Glycoproteomic Analysis of Human Fibrinogen Reveals Novel

Article pubs.acs.org/jpr

Glycoproteomic Analysis of Human Fibrinogen Reveals Novel Regions of O‑Glycosylation Gerhild Zauner,*,†,# Marcus Hoffmann,#,‡ Erdmann Rapp,‡ Carolien A. M. Koeleman,† Irina Dragan,† André M. Deelder,† Manfred Wuhrer,† and Paul J. Hensbergen† †

Department of Parasitology, Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, The Netherlands Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany

‡

S Supporting Information *

ABSTRACT: Human fibrinogen is a 340 kDa, soluble plasma glycoprotein composed of paired sets of three subunits (α, β, γ). The protein plays a crucial role in protecting the vascular network against the loss of blood after tissue injury. The beta and gamma subunits each contain one N-glycosylation site, each of which is occupied by a biantennary N-glycan. So far O-linked oligosaccharides have rarely been described. Here, we make use of tryptic- and proteinase K-generated fibrinogen glycopeptides for the detailed analysis of the protein’s O-glycosylation by combining information obtained from both one- and two-dimensional nanoLC−ESI-ion trap (IT)−MS approaches. Glycopeptides were analyzed by ion trap-MS/MS which displayed fragmentations of glycosidic linkages and some peptide backbone cleavages. MS3 spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N-glycan attachment sites of human fibrinogen could be confirmed. Moreover, we describe seven novel O-glycosylation regions in human fibrinogen, all occupied by a monosialylated T-antigen. Our findings may help to improve the general understanding of human fibrinogen in the blood clotting process. KEYWORDS: fibrinogen, O-glycosylation, glycopeptide, trypsin, proteinase K

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growth factor-2, vascular endothelial growth factor, and interleukin-1.1 Through the combination of polymerization of fibrin monomers after thrombin-mediated cleavage of the α and

INTRODUCTION Human fibrinogen is a 340 kDa, fibrous plasma protein composed of paired sets of three subunits (α, β, γ) linked together by 29 disulfide bridges.1,2 Fibrin(ogen) specifically binds a variety of other proteins, including fibronectin, albumin, thrombospondin, von Willebrand factor, fibulin, fibroblast © 2012 American Chemical Society

Received: July 2, 2012 Published: October 10, 2012 5804

dx.doi.org/10.1021/pr3005937 | J. Proteome Res. 2012, 11, 5804−5814

Journal of Proteome Research

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β subunits (releasing fibrinopeptides A and B) and the binding of fibrin(ogen) to activated αIIbβ3 integrin on the surface of platelets, it plays a central role in the formation of a hemostatic plug after tissue injury.2 Fibrinogen is one of the major glycoproteins in plasma, and normal levels in venous blood are between 1.5 and 3.5 g/L.3 There are two biantennary N-glycans on human fibrinogen: one linked to asparagine 364 on the β-chain and one linked to aparagine 52 on the γ-chain.1 The α-chain of human fibrinogen is considered to be free of glycosylation, although a variant of the α-chain, αE, which has an extended C-terminal sequence containing several N-glycosylation consensus sequences, was shown to be N-glycosylated when expressed in monkey cells (COS cells).4 Several studies have shown the importance of fibrinogen N-glycosylation in the structural and biophysical characteristics of a fibrin clot.5−7 In addition, fibrinogen is sensitive to nonenzymatic glycation, and in vitro studies have shown that highly glycated fibrin is less accessible to cleavage by plasmin resulting in poor fibrinolysis of such clots,8 which may be particularly relevant in patients suffering from diabetes.9,10 Fibrinogen O-linked oligosaccharides are less well described and only one group has reported on O-glycans on human fibrinogens detected by lectin binding studies, but this finding has hithterto not been confirmed by structural analysis.11,12 Interestingly, in a comparative urinary metabolomic study of patients suffering from urinary tract infection (UTI), Pacchiarotta et al. identified an O-glycopeptide as a C-terminal peptide of the fibrinogen α-chain carrying a disialylated T-antigen. This novel glycopeptide marker was found to be the major discriminator between UTI patients and controls.13 Interestingly, the peptide moiety originates from a 15-amino acid C-terminal region of the fibrinogen α-chain which is not present in the full length circulating isoform.14 These studies prompted us to study fibrinogen’s glycosylation in more detail. To do so, we decided to digest two batches of fibrinogen using a specific enzyme (trypsin) and a nonspecific enzyme (proteinase K) and analyze the resulting glycopeptides by 1D and 2D nanoLC− ESI-IT−MS. Unspecific proteinases have proven to allow a detailed analysis of the N- and O-glycosylation of several proteins.15−20 For instance, in the study performed by Nwosu et al. the MS analysis of a mixture of glycopeptides generated by immobilized-Pronase digestion of a cocktail of glycoproteins allowed the identification of N- and O-glycosylation sites in this mixture with extensive site heterogeneity.17 In addition to confirming the two known N-glycosylation sites substituted with biantennary N-glycans, our data provide strong evidence for the O-glycosylation of several regions of fibrinogen’s α- and β-chain by a monosialylated T-antigen. Hence, the present study provides for the first time a comprehensive analysis of the overall glycosylation of human fibrinogen.

