Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins
2015; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês
10.1074/mcp.m115.053546
ISSN1535-9484
AutoresMarcus Hoffmann, Kristina Marx, Udo Reichl, Manfred Wuhrer, Erdmann Rapp,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoSite-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270. Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270. Human blood plasma harbors arguably the most complex yet also the most informative proteome present in the human body (1.Anderson N.L. The Human Plasma Proteome: History, Character, and Diagnostic Prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3559) Google Scholar). 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Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (916) Google Scholar). To better understand these protective and regulatory capabilities and to move the mucin-type O-glycoproteome from form to function comprehensive site-specific O-glycosylation analyses are required. One of the main obstacles in site-specific mucin-type O-glycosylation analyses relates to the lack of a predictable O-glycan consensus-motif within the peptide backbone as it can be found for N-glycans (28.Jensen P.H. Kolarich D. Packer N.H. Mucin-type O-glycosylation–putting the pieces together.FEBS J. 2010; 277: 81-94Crossref PubMed Scopus (184) Google Scholar). The initial attachment of the N-acetylgalactosamine monosaccharide to the hydroxyl group of either serine or threonine, but also to tyrosine or hydroxylysine, is governed by a family of 20 distinct polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with different but partially overlapping peptide specificities and tissue expression patterns. This dynamic regulation, in turn, contributes to the complexity of the mucin-type O-glycoproteome. However, previous studies could show that mucin-type O-glycans are primarily attached to serine or threonine in regions with a high content of serine, threonine and proline (Ser/Thr-X-X-Pro, Ser/Thr-P and Pro-Ser/Thr) (29.Strous G.J. Dekker J. Mucin-type glycoproteins.Crit. Rev. Biochem. Mol. Biol. 1992; 27: 57-92Crossref PubMed Scopus (770) Google Scholar, 30.Halim A. Ruetschi U. Larson G. Nilsson J. LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.J. 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Mass spectrometry has proven to be the core technique in site-specific N- and O-glycosylation analyses. A generic O-glycoproteomic workflow usually starts with the isolation, enrichment or prefractionation of a single glycoprotein or a group of glycoproteins. In subsequent steps, (glyco)peptides are generated by proteolytic digestion primarily using specific proteases like trypsin. Apart from this, also broad- and nonspecific proteases like Proteinase K or Pronase E were successfully employed in recent years (32.Zauner G. Koeleman C.A. Deelder A.M. Wuhrer M. Protein glycosylation analysis by HILIC-LC-MS of Proteinase K-generated N- and O-glycopeptides.J. Sep. Sci. 2010; 33: 903-910Crossref PubMed Scopus (87) Google Scholar, 33.Nwosu C.C. Seipert R.R. Strum J.S. Hua S.S. An H.J. Zivkovic A.M. German B.J. Lebrilla C.B. Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.J. 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O-glycoproteomics: Site-specific O-glycoprotein analysis by CID/ETD electrospray ionization tandem mass spectrometry and top-down glycoprotein Sequencing by In-Source Decay MALDI Mass Spectrometry.Methods Mol. Biol. 2012; : 179-189Crossref PubMed Scopus (15) Google Scholar, 38.Alley Jr., W.R. Mechref Y. Novotny M.V. Characterization of glycopeptides by combining collision-induced dissociation and electron-transfer dissociation mass spectrometry data.Rapid Commun. Mass Spectrom. 2009; 23: 161-170Crossref PubMed Scopus (128) Google Scholar), and high resolution orbital mass analyzers, have paved the way for the mapping of thousands of occupied N- and O-glycosylation sites as recently shown (17.Zielinska D.F. Gnad F. Wisniewski J.R. Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.Cell. 2010; 141: 897-907Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 27.Steentoft C. Vakhrushev S.Y. Joshi H.J. Kong Y. Vester-Christensen M.B. Schjoldager K.T. Lavrsen K. Dabelsteen S. Pedersen N.B. Marcos-Silva L. Gupta R. Bennett E.P. Mandel U. Brunak S. Wandall H.H. Levery S.B. Clausen H. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.EMBO J. 2013; 32: 1478-1488Crossref PubMed Scopus (916) Google Scholar). Combined workflows using ETD/ECD fragmentation along with (multistage, MSn) fragmentation with high- and/or low collisional induced dissociation energy (HCD/CID) can provide compositional (structural) information on the glycan moiety as well as information on the peptide sequence and the glycosylation site (39.Saba J. Dutta S. Hemenway E. Viner R. Increasing the Productivity of Glycopeptides Analysis by Using Higher-Energy Collision Dissociation-Accurate Mass-Product-Dependent Electron Transfer Dissociation.Int. J. Proteomics. 2012; 2012: 7Crossref Google Scholar, 40.Singh C. Zampronio C.G. Creese A.J. Cooper H.J. Higher energy collision dissociation (HCD) product ion-triggered electron transfer dissociation (ETD) mass spectrometry for the analysis of N-linked glycoproteins.J. Proteome Res. 2012; 11: 4517-4525Crossref PubMed Scopus (129) Google Scholar). Recent advances in mass spectrometry driven O-glycoproteomics have been reviewed in detail elsewhere (41.Thaysen-Andersen M. Packer N.H. Advances in LC-MS/MS-based glycoproteomics: Getting closer to system-wide site-specific mapping of the N- and O-glycoproteome.Biochim. Biophys. Acta. 2014; 1844: 1437-1452Crossref PubMed Scopus (170) Google Scholar, 42.Levery S.B. Steentoft C. Halim A. Narimatsu Y. Clausen H. Vakhrushev S.Y. Advances in mass spectrometry driven O-glycoproteomics.Biochim. Biophys. Acta. 2014; 1850: 33-42Crossref PubMed Scopus (94) Google Scholar). Owing to the amount and complexity of O-glycoproteomic data a number of bioinformatic software tools for the prediction of mucin-type O-glycosylation sites (27.Steentoft C. 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Moreover, reporting guidelines for collecting, sharing, integrating, and interpreting mass spectrometry based glycomics data have been specified by the MIRAGE consortium (minimum information required for a glycomics experiment) (45.Kolarich D. Rapp E. Struwe W.B. Haslam S.M. Zaia J. McBride R. Agravat S. Campbell M.P. Kato M. Ranzinger R. Kettner C. York W.S. The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting mass-spectrometry-based glycoanalytic data.Mol. Cell. Proteomics. 2013; 12: 991-995Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 46.York W.S. Agravat S. Aoki-Kinoshita K.F. McBride R. Campbell M.P. Costello C.E. Dell A. Feizi T. Haslam S.M. Karlsson N. Khoo K.H. Kolarich D. Liu Y. Novotny M. Packer N.H. Paulson J.C. Rapp E. Ranzinger R. Rudd P.M. Smith D.F. Struwe W.B. Tiemeyer M. Wells L. Zaia J. Kettner C. MIRAGE: the minimum information required for a glycomics experiment.Glycobiology. 2014; 24: 402-406Crossref PubMed Scopus (85) Google Scholar). liquid chromatography Electron-capture dissociation Electron-transfer dissociation Fourier transform ion cyclotron resonance Hydrophilic interaction liquid chromatography Iodoacetamide Ion charge control Linear trap quadrupole. The aim of our study was to develop a glycoproteomic workflow that allows the explorative nontargeted analysis of O-glycosylated human blood plasma proteins, which are known to carry mainly short mono- and disialylated mucin-type core-1 and -2 O-glycans. To achieve this, we have combined O-glycopeptide selective offline-HILIC fractionation of Proteinase K digested peptides with nano-reversed-phase liquid chromatography coupled online to multistage ion-trap mass spectrometry (nanoRP-LC-ESI-IT-MS: CID-MS2/-MS3, ETD-MS2). The workflow has been applied to investigate the mucin-type O-glycoproteome of a pooled blood plasma sample derived from 20 healthy donors. Based on the mass spectrometric analysis of intact O-glycopeptides, we were able to characterize the O-glycosylation (i.e. peptide, site, and attached O-glycans) of a number of major human blood glycoproteins, including many acute phase proteins such as fibrinogen and plasminogen. Overall, the site-specific glycosylation analysis of human blood plasma glycopeptides revealed exclusively mono- and disialylated core-1 mucin-type O-glycopeptides. Interestingly, also a few novel O-glycosylation sites could be identified. All chemicals and solvents were of the highest purity available. Purified water used for sample preparation and HILIC fractionation was freshly prepared using a Milli-Q water purification system (referred to as "Milli-Q water", 18.2 mΩ × cm−1 at 25 °C, Total Organic Carbon 3 ppb; Merck Millipore, Darmstadt, Germany). For preparation of LC-MS solvents ultrapure water was used, which was freshly prepared