was also identified by CID-MS2 (Fig. 3C) and ECD-MS2 (Fig. 3D). Further, three core 2-like structures with Hex(HexNAc)HexNAc (Fig. 3E), Hex(HexHexNAc)HexNAc (Fig. 3G) and dHexHex- (HexHexNAc)HexNAc (Fig. 3I) glycans were also identified. One glycosylation site on the Thr346 residue was mapped for these O-linked glycopeptides by ECD-MS2 (Fig. 3F, 3H, and 3J). The CID-MS2 fragmentation spectrum of the HexHexNAc glycosylated P52ATDETVLA60 peptide (Microfibrillar-associated protein 5, UniProt/KB accession Q13361) (Fig. 4A) showed an initial loss of 80 Da (m/z 641.0), which we tentatively assigned as a sulfate group (79.9568 Da), but which could in theory also be a phosphate group (79.9663 Da). Co-eluting with the sulfated precursor, we also observed the nonsulfated glycoform, i.e. the HexHexNAc modified peptide, which was also characterized by CID-MSn and ECD-MS2 fragmentation (supplemental Fig. S5). The CID-MS2 spectrum of the HexHexNAc glycosylated T19PAPLDSVFSSSER32 peptide (Vitamin K-dependent pro- tein C, UniProt/KB accession P04070) is shown in Fig. 4C. ECD-MS2 fragmentation (sup- plemental Fig. S5) allowed us to pinpoint the HexHexNAc-O-sequence to Thr19 but the phosphorylated serine residue, among the four possible, was not identified (Table I and supplemental Fig. S5). Firstly, the FTICR-MS1 spectrum (Fig. 5A) showed the ADGTVNQIEGEATPVN98LTEPAK peptide from Apolipopro- tein D (UniProt/KB accession P05090) with N-linked glycans corresponding to the complex type biantennary and fucosy- lated bi-, tri-, and tetraantennary structures. Second, the CID-MS2 fragmentation of a precursor at m/z 1394.9, corresponded to a fucosylated tetra-antennary com- plex type N-glycopeptide from uromodulin (UniProt/KB ac- cession P07911) and resulted in a prominent charge reduced fragment ion at m/z 1909.8 because of the loss of a terminal HexHexNAc moiety and a proton (Fig. 5D). The second most intense fragment (m/z 1017.4) corresponded to [peptide dHexHexNAc 2H]2, indicating that the fucose re- sided on the asparagine linked GlcNAc. We observed an intense peak at m/z 943.9 corresponding to the loss of dHex together with minor peaks corresponding to peptide fragmentation and the MS3 spectrum was matched to the tryptic QDFN322ITDISLLEHR peptide of uromodulin, with a Mascot score of 13 (p 0.05 threshold; 10). However, considering the high charge state, and thus the relatively low m/z ratio, this glycopeptide was efficiently fragmented into c- and z-type ions by ECD-MS2 (Fig. 5I) and the peptide sequence was identified to originate from the tryptic YPHKPEIN143STTHPGADLQENFCR peptide from prothrombin (UniProtKB accession P00734). The combination of CID- MSn with ECD-MS2 was found to be useful in the identifi- cation of an additional N-linked glycopeptide (sup- plemental Fig. S6), namely the tryptic LHEITN117ETFR peptide of vasorin (UniProt/KB accession Q6EMK4). We were also able to observe extensive microheteroge- neity for specific O-glycopeptides, as demonstrated for the A342VAVTLQSH350 peptide of protein YIPF3 in Fig. 3. This O-glycopeptide was identified in five different core glycoforms ranging from a single HexNAc residue to a fucose containing pentasaccharide, clearly showing that our approach is not selective for O-glycopeptides occupied only by core 1-like glycans. Several urinary glycoproteins, e.g. CD44, macrophage col- ony-stimulating factor 1, vasorin, complement component 7 and protein HEG homolog, identified as enriched glycopep- tides in Table I, are each estimated to constitute less than 0.1–0.02% (by mass) of the core urinary proteome (10). Figure Legends FIG. 2. LTQ-FTICR mass spectrometry of urinary O-linked glycopeptides derived from Insulin-like growth factor II. ECD-MS2 spectrum of the triply charged Hex2HexNAc2 glycoform (m/z 795.70853 ) showing the effective dissociation of the precursor, which revealed the novel glycosylation site at Thr96. ECD-MS2 spectrum of the triply charged HexHexNAc