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Safe Stain, Invitrogen). Protein bands were cut into small pieces and washed with 25 mM NH4HCO3 (Biosolve, Valkenswaard, The Netherlands) followed by two rounds of dehydration with 100% acetonitrile (ACN) for 10 min. For reduction and alkylation, gel particles were first incubated with 10 mM dithiothreitol dissolved in 25 mM NH4HCO3 for 30 min at 56 °C. Following dehydration with ACN, gel plugs were incubated in 55 mM iodoacetamide dissolved in 25 mM NH4HCO3 for 20 min at room temperature in the dark. After two rounds of washing with 25 mM NH4HCO3 and dehydration with 100% ACN, the gel particles were completely dried in a centrifugal vacuum concentrator (Eppendorf, Hamburg, Germany). Dried gel particles were reswollen for 15 min on ice by addition of 15 μL of a trypsin solution (12.5 ng/μL in 25 mM NH4HCO3, sequencing grade modified trypsin, Promega, Madison, WI). Following this, 20 μL of 25 mM NH4HCO3 was added and samples were kept on ice for additional 30 min. Tryptic digestion was subsequently performed overnight at 37 °C. Following tryptic digestion, the overlaying digestion-solution was collected. Two additional rounds of extraction with 20 μL of 0.1% TFA in ACN were used to retrieve peptides from the gel plugs and all extracts were pooled. Proteinase K Digestion

Five hundred micrograms of fibrinogen 1 and 2, both dissolved in 50 mM NH4HCO3, were reduced with 5 mM of dithiothreitol (Sigma-Aldrich) for 30 min at 60 °C after which samples were allowed to cool down to room temperature. Then, iodoacetamide (Sigma-Aldrich) dissolved in 50 mM NH4HCO3 was added to a final concentration of 15 mM, and the reaction mix was left for 30 min in the dark. The alkylation reaction was stopped by putting the sample under a fluorescent lamp (gas discharge lamp). Reduced and alkylated fibrinogen samples were subsequently treated overnight with proteinase K (from Tritirachium album; Sigma-Aldrich) at different enzyme/substrate ratios (1:30 and 1:3) in 50 mM NH4HCO3 at 37 °C. HILIC−HPLC

The proteinase K digests from the same fibrinogen batch (enzyme/substrate ratios 1:3 and 1:30) were pooled, dried and dissolved in 70% ACN. The samples were subsequently centrifuged for 1 min at 13 000 rpm in order to remove any particles from the sample solution. The resulting solutions of peptides and glycopeptides were fractionated by hydrophilic interaction liquid chromatography (HILIC)− HPLC (TSK amide 80, 3 μm, 150 mm × 4.6 mm inner diameter column; Tosoh Bioscience, Stuttgart, Germany) at a 1 mL/min flow rate. A binary gradient was applied using 100% acetonitrile (ACN; solvent A), and 50 mM ammonium formiate pH 4.4 (solvent B) from 30% solvent B (0 min) to 55% solvent B (40 min) followed by a 5 min isocratic elution prior to a step to 100% solvent B over 1 min. After 5 min at 100% solvent B, the gradient went back to 30% solvent B over 2 min (hold 10 min). With the use of this gradient system, the total analysis time per run was 60 min. Five minute fractions were collected from 3 to 48 min. Fractions were dried by vacuum centrifugation and reconstituted in 100 μL of water.

EXPERIMENTAL SECTION

Materials

Two stocks of human fibrinogen  one obtained from SigmaAldrich (Zwijndrecht, The Netherlands) which will be referred to as fibrinogen 1 and one from Calbiochem/EMD Chemicals (Gibbstown, NJ, US) referred to as fibrinogen 2  were used in this study. Water was purified using a Milli-Q system (Millipore, Billerica, MA).

