Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry
2009; Elsevier BV; Volume: 8; Issue: 9 Linguagem: Inglês
10.1074/mcp.m900088-mcp200
ISSN1535-9484
AutoresWen Ding, Harald Nothaft, Christine M. Szymanski, John F. Kelly,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoGlycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics. Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics. Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1Jaeken J. Matthijs G. Congenital disorders of glycosylation.Annu. Rev. Genomics Hum. Genet. 2001; 2: 129-151Crossref PubMed Scopus (162) Google Scholar, 2Arnold J.N. Wormald M.R. Sim R.B. Rudd P.M. Dwek R.A. 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A novel approach for identification and characterization of glycoproteins using a hybrid linear ion trap/FT-ICR mass spectrometer.J. Am. Soc. Mass Spectrom. 2006; 17: 168-179Crossref PubMed Scopus (58) Google Scholar). Both N- and O-glycans are structurally heterogeneous (i.e. a single site may have different glycans attached or be only partially occupied). Therefore, the MS 1The abbreviations used are:MSmass spectrometryIPRion-pairing reagentsIP-NPLCion-pairing normal-phase liquid chromatographyRTretention timewtwild-typeTFAtrifluoroacetic acetic acidBPCbase peak chromatogramsUDPuridine diphosphateI.D.internal diameter. signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11Dell A. Morris H.R. 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However, when peptides and glycopeptides co-elute, parent ion scanning is required to selectively detect the glycopeptides (14Carr S.A. Huddleston M.J. Bean M.F. Selective identification and differentiation of N-and O-linked oligosaccharides in proteins by liquid chromatography-mass spectrometry.Protein Sci. 1993; 2: 183-196Crossref PubMed Scopus (303) Google Scholar). This can be problematic in terms of sensitivity, especially for detecting glycopeptides in digests of complex protein extracts. mass spectrometry ion-pairing reagents ion-pairing normal-phase liquid chromatography retention time wild-type trifluoroacetic acetic acid base peak chromatograms uridine diphosphate internal diameter. Isolation of glycopeptides from proteolytic digests of complex protein mixtures can greatly enhance the MS signals of glycopeptides using reversed-phase LC-ESI-MS (RPLC-ESI-MS) or MALDI-MS (15Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1275) Google Scholar, 16Sun B. Ranish J.A. Utleg A.G. White J.T. Yan X. Lin B. Hood L. Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics.Mol. Cell. Proteomics. 2007; 6: 141-149Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 17Hill J.J. Moreno M.J. Lam J.C. Haqqani A.S. Kelly J.F. Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.Proteomics. 2009; 9: 535-549Crossref PubMed Scopus (25) Google Scholar, 18Ghosh D. Krokhin O. Antonovici M. Ens W. Standing K.G. Beavis R.C. Wilkins J.A. Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins.J. 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Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry.Mol. Cell. Proteomics. 2005; 4: 107-119Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 23Hägglund P. Bunkenborg J. Elortza F. Jensen O.N. Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.J. Proteome Res. 2004; 3: 556-566Crossref PubMed Scopus (410) Google Scholar, 24Hagglund P. Matthiesen R. Elortza F. Højrup P. Roepstorff P. Jessen O.N. Bunkenborg J. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins.J. Proteome Res. 2007; 6: 3021-3031Crossref PubMed Scopus (104) Google Scholar). Hydrazide chemistry is used to isolate, identify, and quantify N-linked glycopeptides effectively, but this method involves lengthy chemical procedures and does not preserve the glycan moieties thereby losing valuable information on glycan structure and site occupancy (15Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1275) Google Scholar, 16Sun B. Ranish J.A. Utleg A.G. White J.T. Yan X. Lin B. Hood L. Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics.Mol. Cell. Proteomics. 2007; 6: 141-149Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 17Hill J.J. Moreno M.J. Lam J.C. Haqqani A.S. Kelly J.F. Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.Proteomics. 2009; 9: 535-549Crossref PubMed Scopus (25) Google Scholar). Capturing glycopeptides with lectins has been widely used, but restricted specificities and unspecific binding are major drawbacks of this method (18Ghosh D. Krokhin O. Antonovici M. Ens W. Standing K.G. Beavis R.C. Wilkins J.A. Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins.J. Proteome Res. 2004; 3: 841-850Crossref PubMed Scopus (87) Google Scholar, 19Zhao J. Simeone D.M. Heidt D. Anderson M.A. Lubman D.M. Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis: Application to pancreatic cancer serum.J. Proteome Res. 2006; 5: 1792-1802Crossref PubMed Scopus (199) Google Scholar, 20Durham M. Regnier F.E. Targeted glycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome.J. Chromatogr. A. 2006; 1132: 165-173Crossref PubMed Scopus (88) Google Scholar, 21Yang Z. Hancock W.S. Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography.J. Chromatogr. A. 2005; 1070: 57-64Crossref PubMed Scopus (95) Google Scholar). Under reversed-phase LC conditions, glycopeptides from tryptic digests of gel-separated glycoproteins have been enriched using graphite powder medium (22Larsen M.R. Højrup P. Roepstorff P. Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry.Mol. Cell. Proteomics. 2005; 4: 107-119Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In this case, however, a second digestion with proteinase K is required for trimming down the peptide moieties of tryptic glycopeptides so that the glycopeptides (typically <5 amino acid residues) essentially resemble the glycans with respect to hydrophilicity for subsequent separation. Moreover, the short peptide sequences of the proteinase K digest are often inadequate for de novo sequencing of the glycopeptides. Glycopeptide enrichment under normal-phase LC (NPLC) conditions has been demonstrated using various hydrophilic media and different capture and elution conditions (23Hägglund P. Bunkenborg J. Elortza F. Jensen O.N. Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.J. Proteome Res. 2004; 3: 556-566Crossref PubMed Scopus (410) Google Scholar, 24Hagglund P. Matthiesen R. Elortza F. Højrup P. Roepstorff P. Jessen O.N. Bunkenborg J. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins.J. Proteome Res. 2007; 6: 3021-3031Crossref PubMed Scopus (104) Google Scholar, 25Thaysen-Andersen M. Thøgersen I.B. Nielsen H.J. Lademann U. Brünner N. Enghild J.J. Højrup P. Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1.Mol. Cell. Proteomics. 2007; 6: 638-647Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 26Thaysen-Andersen M. Hojrup P. Enrichment and characterization of glycopeptides from gel-separated glycoproteins.Am. Biotechnol. Lab. 2006; 24: 14-17Google Scholar, 27Larsen M.R. Jensen S.S. Jakobsen L.A. Heegaard N.H. Exploring the sialiome using titanium dioxide chromatography and mass spectrometry.Mol. Cell. Proteomics. 2007; 6: 1778-1787Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 28Wada Y. Tajiri M. Yoshida S. Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics.Anal. Chem. 2004; 76: 6560-6565Crossref PubMed Scopus (287) Google Scholar). NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZIC®-HILIC column (23Hägglund P. Bunkenborg J. Elortza F. Jensen O.N. Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.J. Proteome Res. 2004; 3: 556-566Crossref PubMed Scopus (410) Google Scholar, 24Hagglund P. Matthiesen R. Elortza F. Højrup P. Roepstorff P. Jessen O.N. Bunkenborg J. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins.J. Proteome Res. 2007; 6: 3021-3031Crossref PubMed Scopus (104) Google Scholar, 25Thaysen-Andersen M. Thøgersen I.B. Nielsen H.J. Lademann U. Brünner N. Enghild J.J. Højrup P. Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1.Mol. Cell. Proteomics. 2007; 6: 638-647Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 26Thaysen-Andersen M. Hojrup P. Enrichment and characterization of glycopeptides from gel-separated glycoproteins.Am. Biotechnol. Lab. 2006; 24: 14-17Google Scholar, 27Larsen M.R. Jensen S.S. Jakobsen L.A. Heegaard N.H. Exploring the sialiome using titanium dioxide chromatography and mass spectrometry.Mol. Cell. Proteomics. 2007; 6: 1778-1787Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28Wada Y. Tajiri M. Yoshida S. Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics.Anal. Chem. 2004; 76: 6560-6565Crossref PubMed Scopus (287) Google Scholar). However, NPLC is neither effective for enriching less hydrophilic glycopeptides, e.g. the five high mannose type glycopeptides modified by 7–11 monosaccharide units from a tryptic digest of ribonuclease b (RNase B), nor for enriching O-linked glycopeptides of bovine fetuin using a ZIC-HILIC column (23Hägglund P. Bunkenborg J. Elortza F. Jensen O.N. Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.J. Proteome Res. 2004; 3: 556-566Crossref PubMed Scopus (410) Google Scholar). The use of Sepharose medium for enriching glycopeptides yielded only modest recovery of glycopeptides (28Wada Y. Tajiri M. Yoshida S. Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics.Anal. Chem. 2004; 76: 6560-6565Crossref PubMed Scopus (287) Google Scholar). In addition, binding of hydrophilic non-glycopeptides with these hydrophilic media contaminates the enriched glycopeptides (23Hägglund P. Bunkenborg J. Elortza F. Jensen O.N. Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.J. Proteome Res. 2004; 3: 556-566Crossref PubMed Scopus (410) Google Scholar, 28Wada Y. Tajiri M. Yoshida S. Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics.Anal. Chem. 2004; 76: 6560-6565Crossref PubMed Scopus (287) Google Scholar). We have recently developed an ion-pairing normal-phase LC (IP-NPLC) method to enrich glycopeptides from complex tryptic digests using Sepharose medium and salts or bases as ion-pairing reagents (29Ding W. Hill J.J. Kelly J. Selective enrichment of glycopeptides from glycoprotein digests using ion-pairing normal-phase liquid chromatography.Anal. Chem. 2007; 79: 8891-8899Crossref PubMed Scopus (43) Google Scholar). Though reasonably effective the technique still left room for significant improvement. For example, the method demonstrated relatively modest glycopeptide selectivity, providing only 16% recovery for high mannose type glycopeptides (29Ding W. Hill J.J. Kelly J. Selective enrichment of glycopeptides from glycoprotein digests using ion-pairing normal-phase liquid chromatography.Anal. Chem. 2007; 79: 8891-8899Crossref PubMed Scopus (43) Google Scholar). Here we report on a new IP-NPLC method using acids as ion-pairing reagents and polyhydroxyethyl aspartamide (A) as the stationary phase for the effective isolation of tryptic glycopeptides. The method was developed and evaluated using a tryptic digest of RNase B and fetuin mixture. In addition, we demonstrate that O-linked glycopeptides can be effectively isolated from a fetuin tryptic digest by IP-NPLC after removal of the N-linked glycans by PNGase F. The new IP-NPLC method was used to enrich N-linked glycopeptides from the tryptic digests of protein extracts of wild-type (wt) and PglD mutant strains of Campylobacter jejuni NCTC 11168. C. jejuni has a unique N-glycosylation system that glycosylates periplasmic and inner membrane proteins containing the extended N-linked sequon, D/E-X-N-X-S/T, where X is any amino acid other than proline (30Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. Structure of the N-linked glycan present on multiple glycoproteins in the gram-negative bacterium Campylobacter jejuni.J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 31Wacker M. Linton D. Hitchen P.G. Nita-Lazar M. Haslam S.M. North S.J. Panico M. Morris H.R. Dell A. Wren B.W. Aebi M. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli.Science. 2002; 298: 1790-1793Crossref PubMed Scopus (628) Google Scholar, 32Kowarik M. Young N.M. Numao S. Schulz B.L. Hug I. Callewaert N. Mills D.C. Watson D.C. Hernandez M. Kelly J.F. Wacker M. Definition of the bacterial N-glycosylation site consensus sequence.EMBO J. 2006; 25: 1957-1966Crossref PubMed Scopus (274) Google Scholar). The N-linked glycan of C. jejuni has been previously determined to be GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3]-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1 (BacGalNAc5Glc residue mass: 1406 Da), where Bac is 2,4-diacetamido-2,4,6-trideoxyglucopyranose (30Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. Structure of the N-linked glycan present on multiple glycoproteins in the gram-negative bacterium Campylobacter jejuni.J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). In addition, the glycan structure of C. jejuni is conserved, unlike in eukaryotic systems (30Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. Structure of the N-linked glycan present on multiple glycoproteins in the gram-negative bacterium Campylobacter jejuni.J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 31Wacker M. Linton D. Hitchen P.G. Nita-Lazar M. Haslam S.M. North S.J. Panico M. Morris H.R. Dell A. Wren B.W. Aebi M. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli.Science. 2002; 298: 1790-1793Crossref PubMed Scopus (628) Google Scholar, 32Kowarik M. Young N.M. Numao S. Schulz B.L. Hug I. Callewaert N. Mills D.C. Watson D.C. Hernandez M. Kelly J.F. Wacker M. Definition of the bacterial N-glycosylation site consensus sequence.EMBO J. 2006; 25: 1957-1966Crossref PubMed Scopus (274) Google Scholar). IP-NPLC recovered close to 100% of the bacterial N-linked glycopeptides with virtually no contamination of non-glycopeptides. Furthermore, we demonstrate for the first time that acetylation of bacillosamine is incomplete in the wt using IP-NPLC and label-free MS. Bovine RNase B, bovine fetuin, dithiothreitol, iodoacetamide, and the ion-pairing reagents were acquired from Sigma. Modified trypsin was purchased from Promega. The polyhydroxyethyl ATM Javelin® guard column (1 cm × 1 mm I.D., 5 µm) and the ZIC®-HILIC guard column (0.5 cm × 1 mm I.D., 5 µm) were purchased from Nest Group (Southborough, MA). The Luna NH2 column (1 cm × 0.3 mm I.D., 5 µm) was purchased from Bodman Industries (Aston, PA). PNGase F was purchased from Roche Applied Sciences (Mannheim, Germany). C. jejuni