How to Dig Deeper? Improved Enrichment Methods for Mucin Core-1 Type Glycopeptides
2012; Elsevier BV; Volume: 11; Issue: 7 Linguagem: Inglês
10.1074/mcp.o111.016774
ISSN1535-9484
AutoresZsuzsanna Darula, Jamie Sherman, Katalin F. Medzihradszky,
Tópico(s)RNA and protein synthesis mechanisms
ResumoTwo different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to ∼25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515–2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified. Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to ∼25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515–2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified. Glycosylation is one of the most frequent post-translational modifications of proteins. It is estimated that over 50% of all proteins undergo glycosylation during their lifespan (1Apweiler R. Hermjakob H. Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.Biochim. Biophys. Acta. 1999; 1473: 4-8Crossref PubMed Scopus (1491) Google Scholar). Apart from the regulatory O-GlcNAc modification, glycosylation occurs mostly on secreted proteins and extracellular domains of membrane proteins. Altered physiological conditions such as pregnancy (2Kenan N. Larsson A. Axelsson O. Helander A. Changes in transferrin glycosylation during pregnancy may lead to false-positive carbohydrate-deficient transferrin (CDT) results in testing for riskful alcohol consumption.Clin. Chim. Acta. 2011; 412: 129-133Crossref PubMed Scopus (52) Google Scholar) or disease including cancer (3Brockhausen I. Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions.EMBO Rep. 2006; 7: 599-604Crossref PubMed Scopus (417) Google Scholar, 4Arnold J.N. Saldova R. Hamid U.M. Rudd P.M. Evaluation of the serum N-linked glycome for the diagnosis of cancer and chronic inflammation. Review.Proteomics. 2008; 8: 3284-3293Crossref PubMed Scopus (266) Google Scholar) may result in different glycosylation of target proteins involved. Hence, glycoprofiling is an indispensable part of biomarker research. Unfortunately, characterization of protein glycosylation of complex samples such as serum is a rather challenging task mainly because of two factors. First, bodily fluids usually feature a high background of nonglycosylated proteins. Moreover, modified sequences are frequently also present unmodified (heterogeneity), and when occupied, the same site may be modified with different carbohydrate structures (microheterogeneity). Second, up to now there is no single analytical approach that can readily identify both the glycosylation sites and the modifying sugar structures. Glycosylation analysis of complex mixtures is usually restricted to N-glycosylation. This is because O-glycosylation lacks those features that facilitate N-glycosylation analysis; namely, a consensus sequence for modification and a single core structure for modification. Single sugar units as well as short or complex extended structures can modify Ser, Thr, and as recently reported Tyr residues (5Halim A. Brinkmalm G. Rüetschi U. Westman-Brinkmalm A. Portelius E. Zetterberg H. Blennow K. Larson G. Nilsson J. Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid beta-peptides in human cerebrospinal fluid.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 11848-11853Crossref PubMed Scopus (171) Google Scholar, 6Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (263) Google Scholar). For this reason there is no universal enzyme that can cleave all the O-linked carbohydrates (in the way e.g. PNGaseF can for N-linked glycopeptides), and those glycosidases that eliminate certain, well specified sugar structures leave an unmodified amino acid and thus no trace of the previous modification site. Sugar-elimination under basic conditions followed by Michael-addition has been used for the characterization of O-glycosylation (7Wells L. Vosseller K. Cole R.N. Cronshaw J.M. Matunis M.J. Hart G.W. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications.Mol. Cell. Proteomics. 2002; 1: 791-804Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar), but its efficiency varies for the different sugar structures, definitely slower for Thr, and phosphorylated, sulfated, even unmodified Ser residues may also undergo the same reactions as well as alkylated Cys residues (7Wells L. Vosseller K. Cole R.N. Cronshaw J.M. Matunis M.J. Hart G.W. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications.Mol. Cell. Proteomics. 2002; 1: 791-804Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 8McLachlin D.T. Chait B.T. Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides.Anal. Chem. 2003; 75: 6826-6836Crossref PubMed Scopus (224) Google Scholar, 9Medzihradszky K.F. Darula Z. Perlson E. Fainzilber M. Chalkley R.J. Ball H. Greenbaum D. Bogyo M. Tyson D.R. Bradshaw R.A. Burlingame A.L. O-sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes.Mol. Cell. Proteomics. 2004; 3: 429-440Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In the last two decades mass spectrometry has inevitably become the method of choice for protein characterization including post-translational modification analysis. However, MS characterization of O-glycopeptides by collision-induced dissociation (CID) 1The abbreviations used are:CIDcollision-induced dissociationAGCautomatic gain controlCVcolumn volumeECDelectron capture dissociationERLICelectrostatic repulsion hydrophilic interaction chromatographyETDelectron transfer dissociationGlcNAcN-acetyl glucosamineGalNAcN-acetyl galactosamineHCDhigher-energy collision-dissociationHexhexoseHexNAcN-acetyl-hexosaminemixedIEXion exchange on a mixed-bed columnSAsialic acidTEAPtriethylammonium phosphate. 1The abbreviations used are:CIDcollision-induced dissociationAGCautomatic gain controlCVcolumn volumeECDelectron capture dissociationERLICelectrostatic repulsion hydrophilic interaction chromatographyETDelectron transfer dissociationGlcNAcN-acetyl glucosamineGalNAcN-acetyl galactosamineHCDhigher-energy collision-dissociationHexhexoseHexNAcN-acetyl-hexosaminemixedIEXion exchange on a mixed-bed columnSAsialic acidTEAPtriethylammonium phosphate. activation is ineffective at identifying the peptide as sugar oxonium ions and fragment ions corresponding to carbohydrate fragmentation dominate MS/MS spectra. On the other hand, electron capture dissociation (10Zubarev R.A. Horn D.M. Fridriksson E.K. Kelleher N.L. Kruger N.A. Lewis M.A. Carpenter B.K. McLafferty F.W. Electron capture dissociation for structural characterization of multiply charged protein cations.Anal. Chem. 2000; 72: 563-573Crossref PubMed Scopus (848) Google Scholar) and electron transfer dissociation (ETD) (11Syka J.E. Coon J.J. Schroeder M.J. Shabanowitz J. Hunt D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9528-9533Crossref PubMed Scopus (1997) Google Scholar) analysis of glycopeptides is a more successful approach in this respect [ECD: 5Halim A. Brinkmalm G. Rüetschi U. Westman-Brinkmalm A. Portelius E. Zetterberg H. Blennow K. Larson G. Nilsson J. Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid beta-peptides in human cerebrospinal fluid.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 11848-11853Crossref PubMed Scopus (171) Google Scholar,12Takahashi K. Wall S.B. Suzuki H. Smith 4th, A.D. Hall S. Poulsen K. Kilian M. Mobley J.A. Julian B.A. Mestecky J. Novak J. Renfrow M.B. Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation.Mol. Cell. Proteomics. 2010; 9: 2545-2557Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar; ETD: 6Steentoft C. Vakhrushev S.Y. Vester-Christensen M.B. Schjoldager K.T. Kong Y. Bennett E.P. Mandel U. Wandall H. Levery S.B. Clausen H. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines.Nat. Methods. 2011; 8: 977-982Crossref PubMed Scopus (263) Google Scholar,13Scott N.E. Parker B.L. Connolly A.M. Paulech J. Edwards A.V. Crossett B. Falconer L. Kolarich D. Djordjevic S.P. Højrup P. Packer N.H. Larsen M.R. Cordwell S.J. Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni.Mol. Cell. Proteomics. 2011; 10: M000031-MMCP201PubMed Google Scholar,14Darula Z. Medzihradszky K.F. Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum.Mol. Cell. Proteomics. 2009; 8: 2515-2526Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar], despite the fact that these activation techniques are less efficient compared with CID, work considerably better on higher charge state peptide precursors and have significant precursor m/z limitations (15Good D.M. Wirtala M. McAlister G.C. Coon J.J. Performance characteristics of electron transfer dissociation mass spectrometry.Mol. Cell. Proteomics. 