NanoLC−ESI-IT−MS/MS

HILIC fractions of proteinase K digests and in-gel tryptic digests were analyzed by reversed phase nanoLC−MS on an Ultimate3000 system (Dionex/Thermo Scientific, Sunnyvale, CA) nanoESI- coupled to MS. After sample injection (20 μL of each HILIC fraction or tryptic digest) (glyco)peptides were trapped on a guard column (5 μm PepMap particles, 300 μm × 5 mm; Dionex/Thermo Scientific) followed by a 5 min wash with

SDS-PAGE and In-Gel Tryptic Digestion of Human Fibrinogen

Twenty micrograms of fibrinogen 1 and 2 were analyzed by SDSPAGE on a NuPage 4−12% gradient Bis-Tris gel (Invitrogen, Carlsbad, CA) and stained with Coomassie G-250 (SimplyBlue 5805



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tryptic digest of fibrinogen (alpha chain: P02671−1 Isoform 1 (α-E) and P02671−2 Isoform 2; beta chain: P02675; gamma chain: P02679). For the proteinase K generated glycopeptides, the list of obtained peptide masses of the samples was used for matching to theoretical masses of randomly cleaved peptide moieties of human fibrinogen using the FindPept tool (http:// www.expasy.org/tools/findpept.html). Potential peptide masses were entered into the FindPept tool and searched using the following parameters: cysteines as carbamidomethylcysteine, mass tolerance of ±0.5 Da and “no enzyme”. Peptide sequences with potential N- or O-glycosylation sites, which were indicated by this tool, were then compared to the peptide sequence information obtained from the MS3 spectra of the glycopeptides species. Registered b-ions and y-ions were assigned using Protein Prospector (http://prospector.ucsf.edu/prospector/mshome.htm).

0.1% formic acid at a flow rate of 25 μL/min. Subsequently, the guard column was switched in line with the nano column (PepMap, 3 μm; 75 μm × 150 mm; Dionex/Thermo Scientific) running at a flow rate of 300 nL/min. Solvent A was 0.1% formic acid in water, solvent B was 95% ACN. The following gradient conditions were used: t = 0 min, 0% solvent B; t = 15 min, 25% solvent B; t = 25 min, 70% solvent B; t = 30 min, 70% solvent B. In addition, the O-glycopeptide-containing HILIC fractions were subjected to carbon SPE,21 dried, taken up in 100 μL of water and subsequently analyzed by porous graphitized carbon (PGC) nanoLC−MS on an Ultimate3000 system (Dionex/ Thermo Scientific), nanoESI coupled to MS. After sample injection (10 μL of each fraction) (glyco)peptides were trapped on a guard column (5 μm Hypercarb particles, 170 μm × 10 mm; packed by Alltech Grom GmbH, Rottenburg, Germany) followed by a 5 min wash with 0.1% formic acid at a flow rate of 8 μL/min. Subsequently the guard column was switched in line with the nano column (5 μm; 75 μm × 100 mm, Alltech Grom GmbH) operated at a flow rate of 400 nL/min. Solvent A was 0.1% formic acid in water, solvent B was 95% ACN. The following gradient conditions were used: t = 0 min, 0% solvent B; t = 15 min, 25% solvent B; t = 25 min, 70% solvent B; t = 30 min, 70% solvent B. For both reversed phase (RP) and porous graphitized carbon (PGC) phase separation, the nanoLC-system was directly coupled to an HCTultra ESI-IT-MS (Bruker Daltonics, Bremen, Germany) equipped with an online nanospray source operating in the positive ion mode. For electrospray (900−1200 V), stainless steel capillaries with an inner diameter of 30 μm (from Proxeon, Odense, Denmark) were used. The solvent was evaporated at 165 °C with a nitrogen stream of 6 L/min. Ions from m/z 300−1800 were detected. With RP separation, automatic fragment ion analysis was enabled, resulting in MS/MS spectra of the eight most abundant peaks. For further identification/confirmation of glycopeptides that were identified in the MS2 mode, another LC−MS run was performed where the fragment ion corresponding to the peptide moiety was subjected to an additional ion isolation/fragmentation cycle by manual MS3. With PGC separation, fragment ion analysis in MS2 mode was performed as mentioned above, whereas an additional automatic ion isolation/fragmentation cycle of the three most abundant MS2 peaks was enabled to obtain MS3 spectra.

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RESULTS Two different fibrinogen samples were analyzed by SDS-PAGE and visible protein bands (Figure 1) were in-gel digested with trypsin and analyzed with nanoLC−ESI-IT−MS. As expected, this analysis revealed that the major proteins in these samples were fibrinogen α, β and γ chain, although some minor contaminants were also identified (Figure 1, Table S1, Supporting Information).