glycoform (m/z 673.99793 ), which confirms the previously reported glycosylation site at Thr99. FIG. 3. Microheterogeneity of the A342VAVTLQSH350 O-linked glycopeptide from urinary protein YIPF3. (A) CID-MS2 (m/z 564.79912 ) and (B) ECD-MS2 spectra (m/z 564.79872 ) of the Hex- NAc glycoform which pinpoint the novel glycosylation site to Thr346. (E) CID-MS2 spec- trum of the HexHexNAc2 glycoform (m/z 747.36582 ) with a diagnos- tic ion at m/z 407, indicating a branched core 2-like structure and (F) ECD-MS2 spectrum (m/z 747.36552 ) for the same HexHexNAc2 glycoform showing that the entire glycan moiety resides on Thr346. (I) CID- MS2 spectrum of the dHexHex2HexNAc2 glycoform (m/z 901.42242 ) which shows a complex glycosidic fragmentation pat- tern and (J) ECD-MS2 of the same fucosylated glycoform (m/z 901.42212 ) showing once again that the entire glycan moiety is attached to Thr346. FIG. 4. Modifications of O-linked glycopeptides. A, CID-MS2 spectrum (m/z 681.28042 ) of P52ATDETVLA60 peptide (Microfibrillar- associated protein 5) with a tentative sulfate group on the HexHex- NAc component. CID-MS2 spectrum (m/z 969.42002 ) of the HexHexNAc glycosylated T19PAPLDSVFSSSER32 peptide with a tentative phosphate group attached to the peptide. Fig 5. A, MS1 acquisition of individual peptide glycoforms for the ADGTVNQIEGEATPVN98LTEPAK peptide from apolipoprotein D. CID-MS2 spectrum of the bi-antennary N-linked glycopeptide from apolipoprotein D (m/z 1292.91043 ). D, CID-MS2 spectrum of the fucosylated tetra-antennary N-linked glycopeptide (QDFN322ITDISLLEHR) from uromodulin (m/z 1394.92253 ) G, CID-MS2 spectrum of the pentuply charged bi-antennary N-linked glycopeptide (YPHKPEIN143STTHPGADLQENFCR) from prothrombin (m/z 867.57705 ). *************** PMC4035941 Detailed glycosylation analysis exhibited com- plex and oligomannosidic N-glycans in a site-specific manner on human-serum IgM and on plant- and human-cell-line–produced SM6. Human-serum IgM contains five N-glycosylation sites (GS) in the constant region of the heavy chain (GS1–5; Asn171, Asn332, Asn395, Asn402, and Asn563) (22). Previous data from total glycan release experiments showed the presence of complex and oligomannosidic N-glycans (22). Liquid chromatography-electrospray ionization-mass spectrom- etry (LC-ESI-MS) (Fig. 2) exhibited the presence of mainly sialylated complex-type N-glycans on the three N-terminally located GS1–3 (i.e., Asn171, Asn332, and Asn395). Overall two domi- nant species, monosialylated and fucosylated ANaF and ANaFbi, were present (Table 1 and Fig. 2). In contrast, the C-terminally located GS4 and GS5 (Asn402, Asn563) carry oligomannosidic structures (Fig. 2). Deviating from that study, which suggested incomplete occupation of GS5 (22), our analyses exhibited full glycosylation of this site (Fig. S2). As a next step the glycosylation profile of SM6 produced in the human PER.C6 cell line (SM6PER) was determined (5). In general, the glycosylation pattern largely resembled that of human-serum– derived IgM: the three N-terminal glycosylation sites are occupied by complex-type N-glycans and the two C-terminal sites carried oligomannosidic structures. Although the monosialylated glycan ANaF emerged as the main glycan structure in both samples (Table 1), SM6PER showed a larger number of complex N-glycan species. Other dif- ferences are the presence of difucosylated glycans (i.e., ANaF+F) and the lack of bisected structures (N-acetylglucosamine at- tached in β-1,4-position to the innermost mannose residue) in SM6PER. Glycosylation profiling of SM6 produced in WT plants (SM6wt) showed the same distribution of complex and oligomannosidic N-glycans as for serum-derived IgM and SM6PER: the N-terminally located GS1–3 were mainly decorated with complex type N-glycans and the C-terminal GS4 and GS5 with oligomannosidic structures. However, in contrast to the human-cell–derived IgMs, GS1–3 carried a single dominant glycan species