2007; 6: 1942-1951Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). collision-induced dissociation automatic gain control column volume electron capture dissociation electrostatic repulsion hydrophilic interaction chromatography electron transfer dissociation N-acetyl glucosamine N-acetyl galactosamine higher-energy collision-dissociation hexose N-acetyl-hexosamine ion exchange on a mixed-bed column sialic acid triethylammonium phosphate. collision-induced dissociation automatic gain control column volume electron capture dissociation electrostatic repulsion hydrophilic interaction chromatography electron transfer dissociation N-acetyl glucosamine N-acetyl galactosamine higher-energy collision-dissociation hexose N-acetyl-hexosamine ion exchange on a mixed-bed column sialic acid triethylammonium phosphate. Currently, for successful ETD-based O-linked glycopeptide characterization one has to know either the protein(s) or the sugar structure to begin with. General glycopeptide enrichments as hydrophilic interaction liquid chromatography (16Christiansen M.N. Kolarich D. Nevalainen H. Packer N.H. Jensen P.H. Challenges of determining O-glycopeptide heterogeneity: a fungal glucanase model system.Anal. Chem. 2010; 82: 3500-3509Crossref PubMed Scopus (41) Google Scholar) or selective capture/release based on the unique properties of sialic acid (17Halim A. Nilsson J. Rüetschi U. Hesse C. Larson G. Human urinary glycoproteomics; attachment site specific analysis of N-and O-linked glycosylations by CID and ECD.Mol Cell Proteomics. 2012; ([Epub ahead of print])Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) presently cannot be combined with large scale automated studies. Although CID data can provide information about sugar structure and ETD can characterize peptide sequence, there is currently no automated way to correlate these two types of data. Hence, only a fraction of glycopeptides enriched in a nonstructure-specific fashion can be characterized and it is done manually (17Halim A. Nilsson J. Rüetschi U. Hesse C. Larson G. Human urinary glycoproteomics; attachment site specific analysis of N-and O-linked glycosylations by CID and ECD.Mol Cell Proteomics. 2012; ([Epub ahead of print])Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Thus, either one can characterize glycosylation within a protein mixture of limited complexity (proteins identified from a strict database search can be subjected to a second search where undefined modifications over a mass range are considered (18Chalkley R.J. Baker P.R. Medzihradszky K.F. Lynn A.J. Burlingame A.L. In-depth analysis of tandem mass spectrometry data from disparate instrument types.Mol. Cell. Proteomics. 2008; 7: 2386-2398Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar)) or one has to apply some oligosaccharide-selective enrichment strategy for the glycopeptides, so that the database search could be restricted to a few sugar compositions. Jacalin, a lectin isolated from Artocarpus integrifolia has been reported binding GalNAcα1-modified glycopeptides in which C6-OH is free, but not recognizing such structures with substitution at the C6 position (19Tachibana K. Nakamura S. Wang H. Iwasaki H. Tachibana K. Maebara K. Cheng L. Hirabayashi J. Narimatsu H. Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography.Glycobiology. 2006; 16: 46-53Crossref PubMed Scopus (91) Google Scholar). Previously we have shown that Jacalin affinity-chromatography combined with MS-analysis by CID and ETD fragmentation is a viable experimental setup for characterization of the core-1 mucin-type glycoproteome of serum (14Darula Z. Medzihradszky K.F. Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum.Mol. Cell. Proteomics. 