Data Processing

For the proteomic analysis of the data of the tryptic peptides, peak lists were generated using DataAnalysis 4.0 software (Bruker Daltonics) and exported as Mascot Generic Files (MGF). These files were searched against the human IPI database using the Mascot (version 2.2.1) search algorithm (Matrix Science, London, UK). An MS tolerance of 0.6 Da (with # 13C = 1) and an MS/MS tolerance of 0.5 Da was used. Trypsin was designated as the enzyme and one missed cleavage site was allowed. Carbamidomethylcysteine was selected as a fixed modification and oxidation of methionine as a variable modification. The data of the proteinase K and trypsin generated glycopeptides were analyzed as described previously.20 Briefly, LC−MS/MS data were screened for glycopeptides using characteristic fragment ions, e.g., of m/z 366 ([HexNAc1Hex1 + H]+). The oligosaccharide composition of the glycopeptides was deduced from the MS/MS data which allowed for the determination of the peptide moiety mass. For the tryptic glycopeptides these could directly be compared to the in-silico

Figure 1. SDS-PAGE and proteomic analysis of two batches of human fibrinogen. Left to right: marker; 20 μg of Fib 1; 20 μg of Fib 2. Protein bands were analyzed for Fib 1 (long arrows), Fib 2 (intermediate-length arrows), or both Fib 1 and Fib 2 (short arrows), and identified proteins are listed on the right. The order of the annotated proteins reflects the order of appearance in the Mascot results file and in case the data clearly showed the presence of one dominating protein (based on the total number of identified MS/MS spectra and manual inspection of the LC− MS run), this protein is indicated in bold. Boxes show the gel bands that were used for MS based glycopeptide identification of the individual fibrinogen chains.

In addition, fibrinogen α, β and γ chains were identified in multiple different bands, most of them probably related to proteolytic degradation, insufficient reduction or differential post-translational modifications. The major bands corresponding to the fibrinogen α, β 5806



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and γ chains (outlined in Figure 1) were selected for further analysis of their glycosylation. For this purpose, the MS2 spectra were screened for glycopeptide-derived oxonium fragment ions at m/z 292.10 ([NeuAc1 + H]+), m/z 366.13 ([HexNAc1Hex1 + H]+), m/z 657.23 ([HexNAc1Hex1NeuAc1 + H]+) and the N-glycan-indicative signal at m/z 528.19 ([HexNAc1Hex2 + H]+).22 First fibrinogen N-glycosylation was investigated. In accordance with the literature,1,23−26 we identified the known biantennary glycosylation on tryptic peptides covering N364 of the fibrinogen β-chain and N52 of the γ-chain, in mono-, di- and non-sialylated form from both batches of fibrinogen (Table 1, Suppl. Figure S1, Supporting Information). The elution of the differently glycosylated glycopeptides species from the nanoRP column follows a specific order: the non-sialylated glycopeptides (347GTAGNALMoxDGASQLMoxGENR365) eluted prior to the mono- and the disialylated species (elution around 27 min, 39 and 43 min, respectively, Suppl. Figure S1, Supporting Information). Our analysis showed that for both tryptic N-glycopeptides the monoand disialylated forms are the most dominant species followed by relatively small amounts of the asialylated form (see Suppl. Figure S1, Supporting Information). In contrast, Townsend et al.25,26 found equal amounts of mono- and disialylated biantennary glycans but no asialylated species on human plasma fibrinogen. We did not observe the non-glycosylated tryptic peptides in the overall sequence coverage of these two fibrinogen chains after standard database searching (Figure S2, Supporting Information). This indicates that these sites are most likely fully occupied within these major protein species. Furthermore, we also did not observe such peptides at other positions on the gel where fibrinogen β-chain and γ-chain were identified. Although fibrinogen α-chain contains a potential N-glycosylation site, this appears not to be glycosylated.1 However, we observed tryptic O-glycopeptides from fibrinogen α-chain carrying a glycan of composition HexNAc1Hex1NeuAc1 which is interpreted as a monosialylated T-antigen (Galβ1−3GalNAcα1-). This particular glycan feature has been described on other plasma glycoproteins such as apolipoprotein C III.27 As an example, an MS/MS spectrum of m/z 750.64 [M + 3H]3+, demonstrating clear consecutive losses of N-acetylneuraminic acid (at m/z 980.28), hexose (at m/z 899.27) and N-acetylhexosamine (at m/z 797.47), is shown (Figure 2). On the basis of the derived peptide mass (m/z 797.47 [M + 2H]2+ see Table 1) the glycopeptide was tentatively assigned as 494HPDEAAFFDTASTGK508, corresponding to amino acid (aa) 494-aa508 of the α-chain, modified by an O-glycan (see Figure 3 for an overall scheme of identified O-glycosylation). Potential O-glycosylation sites are the two threonines and the serine included in this sequence stretch. In addition, in both fibrinogen samples we observed several O-glycosylated peptides pointing to O-glycosylation of the region from aa529-aa580 of the α-chain (Figure 3A,B). Details about these peptides are listed in Table 1 and Suppl. Figure S3, Supporting Information. Again, all of them solely contained a sialylated T-antigen. The assignment of the peptide portion was based on the uniqueness within the in silico digest of fibrinogen α-chain and/or the relative elution with respect to the nonglycosylated counterpart (we observed a constant difference between 6 and 8 min; see Suppl. Figure S3, Supporting Information). For one of the O-glycopeptides, no non-glycosylated counterpart was observed (possibly because cleavage was hampered due to steric hindrance) but the tentative assignment is supported by the fact that it partially overlaps with one of the other tryptic O-glycosylated peptides. In contrast to the N-glycosylation of the β- and γ-chain described above, the level of O-glycosylation was substoichiometric