glycoprotein extracts were prepared from 4 liters of culture as described (30Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. Structure of the N-linked glycan present on multiple glycoproteins in the gram-negative bacterium Campylobacter jejuni.J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Proteins were quantified spectrophotometrically (Nanodrop ND-1000 Spectrophotometer; Thermo Fisher Scientific), adjusted to a concentration of 20 µg/µl using pure water (Milli-Q system, Millipore Corp.) and were either processed immediately or stored at −20 °C. Bovine RNase B or fetuin at 1 mg/ml was reduced, alkylated, and digested with trypsin, as described previously (29Ding W. Hill J.J. Kelly J. Selective enrichment of glycopeptides from glycoprotein digests using ion-pairing normal-phase liquid chromatography.Anal. Chem. 2007; 79: 8891-8899Crossref PubMed Scopus (43) Google Scholar). A 100-µl solution of periplasmic protein extracts of C. jejuni 11168 or the pglD mutant at 320 µg/µl in 50 mm NH4HCO3 was reduced with 8 mm dithiothreitol at 37 °C for 1 h and alkylated with 100 mm iodoacetamide at 37 °C for 30 min. The reagents used for reduction and alkylation were removed by centrifugal ultrafiltration (3000 MWCO) until the samples were at pH 5–6. After the addition of 60 µg of trypsin, the protein solution (120 µl with 50 mm NH4HCO3) was incubated at 37 °C for 16 h. For the O-glycopeptide isolation experiments, half a unit of PNGase F was added to 40 µl of 69 pmol/µl of a fetuin tryptic digest with 50 mm NH4HCO3 and then incubated at 37 °C for 14 h. All IP-NPLC experiments were performed either on a CapLCTM capillary LC system or nanoAcquity UPLC® system coupled to a Q-TOF-2TM hybrid quadrupole/TOF mass spectrometer (Waters). The NPLC and IP-NPLC experiments were performed as follows: 1) The sample was suspended in 7.5 µl of 80% ACN + 20% H2O (pH 4.7), if not otherwise stated, with/without addition of ion-pairing reagents. All pHs in this report were measured in ACN/H2O with electrodes calibrated in water (33Gagliardi L.G. Castells C.B. Clarra R. Ràfols C. Rosés M. Bosch E. δ Conversion parameter between ph scales (swpH and sspH) in acetonitrile/water mixtures at various compositions and temperatures.Anal. Chem. 2007; 79: 3180-3187Crossref PubMed Scopus (66) Google Scholar). 2) A gradient of 15% to 30% solvent B (100% HPLC grade H2O) for 5 min, then 30% to 50% solvent B for 5 min was used at a flow rate of 12 µl/min. The column was equilibrated at 15% solvent B for 4 min after each gradient. Solvent A is 100% ACN. The flow was split after the column so that ∼400 nL/min was directed to the ESI source to obtain NPLC-ESI-MS or IP-NPLC-ESI-MS chromatograms for each sample. The remainder of the column eluate was directed to a fraction collector or discarded. 3) The peptides eluted from the column between ∼1-x min are referred to below as the "non-glycopeptide fractions" whereas the peptides eluted between ∼ x-13 min are referred to as the "glycopeptide fraction". The time x refers to the elution time of the least hydrophilic glycopeptide observed from IP-NPLC-MS of the RNase B tryptic digest and was used in all IP-NPLC experiments as a reference time for determination of the starting elution time of glycopeptides. If not otherwise stated, the IP-NPLC column used in this study was polyhydroxyethyl A, which was used for months without significant deterioration of performance. The tryptic digest of periplasmic protein extracts of the C. jejuni 11168 and the pglD mutant (40 µg of each sample suspended in 7.5 µl of 80% ACN + 20% H2O + 1% HCl + 10 mm NH4HCO3) were repetitively subjected to IP-NPLC for off-line isolation of glycopeptides. The glycopeptide and non-glycopeptide fractions (∼90 µl for each fraction) were collected and dried to ∼1 µl, which was then resuspended in 100 µl of 0.1% formic acid (aq) and analyzed by label-free nano-flow RPLC-ESI-MS (nanoRPLC-ESI-MS) using a nanoAcquity UPLC system coupled to a Q-TOF UltimaTM hybrid quadrupole/TOF mass spectrometer (Waters). The peptides were first loaded onto a 180 µm I.D. × 20 mm 5-µm symmetry® C18 trap (Waters), then eluted to a 100 µm I.D. × 10 cm 1.7-µm BEH130C18 column (Waters) using a linear gradient from 0% to 36% solvent B (ACN + 0.1% formic acid) in 36 min, 36–90% solvent B for 2 min. Solvent A was 0.2% formic acid in water. The peak areas (signal/noise ≥ 3) from extracted ion chromatograms of label-free nanoRPLC-ESI-MS of the glycopeptides isolated by IP-NPLC were used for differential expression analysis of periplasmic glycoproteins between the wt and mutant strains. NanoRPLC-ESI-MS/MS analyses were also performed with data-dependent analysis with a survey intensity threshold of 20 for the total digest and the glycopeptide and non-glycop
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