2009; 8: 2515-2526Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, our findings were restricted to the more abundant proteins of serum. In order to gain a deeper insight, we have now combined the affinity-enrichment with other protein- or peptide-level fractionations, and tested two different workflows. In the protein-level fractionation approach, ion-exchange chromatography was implemented for fractionation of the glycoprotein mixture isolated by Jacalin lectin affinity-chromatography. Because of its high sample capacity, ion exchange is a popular method for separation of protein samples. Using a mixed-bed ion-exchange column that contains anion-exchange and cation-exchange material in equal amounts enables the retention and fractionation of proteins over the entire pI range (20Lee S. Chen Y. Luo H. Wu A.A. Wilde M. Schumacker P.T. Zhao Y. The first global screening of protein substrates bearing protein-bound 3,4-Dihydroxyphenylalanine in Escherichia coli and human mitochondria.J. Proteome Res. 2010; 9: 5705-5714Crossref PubMed Scopus (14) Google Scholar). A further advantage of this separation step is that even abundant proteins are expected to be restricted to a few fractions, thus increasing the chances for the identification of less abundant glycoproteins. In the peptide-level approach, the tryptic digest of the glycoprotein mixture isolated by Jacalin lectin affinity-chromatography was subjected to further separation applying the ERLIC (electrostatic repulsion hydrophilic interaction chromatography) principle (21Alpert A.J. Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides.Anal Chem. 2008; 80: 62-76Crossref PubMed Scopus (444) Google Scholar). ERLIC is a mixed mode chromatography where the retention of any given compound depends on the combination of electrostatic repulsion from and hydrophylic interaction with the solid support (21Alpert A.J. Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides.Anal Chem. 2008; 80: 62-76Crossref PubMed Scopus (444) Google Scholar). In the case of tryptic digests, unmodified peptides are expected to be protonated at pH 2 and therefore elute in the flow-through or early eluting fractions, whereas peptides modified by highly acidic groups such as phospho- and sulfopeptides, and sialylated glycopeptides are retained longer. As a result, sialylated glycopeptides can be selectively isolated from unmodified peptides. Although this workflow was expected to be limited to the selective isolation of sialylated glycopeptides, in our pilot studies the majority of the glycopeptides bore sialic acid residues. Therefore we did not consider this as a major limitation. In this study glycopeptide enrichment results are compared from the two above described workflows. In order to ensure higher identification rates, i.e. to overcome the charge-density limits for successful ETD experiments, glycopeptides were treated with neuraminidase and β-galactosidase, and the sequences retaining only the core GalNAc units were subjected to MS/MS analysis using both HCD and ETD activation. We identified 124 glycosylation sites in 51 glycoproteins; an ∼6-fold improvement in comparison to our previous results, when only lectin affinity-chromatography was used. Thirty-five of the proteins were previously not known to be glycosylated. Similarly, more than half of the sites determined represent novel glycosylation sites. Chromatography was performed on a Jasco semimicro HPLC system complete with a four-line degasser (DG-2080–54, Jasco), two pumps (PU2085, Jasco), a dynamic mixer (MX 2080–32, Jasco), a UV-VIS detector (Spectra-Flow 501, Sunchrom), and a fraction collector (CHF 122 SC, Advantec). Chromatography was performed as previously published (14Darula Z. Medzihradszky K.F. Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum.Mol. Cell. Proteomics. 2009; 8: 2515-2526Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), 2 ml of fetal calf serum was injected onto a 1 mm × 2000 mm (CV:1.57 ml) column packed with agarose-bound Jacalin (VectorLabs AL1153). After introducing the sample (flow rate: 50 μl/min), the column was washed with eight CV of solvent A (175 mm Tris.HCl, pH 7.5; flow rate:150 μl/min) then the species bound were eluted with five CV of solvent B (0.8 m galactose/175 mm Tris.HCl, pH 7.5; flow rate:150 μl/min) collecting 8-min fractions. The tryptic digest of the protein mixture isolated by Jacalin affinity-chromatography was fractionated on a weak anion-exchange column (PolyWAX LP, PolyLC Inc, 4.6 mm ID × 20 cm, 5 μm particle size, 300A pore size) applying the following gradient program (flow rate: 1 ml/min, UV-detection at 215 nm): 0–5 min: 0% B, 5–15 min: 0–10% B, 15–35 min: 10–60% B, 35–45 min: 60–100% B, 45–55 min:100% B (solvent A: 20 mm methyl-phosphonic acid pH:2/70% acetonitrile, solvent B: 200 mm TEAP (triethylammonium phosphate) pH 2/60% ACN; the pH of solvent A and solvent B were adjusted using 10 m aqueous NaOH and triethylamine, respectively). 