because we observed higher levels of the non-glycosylated species. We estimated the degree of site occupancy by comparing the relative intensity of the O-glycopeptide with that of the nonglycosylated counterpart and for the four peptides for which this was possible we found levels of approximately 1, 5, 7 and 13%, respectively (see Suppl. Figure S3, Supporting Information). This region of fibrinogen alpha chain contains a lot of serine and threonine residues, and the CID tandem mass spectra did not allow assignment of the glycosylation site. However, these results triggered us to look into the O-glycosylation of human fibrinogen in more detail pursuing another strategy that is based on use of an unspecific enzyme in combination with various (glyco)peptide separation techniques. For this purpose, both batches of fibrinogen were enzymatically cleaved by proteinase K at two different enzyme to substrate ratios (1:30 and 1:3), and the resulting glycopeptides were analyzed by nanoRP−ESI-IT− MS/MS and by nanoPGC−ESI-IT−MS/MS. An additional 2D separation (preparative HILIC−HPLC fractionation prior to nanoRP−ESI-IT−MS) on pooled samples (the two digests prepared at different enzyme to substrate ratios) was performed for further in-depth analysis. All the information on the identified glycopeptides is summarized in Table 2. With this approach we found in total 18 O-glycopeptides all of which contained a sialylated T-antigen. The identified O-glycopeptides exhibited different peptide moieties, the majority of which (13 out of 18) could be allocated to fibrinogen α-chain, while 5 originated from fibrinogen β-chain. An overview of our findings is presented in Figure 3. As an example, we observed the O-glycopeptide 2+ 505STGKTFPG512 (m/z 725.83 [M + 2H] ) of the α-chain 3 (Figure 4A). The MS spectrum of the ion corresponding to the peptide moiety (m/z 794.39 [M + H]+) clearly demonstrates the identity of this peptide (Figure 4B). This proteinase K generated O-glycopeptide matches with one of the tryptic O-glycopeptides identified in the SDS-PAGE band of the α-chain described above (494HPDEAAFFDTASTGK508) and collectively indicates that either S505 or T506 is glycosylated. In addition to this, three series of O-glycopeptides were identified in close vicinity to each other corresponding to the middle region of the α-chain (between Gly-259 and Ser-271; Asn-290 and Gly-305; Thr-325 and Gly-337, respectively). All of the possible O-glycosylation sites were covered by more than one peptide. The first series of glycopeptides exhibited peptide moieties differing slightly in length with the shared peptide portion 262SETESPRNPS271 (Figure 3B) which contains four potential O-glycosylation sites. The MS/MS analysis of the glycopeptide 2+ 262SETESPRNPS271 at m/z 880.34 [M+2H] clearly showed the fragmentation of the oligosaccharide moiety, resulting in a major signal of the intact peptide at m/z 1103.58 [M+H]+ (Figure 4C). In addition a few peptide backbone cleavages were apparent. The MS3 spectrum of the peptide moiety allowed the unambiguous assignment of this peptide (Figure 4D). The other two glycopeptides of this series exhibited the same C-terminus (Ser271) and consequently shared a series of y-ions in their respective MS3 spectra (y4, y5, y6 and y8; Table 2). The O-glycopeptides corresponding to Asn290-Gly305 and Thr301Gly305 formed a second series which share the sequence 301TWKPG305, indicating Thr301 to be the O-glycosylated amino acid. The fragmentation of m/z 622.39 [M + 2H]2+ clearly demonstrated the fragmentation of the sialylated T antigen and the subsequent MS3 of the peptide moiety (m/z 588.29 [M + H]+) unambiguously identified the peptide moiety (Figure 4E,F). 5807



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Table 1. Information Gathered from MS Analysis of Identified Fibrinogen Tryptic N- and O-Glycopeptidesa

a N-Glycosylation sites and (potential) O-glycosylation sites are marked in bold and underlined. N, N-Acetylhexosamine; H, hexose; S, N-acetylneuraminic acid; Mox, oxidized methionine, Fib 1, batch 1 of fibrinogen; Fib 2, batch 2 of fibrinogen.

