Tes, and 114 have been unknown either mainly because the web pages weren’t annotated or for the reason that the corresponding proteins did not have a SWISS-PROT entry (Supplementary Table 1). Twenty-six peptides had more than one particular putative N-glycosylation web page. Two peptides were identified with three putative websites, and all of those sites have been annotated in SWISS-PROT as identified or probable N-glycosylation websites. The XIAP Compound peptide R.ETIYPNASLLIQNVTQNDTGFYTLQVIK.S, with all 3 sites annotated as identified glycosylation internet sites, was identified from carcinoembryonic antigen-related cell adhesion molecule 1, which features a total of five identified web sites and 15 potential web sites. The other triply Nglycosylated peptide K.NNMSFVVLVPTHFEWNVSQVLANLSWDTLHPPLVWERPTK.V was identified from -2-antiplasmin, and all 3 on the identified websites had been annotated as possible web-sites. The capacity to identify a sizable 5-LOX Antagonist Purity & Documentation number of doubly or triply glycosylated peptides suggests that the glycopeptide capture-and-release process applied within this study delivers fantastic coverage for abundant N-glycopeptides that originate from plasma proteins, though in situ protein digestion may very well be sterically hindered by the presence of large, covalently-bound carbohydrate moieties. In LC-MS/MS evaluation, the assignment with the glycosylation web sites by SEQUEST was performed by searching the protein database working with deamidation of asparagine as a dynamic modification (a monoisotopic mass increment of 0.9840 Da). Such a small mass difference may well make the accurate assignment of glycosylation internet sites challenging because of the restricted mass measurement accuracy of ion-trap instrumentation. This difficulty in web-site assignment is specifically accurate when the peptide has greater than one particular NXS/T motif, because it can be not necessarily usually a 1 motif-one website scenario (e.g., a single peptide which has two NXS/T motifs may have just one N-glycosylation web site). Hence, to assess the LC-MS/MS glycosylation internet site identifications, the same deglycosylated peptide sample (without having SCX fractionation) was measured working with a single LC-FTICR analysis,NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Proteome Res. Author manuscript; offered in PMC 2007 April 10.Liu et al.Pageand the results are summarized in Table three. A total of 246 distinctive peptides covering 95 proteins have been identified applying the accurate mass measurements offered by LC-FTICR; the information of those site-confirmed glycopeptide identifications are obtainable on the web in Supplementary Table three. An AMT tag database was generated that contained the calculated masses (primarily based around the unmodified peptide sequences) and NETs of all peptide identifications with at least one particular NXS/ T motif from the LC-MS/MS analyses. Dynamic modification, corresponding to diverse numbers of deamidation of asparagine residues (i.e., monoisotopic mass increment of n.9840 Da, n=1 to 3), was applied when attributes have been matched to this AMT tag database. Note that peptides that include the NPS/T motif (which cannot be N-glycosylated) have been also incorporated in the AMT tag database to test the accuracy of this approach. Amongst the 229 peptides containing one NXS/T motif, 225 peptides have been determined to possess only one glycosylation web site, and four peptides have been determined to not be glycosylated (1.three , excluding a single NPS/T motif-containing peptide incorporated for test purposes). For the 225 one-site peptides confirmed by LC-FTICR, 169 websites have been annotated as known N-glycosylation web pages in SWISS-PROT and 49 websites were annotated as possible sites (Supplementary table three).