1-min fractions were collected, dried down to ∼200 μl and desalted on 100 μl C-18 tips (Omix, Varian) and concentrated. Protein mixture isolated by Jacalin affinity-chromatography from 2 ml fetal calf serum was fractionated on a mixed-bed ion exchanger column (PolyCATWAX, PolyLC Inc, 4.6 mm ID × 20 cm, 5 μm particle size, 1000 Å pore size) applying the following gradient program (flow rate: 0.5 ml/min, UV-detection at 275 nm): 0–5 min: 0% B, 5–15 min: 0–10% B, 15–35 min: 10–60% B, 35–45 min: 60–100% B, 45–55 min:100% B (solvent A: 20 mm ammonium acetate pH:7, solvent B: 800 mm ammonium acetate pH:7). 2-min fractions were collected and dried down before further treatment. Samples were supplemented with guanidine hydrochloride to give a final concentration of 6 m. Disulfide bridges were reduced using dithiothreitol (56 °C for 30 min) and the resultant free sulfhydryl groups were derivatized using iodoacetamide (1.1x equivalent to dithiothreitol, 30 min in the dark at room temperature). Samples were then diluted eightfold with 100 mm ammonium bicarbonate to reduce the guanidine hydrochloride concentration, and incubated with porcine trypsin (Fluka 93614; 1% (w/w) of the estimated protein content) at 37 °C for 4 h. Digestion was stopped by adding trifluoroacetic acid (final pH ≤3). The resulting peptide mixtures were desalted on C18 reversed phase and concentrated. Chromatography was performed as previously described (14Darula Z. Medzihradszky K.F. Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum.Mol. Cell. Proteomics. 2009; 8: 2515-2526Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The tryptic digest of a glycoprotein mixture was injected onto a 1 mm × 200 mm (CV:0.157 ml) column packed with agarose-bound Jacalin. After introducing the sample (flow rate: 50 μl/min), the column was washed with 20 CV of solvent A (175 mm Tris HCl, pH 7.5; flow rate:150 μl/min) then the species bound were eluted with 20 CV of solvent B (0.8 m galactose/175 mm Tris.HCl, pH:7.5; flow rate:150 μl/min) collecting 4-min fractions. Fractions of interest were acidified and desalted on 100 μl C-18 tips (Omix, Varian) prior to further treatment. The fractions to be purified were pulled up onto pipette-tips pretreated following the manufacturer's instructions, the galactose and salt were removed with 0.1% formic acid in water (5 × 200 μl). Peptides and glycopeptides were eluted with 200 μl 0.1% formic acid/50% acetonitrile/water. Samples were concentrated in a vacuum centrifuge. Sialic acid and β-galactose units of glycopeptides were removed by incubation with neuraminidase (5–10 U/sample, New England Biolabs P0720; in 100 mm sodium citrate, pH 6.0) for 1 h at 37 °C followed by overnight treatment with β-galactosidase (10 U/sample, New England Biolabs P0726; in 100 mm sodium citrate, pH 4.5) at 37 °C. Enzymatic deglycosylation was stopped by acidification to pH ≤3 with 10% trifluoroacetic acid solution, and the resulting peptide mixtures were desalted on 10 μl C-18 tips (Millipore ZTC18S960). Glycopeptide mixtures were separated on nanoflow reversed phase HPLC (nanoAcquity, Waters, Milford, MA) directing the eluent to nanospray sources of a linear ion trap-Orbitrap (Velos-Orbitrap, Thermo Fisher Scientific) mass spectrometer operating in positive ion mode. Samples were injected onto a UPLC trapping column (Symmetry, C18 5 μm, 180 μm × 20 mm; Waters) (15 μl/min with 3% solvent B) followed by a linear gradient of solvent B (5 to 35% in 35 min, followed by a short wash at 50% solvent B, before returning to starting conditions; flow rate: 400 nl/min; nanoACQUITY UPLC BEH C18 Column, 1.7 μm, 75 μm × 200 mm; solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile). MS data acquisition was carried out in data-dependent fashion acquiring sequential HCD and ETD spectra of the three most intense, multiply charged precursor ions identified from each MS survey scan. ETD experiments were performed in the linear trap, whereas HCD activation was carried out in the collision cell. MS and HCD spectra were acquired in the Orbitrap, and ETD spectra in the linear ion trap. Ion populations