The third series of O-glycopeptides corresponding to the middle region of the α-chain have the sequence stretch 328QNPGSPRPG337 in common. This sequence motif indicates that Ser332 is O-glycosylated. Notably, some peptides from this

series showed some deamidation of either a glutamine or asparagine residue. The fragmentation of the O-glycosylated Thr325-Gly337 peptide at m/z 919.39 [M + 2H]2+ in combination with the MS3 spectrum of the peptide moiety clearly reveals the 5808



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Figure 2. Fragment ion spectrum of a tryptic O-glycopeptide from human fibrinogen. The protonated precursor (inset) and the MS2 spectrum are shown. The glycopeptide precursor at m/z 750.64 [M + 3H]3+ was assigned to peptide H494−K508; of the fibrinogen α-chain carrying the O-linked oligosaccharide Hex1HexNAc1NeuAc1. Yellow square, N-Acetylgalactosamine; yellow circle, galactose; purple diamond, N-Acetylneuraminic acid.

full glycan composition was elucidated due to the use of exoglycosidases prior to mass spectrometric analysis, resulting only in the identification of the residual GalNAc residue. The only evidence for O-glycosylation of human fibrinogen originates from a recent comparative metabolomics analysis of urines from patients suffering from urinary tract infection (UTI) versus controls.13 One of the major classifiers between UTI patients and controls was identified as a peptide from human fibrinogen α-chain (619GKPSLSP625) carrying a glycan structure most probably corresponding to a disialylated T-antigen. Worthwhile noticing, this peptide is part of the C-terminal region of the α-chain (611RGIHTSPLGKPSLSP625) that is not present in the α-chain of circulating mature fibrinogen and is putatively cleaved off prior to or during secretion.14 Accordingly, the corresponding tryptic peptide was not identified in our proteomic analysis (see Figure S1, Supporting Information). Moreover, in the current study, we have only identified O-glycan structures corresponding to a monosialylated, instead of a disialylated, T-antigen on fibrinogen α-and β-chain. Similar to other major serum glycoproteins, fibrinogen is primarily synthesized in the liver, although extra-hepatic synthesis has been described.1 It could be that the difference in O-glycan structure observed on the urinary endogenous peptide versus the plasma-derived fibrinogen described in this paper, reflects tissue specific differences in glycosylation. Alternatively, it might be related to site specific differences in glycosylation, possibly reflecting different accessibility of protein regions to the glycosylation machinery. Interestingly, the C-terminus of another major serum glycoprotein, apolipoprotein C−III harbors an O-glycosylation site occupied both by mono and disialylated T-antigens.27 In this comprehensive study, we have identified seven novel O-glycosylated regions within the fibrinogen α- and β-chains, while no O-glycosylation was observed on the γ-chain. Two glycosylation sites could be inferred from the presence of a single threonine or serine residue within the identified glycopeptide (Thr301 and Ser332 in fibrinogen α-chain). In three other cases the glycosylation site could be “restricted” to a set of candidate residues (Ser262, Ser266, Thr264 or Ser271 in fibrinogen α-chain, Ser505 or Thr506 in fibrinogen α-chain, and Ser28 or Ser37 in

nature of this glycopeptide (Figure 4G,H). Among others, the prominent y7-ion was apparent within the MS3 spectra of all seven glycopeptides covering this region (Table 2). Interestingly, in addition to O-glycopeptides of fibrinogen α-chain, the proteinase K digest also revealed O-glycosylation of the β-chain (Figure 3A,B). A set of five glycopeptides was identified which shared the peptide moiety 25EAPSLRPAPPPIS37. The MS2 and MS3 spectra for the O-glycopeptide at m/z 994.49 [M + 2H]2+ and the derived peptide moiety Glu25-Ser37 at m/z 1331.80 ([M + 2H]2+) are shown in Figure 4, panels I and J, respectively. Of note, two of the peptides covering this region contained a hydroxyproline (P*; Table 2) which has been described previously.28