using the same system but equipped with an additional filter (referred to as "Milli-Q water MS"; LC-Pak Polisher, Merck Millipore). Human blood plasma (pooled sample, derived from 20 healthy donors) was purchased from Affinity Biologicals Inc. (VisuCon-F, Frozen Normal Control Blood, FRNCP0125; Ancaster, ON, Canada). To 25 μl of the sample (about 2 mg protein) 25 μl 100 mm ammonium bicarbonate(aq) (NH4HCO3, pH 8.0) (Sigma Aldrich, Steinheim, Germany) was added to obtain a final concentration of 50 mm NH4HCO3(aq) (pH 8.0). Disulfide bonds were reduced by addition of 6.25 μl 100 mm 1,4-dithiothreitol (DTT; Sigma Aldrich) dissolved in 50 mm NH4HCO3(aq) (pH 8.0), to a final concentration of 10 mm DTT. The sample was incubated for 45 min at 60 °C, and subsequently allowed to cool down to room temperature. Cystein alkylation was achieved by addition of 12.5 μl 100 mm iodoacetamide (IAA; Sigma Aldrich) dissolved in 50 mm NH4HCO3(aq) (pH 8.0), to a final concentration of 16.67 mm IAA. The sample was incubated at room temperature for 20 min under light exclusion. The alkylation reaction was quenched by addition of 2.5 μl 100 mm DTT dissolved in 50 mm NH4HCO3(aq) (pH 8.0), followed by addition of 3.75 μl 50 mm NH4HCO3(aq) (pH 8.0), before placing the sample under a fluorescent lamp (gas-discharge lamp) for 15 min to decompose the light-sensitive IAA. By adding 169 μl 50 mm NH4HCO3(aq) (pH 8.0) the sample was brought to a final volume of 250 μl. Proteolytic digestion was achieved by addition of Proteinase K (Sigma Aldrich), a serine protease with a broad specificity that cleaves primarily after aliphatic, aromatic and hydrophobic amino acids. The pooled blood plasma sample (about 2 mg protein in 250 μl buffer) was supplemented with 66 μg Proteinase K dissolved in 122 μl 50 mm NH4HCO3(aq) (pH 8.0) in order to obtain a final enzyme/protein ratio of 1:30 (w/w, 0.033 mg enzyme per mg protein). The sample was incubated for 16 h at 37 °C with gentle agitation (200 rpm). For post-digestion cleanup the sample was precipitated using acetonitrile (ACN; Sigma Aldrich). To this end four volumes of ACN were added and the sample was centrifuged for 10 min at 2880 × g (Centrifuge 5804 R; Eppendorf, Hamburg, Germany). The supernatant was transferred and dried by vacuum centrifugation (RVC 2–33 CDplus, ALPHA 2–4 LDplus; Martin Christ GmbH, Osterode am Harz, Germany). The dried Proteinase K digest was resuspended in 500 μl 80% ACN in 50 mm NH4HCO3(aq) (v/v, pH 8.0) and subsequently centrifuged for 10 min at 20,238 × g to remove any particles (Centrifuge 5424; Eppendorf). The supernatant, containing about 2 mg peptides and glycopeptides, was subjected to HILIC-HPLC (UltiMate™ Nano HPLC-System: Thermo Scientific/Dionex, Dreieich, Germany; HILIC Column: ACQUITY UPLC BEH HILIC Column, 130Å, 1.7 μm, 2.1 mm X 100 mm; Waters, Manchester, UK) for fractionation and glycopeptide enrichment. The HPLC system was operated using a binary gradient of 100% ACN (v/v; solvent A) and 50 mm ammonium formate(aq) (NH4FA, pH 4.4; solvent B, Sigma Aldrich). After sample injection (500 μl) 20% solvent B was applied isocratically for 5 min, followed by a linear gradient to 50% solvent B within 25 min, both using a constant flow rate of 250 μl/min. Subsequently, a linear gradient went to 90% solvent B within 1 min, while reducing the flow rate to 150 μl/min. To wash the column solvent B was kept at 90% for 9 min. Column re-equilibration was achieved by isocratic elution with 20% solvent B for 20 min; (the flow rate was increased to 250 μl/min after 10 min). During the separation the column temperature was kept constant at 40 °C. The elution profile was monitored by UV absorption at 214 nm. Fractions were collected every 2 mins from 0 min to 34 min. The fractions were dried by vacuum centrifugation and reconstituted in 50 μl Milli-Q water. HILIC fractions were analyzed by reversed-phase nano-LC-MSn using an Ultimate3000 nanoHPLC system (Thermo Scientific/Dionex) coupled online to an ion trap mass spectrometer (AmaZon ETD, Bruker Daltonics, Bremen, Germany). Within the first 2 mins after sample injection, (glyco)peptides were loaded isocratically on a C18 μ-precolumn (Acclaim PepMap100, C18, 5 μm, 100 Å, 300 μm i.d. × 5 mm; Thermo Scientific/Dionex). During this pre-concentration and desalting step, "loading pump solvent 1" (98% Milli-Q water MS, 2% ACN, 0.05% trifluoroacetic acid (Sigma Aldrich)) was u
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