that lacked galactose and sialic acid residues, but bore plant-typical xylose and fucose (i.e., GnGnXF; Table 1). Initial expression of SM6wt (without coexpression of GnTII) showed roughly 50% of complex glycans having one truncated glycan arm ending with a mannose residue (e.g., MGnXF). Such incompletely processed structures were not detected in human- cell–derived SM6PER. Upon coexpression of the human glyco- syltransferase GnTII (23), which is responsible for elongating the truncated arm with N-acetylglucosamine residues, we could increase the amount of glycans with fully processed arms (i.e., GnGnFX; Table 1). Using ΔXT/FT plants (21) as expression hosts resulted in the production of SM6ΔXF carrying oligomannosidic N-glycans on GS4 and GS5, and human-type complex GnGn structures lack- ing plant-specific xylose and fucose residues at GS1–3. Notably, the glycosylation profiles of SM6ΔXF GS1–3 are largely homo- geneous showing GnGn as the single major N-glycan species (Table 1). This resulted in the synthesis of mono- and disialylated glycans on GS1–3 (Table 1). GS4 and GS5 were unaffected and decorated with oligomannosidic structures as in SM6wt and SM6ΔXF. One peculiarity of IgM is the distinction between complex (GS1–3) and oligomannosidic (GS4 and GS5) glyco- sylation sites. Here, we confirmed the presence of complex, mainly sialylated carbohydrates on the N-terminally located GS1–3 and oligomannosidic structures on GS4 and GS5. Notably, the glycosylation status of human-cell-line–produced SM6PER largely resembled that of serum IgM, with the main exception that the recombinant pro- tein carried hardly any bisected structures. Plant-derived SM6 variants exhibited the same site-specific distribution of N-glycans as human-serum IgM and SM6PER, mainly complex structures on GS1–3 and oligomannosidic ones on GS4 and GS5. ************* PMC4261947 PSA has a single glycosylation site at Asn69, with glycans constituting approximately 8% of the protein by weight. 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. Peptides containing the glycosylation sequon (N69KS) were not detected in the proteomics analysis, thus suggesting that the N69 residue is glycosylated. 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. One missing glycoform associated with the NKSVILLGR backbone is HexNAc3Hex3dHex1NeuAc1, which was detected on the AVCGGVLVHPQWVLTAAHCIRNK backbone. All of the N-glycans were detected on the NKSVILLGR backbone except HexNAc6Hex4dHex2NeuAc1, which was detected only on the AVCGGVLVHPQWVLTAAHCIRNK backbone. 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. The annotation of this tandem MS demonstrates that this glycopeptide is core fucosylated and monosialylated followed by either a Gal or GlcNAc residue. 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. 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. 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. 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 HexNAc4Hex5d- Hex1NeuAc2 (27%) were observed at higher intensities in the PSAH sample. Glycopeptides possessing HexNAc5- Hex5NeuAc1 (6.8%), HexNAc5Hex4dHex1NeuAc2 (6.6%), HexNAc5Hex5NeuAc2 (5.3%), and HexNAc5Hex4d- Hex1NeuAc1 (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). 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), HexNAc4Hex3d- Hex1NeuAc1 (>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), HexNAc4Hex5d- Hex1NeuAc2 (>4.8-fold), and HexNAc4Hex5dHex1NeuAc1 (>3.2-fold) were observed with higher abundances in PSAH than that in PSA. In particular, the N-glycans HexNAc3Hex4dHex1NeuAc1, HexNAc4Hex5d- Hex1NeuAc2, HexNAc5Hex3dHex1NeuAc1, and HexNAc5Hex4dHex1NeuAc1 were observed with a very high overlap in intensities. The glycopeptide possessing HexNAc3Hex4dHex1NeuAc1 (13.3%) was ob- served with the highest intensity from PSA, whereas the ones with HexNAc4Hex5dHex1NeuAc1 (28.9%) and HexNAc4Hex5dHex1NeuAc2 (27%) were predominantly present from PSAH.