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DISCUSSION Fibrinogen is one of the major glycoproteins in human plasma and forms an essential element within the human blood clotting system following tissue injury. As such, it has been intensively studied but a comprehensive, in-depth glycoproteomic analysis of human fibrinogen has so far not been executed. In this manuscript we have used a combination of different enzymatic digestion strategies (tryptic and proteinase K) in combination with 1D and 2D LC−ESI-multistage-ion trap−MS to study human fibrinogen glycosylation. In addition to the already wellknown N-glycosylation of fibrinogen’s β- and γ-chain,25,26 we describe for the first time O-glycans on several stretches of the α- and β-chain. The glycan composition (Hex1HexNAc1NeuAc1 (H1N1S1)) most probably corresponds to a monosialylated T-antigen which is known to occur on plasma glycoproteins.27 To our knowledge, O-glycans on fibrinogen have only scarcely been studied. The first indications for fibrinogen O-glycosylation came from reports using fibrinogen in lectin binding studies.11 In addition, Darula et al. identified O-glycosylated fibrinogen peptides in bovine serum using a combination of lectin affinity chromatography and mass spectrometry.29 One site was identified as Thr4 of the bovine fibrinogen β-chain, a site not conserved in human fibrinogen. The other two sites were identified in bovine fibrinogen α-chain (Thr-464 and 525).30 For none of these sites the 5809



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Figure 3. Scheme of a fibrinogen heterotrimer indicating the identified O-glycosylation regions found via MS analysis of tryptic and proteinase K generated O-glycopeptides. (A) Above: The three fibrinogen polypeptide chains are schematically represented, and the O-glycosylation regions on the α-chain (green box and red box) and the β-chain (blue box) are indicated. Black lines mark interchain disulfide bonds. Below: The in total seven O-glycosylation regions identified are shown; Green box (α-chain): three regions identified via Proteinase K generated glycopeptides indicated in green. Red box (α-chain): one O-glycosylation site identified via a Proteinase K generated glycopeptide, shown in red. Two additional O-glycosylation regions found via tryptic peptides boxed in black. Blue box (β-chain): one O-glycosylation region identified via a Proteinase K generated glycopeptide, shown in blue. (B) The three O-glycosylation regions of the α-chain (green box and red box) and the β-chain (blue box) were revealed by the proteinase K generated glycopeptides (colored). Tryptic glycopeptides are boxed in black. Possible O-glycosylation sites are indicated in bold. See Figure 4 and Table 1 for details on the identified glycopeptides. P*, hydroxyproline; Q(-17), pyroglutamate.

fibrinogen β-chain) while in the last two regions a multitude of serine and threonine residues were present within the identified peptides. The overall O-glycosylation profile that emerged from our studies could only be achieved by the use of a combination of

different strategies for sample preparation, digestion and analytical workflow. Only the O-glycosylation at Ser505/Thr506 was observed after both tryptic and proteinase K digestion, while the other sites were only observed with one of the two procedures, demonstrating the benefit of applying different 5810



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Table 2. Information Gathered via MS Analysis of Identified Proteinase K Generated Fibrinogen O-Glycopeptidesa

N, N-Acetylhexosamine; H, hexose; S, N-Acetylneuraminic acid; Q(−17), pyroglutamate; Fib 1, batch 1 of fibrinogen; Fib 2, batch 2 of fibrinogen; 2D, 2 dimensional separation; RP of Fr23‑28 min RP LC−MS2 and MS3 analysis of HILIC glycopeptide fraction 23 to 28 min, etc. a

analytical approaches in general and different proteinases in particular.

One aspect that greatly improved the identification of O-glycopeptides was the use of HILIC−HPLC fractionation 5811



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Figure 4. Fragment ion spectra of proteinase K-generated glycopeptides from human fibrinogen. For each of the four different O-glycosylation regions a fragment ion spectrum (left panel; MS2) of the protonated precursor (inset) and a corresponding MS3 spectrum (right panel) are shown. All glycopeptides identified carry the same O-linked oligosaccharide species (Hex1HexNAc1NeuAc1). (A) MS2 of the glycopeptide S505-G512; precursor at m/z 725.83 [M + 2H]2+; (B) the fragment ion at m/z 794.39 [M + H]+ was subjected to MS3. (C) MS2 of the glycopeptide S262−S271; precursor at m/z 880.34 [M + 2H]2+; (D) the fragment ion at m/z 1103.58 [M + H]+ was subjected to MS3. (E) MS2 of glycopeptide T301-G305; precursor at m/z 622.85 [M + 2H]2+; (F) the fragment ion at m/z 791.37 [M + H]+ was subjected to MS3. (G) MS2 of glycopeptide T325-G337; precursor at m/z 919.39 [M + 2H]2+; (H) the fragment ion at m/z 1181.66 [M + H]+ was subjected to MS3. (I) MS2 of glycopeptide E25-S37; precursor at m/z 994.49 [M + 2H]2+; (J) the fragment ion at m/z 1331.80 [M + H]+ was subjected to MS3. Glycan compositions are given in terms of hexose (Hex), N-Acetylhexosamine (HexNAc) and N-Acetylneuraminic acid (NeuAc). Yellow square, N-Acetylgalactosamine; yellow circle, galactose; purple diamond, N-Acetylneuraminic acid. All spectra shown were from α-chain derived glycopeptides, except Figure 4I and 4J which were β-chain derived. 5812