within the trapping instruments were controlled by integrated automatic gain control. For HCD, the AGC target was set to 50,000, with dissociation at 35% of normalized collision energy, activation time: 0.1 ms. For ETD, the automatic gain control target values were set to 10,000 and 200,000 for the isolated precursor cations and fluoranthene anions, respectively, and allowing 100 ms of ion/ion reaction time. Supplemental activation for the ETD experiments was enabled (supplemental activation energy: 15). Dynamic exclusion was also enabled (mass width low: 0.5 Th, mass width high 1.5 Th), exclusion time: 45 s. Some glycopeptides fractions were combined and analyzed on an LTQ-Orbitrap Elite (courtesy of Thermo Scientific, San Jose, CA). A single spectrum from this analysis that enabled unambiguous site assignment for E1BB91 was included in the supplementary Figs. Peaklists from LTQ-Orbitrap raw data files were created by using the UCSF in-house peak-picking program PAVA (22Medzihradszky K.F. Chalkley R.J. Trinidad J.C. Michaelevski A. Burlingame A.L. The utilization of Orbitrap higher collision decomposition device for PTM analysis and iTRAQ-based quantitation.56th ASMS Conference on Mass Spectrometry. Denver, CO, 2008Google Scholar). The software generates separate HCD and ETD peaklists. From the above ETD peaklists "glycopeptide-only" versions were also prepared after HCD-based filtering. An in-house script (supplemental File S1) was used to screen HCD data for the HexNAc specific carbohydrate ion m/z = 204.087 with a mass accuracy of 0.01 Da. Whenever such a fragment was not found, the ETD spectrum of the corresponding precursor ion was deleted from the ETD peaklist. Similar ETD peaklists screened for 204.087 and 366.14; and 204.087 and 407.167 (mass accuracy: 0.01 Da) were also prepared. Database searching was performed by ProteinProspector v.5.8.1 against the UniProt database (07.06.2011), supplemented with a random sequence for each entry, and species specified as Bos taurus (66914/33089872 entries searched). Search parameters were as follows: trypsin was selected as the enzyme, two missed cleavages were permitted, and nonspecific cleavages were also permitted at one of the peptide termini. Mass accuracies of 15 ppm for precursor ions, 20 ppm for HCD fragment ions, and 0.8 Da for ETD fragment ions were considered. Fixed modification was carbamidomethylation of Cys residues. Variable modifications were the acetylation of protein N termini; Met oxidation; and the cyclization of N-terminal Gln residues; plus HexNAc modification on Thr and Ser residues. A maximum of three modifications per peptide were permitted. Search parameters for HCD data also included HexNAc as a variable modification subject to neutral loss; i.e. fragments were assumed to be unmodified. Acceptance criteria were as follows: minimum peptide score: 22, minimum protein score: 22; maximum peptide E-value: 0.1, maximum protein E-value: 0.1; minimum best discriminant score: 1. SLIP score as a measure of reliability of site assignments was set to six (23Baker P.R. Trinidad J.C. Chalkley R.J. Modification site localization scoring integrated into a search engine.Mol. Cell. Proteomics. 2011; 10 (M111. 008078)Abstract Full Text Full Text PDF Scopus (92) Google Scholar). Only the best identification is reported for each unique sequence (considering differently modified sequences as unique). Data was also searched permitting nonspecific cleavages at both termini, which identified a few new glycopeptides that after careful inspection were included in the data set (supplemental Figs.). An additional database search was performed on the subset of identified proteins allowing up to 4 variable modifications per peptide applying the same acceptance criteria as above with manual validation of data providing additional glycosylation information to the original database search results. With the 204 and 366 and 204 and 407-filtered peaklists separate searches were performed. Search parameters were as above, except HexHexNAc and HexHexNAcSA or HexNAc2 on Ser/Thr residues were also permitted as variable modifications. Acceptance criteria reporting those modifications were the same. Novelty of the glycosylatio
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