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where we have identified O-glycosylation are known targets for these proteases.28,35−37 Because of steric hindrance, glycan structures may impair these processes, yet further research would be needed to substantiate this hypothesis. In conclusion, we have performed an in-depth glycoproteomic analysis of human fibrinogen using a combination of different sample preparation, chromatographic and mass spectrometric methods. In addition to the previously reported N-glycans, we have identified novel O-glycosylation sites and regions in the αand β-chain of fibrinogen. We hope that our findings will help to improve the general understanding of the role of human fibrinogen species and isoforms in the blood clotting process.

prior to nanoRP−ESI-IT−MS. Most of the identified glycopeptides (12 out of 18) could be measured and clearly identified using this two-dimensional approach while a 1D approach (RP or PGCMS analysis) resulted in the identification of six O-glycopeptides. This might partly be related to the purity of the glycopeptide fractions using a two-dimensional separation because the N-glycopeptides were nicely separated from the O-glycopeptides using this approach. Moreover, the Proteinase K-generated glycopeptides were found to be hardly contaminated with non-glycosylated peptides. This may be explained in two ways: (1) Nonglycosylated peptide stretches are usually cleaved to the level of single amino acids and will therefore not be detected during the MS analysis of the Proteinase K-generated glycopeptides,20 and (2) non-glycosylated peptides will be separated by HILIC and will not appear in glycopeptide containing fractions that were analyzed in this study. In contrast to the observed N-glycosylation, O-glycosylation on human fibrinogen is substoichiometric. A rough estimate of the level of glycosylation, using intensities of the nonglycosylated tryptic peptides vs the glycosylated species, revealed that, depending on the site, only between 1 and 13% was glycosylated. This may also have resulted in the lack of detection of some of the low abundant O-glycopeptides due to ion suppression or undersampling effects in some of the LC−MS measurements. Likewise, the low intensity of the O-glycopeptides also hampered our efforts to perform ETD experiments to reveal more information on the exact glycosylation sites within the different clusters. One technical aspect noticed within this study is that also O-glycopeptides undergo a hexose rearrangement during fragmentation. It has been reported previously that upon fragmentation of 2AB-labeled milk sugars and O-glycans, individual fucose residues migrate in direction of the labeled end.31 In the case of N-glycans both hexoses and fucoses have been found to migrate within the oligosaccharide or glycopeptide upon fragmentation.32,33 In a very recent study by Halim et al. similar observations were made: During CID-MS2 of urinary tryptic O-glycopeptides, low-intensity fragment ions corresponding to the mass of peptide + hexose were observed suggesting a hexose residue as the internal peptide linked monosaccharide. The authors speculate that these weak peptide + hexose fragment ions generated upon CID fragmentation of protonated O-linked glycopeptides are most likely caused by hexose migrations similar to those observed for N-glycopeptides.34 In the CID-MS2 spectrum in Figure 4C the fragmentation of m/z 880.34 [M + 2H]2+ is shown, and the peak at m/z 1265.61 [M + H]+ indicates a rearrangement product, namely, the peptide moiety with a hexose residue (galactose) attached. This rearrangement may be interpreted as the loss of an internal HexNAc residue (GalNAc) showing resemblance to the rearrangements described upon fragmentation of O-glycopeptides as described by Halim et al.34 Interestingly, our data indicate a transfer of the Gal residue to the peptide moiety upon fragmentation. In contrast, in the case of N-glycopeptide rearrangement it is not clear whether the hexose (mannose) is transferred to the remaining GlcNAc residue or to the peptide moiety.33 Fibrinogen’s N-glycosylation has been shown to be important for the structural and biophysical characteristics of a fibrin clot.8 At present, we can only speculate on the role of O-glycosylation for fibrinogen’s activity or stability. It might be that O-glycosylation characterizes a fraction of fibrinogen involved in a specific function. Fibrin(ogen) is sensitive to a plethora of proteolytic enzymes important in its activation and degradation and several of the regions

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S Supporting Information *

This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Leiden University Medical Center Biomolecular Mass Spectrometry Unit Department of Parasitology Postbus 9600, 2300 RC Leiden, The Netherlands. Tel: +31-71-5268701. Fax: +31-71-5266907. E-mail: [email protected]. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.Z. acknowledges financial support by the Horizon Breakthrough Project funded by The Netherlands Genomic Initiative (Project number: 93518016). We thank Dr. Jos Grimbergen for fruitful discussions.

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REFERENCES

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Glycoproteomic Analysis of Human Fibrinogen Reveals Novel

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