Tes, and 114 had been unknown either simply because the internet sites 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 a single putative N-glycosylation internet site. Two peptides were identified with 3 putative web-sites, and all of these internet sites had been annotated in SWISS-PROT as known or probable N-glycosylation web pages. The peptide R.ETIYPNASLLIQNVTQNDTGFYTLQVIK.S, with all 3 sites annotated as known glycosylation sites, was identified from carcinoembryonic antigen-related cell adhesion molecule 1, which includes a total of five identified web sites and 15 possible internet sites. The other triply Nglycosylated peptide K.NNMSFVVLVPTHFEWNVSQVLANLSWDTLHPPLVWERPTK.V was identified from -2-antiplasmin, and all three of the identified sites have been annotated as possible web-sites. The capacity to identify a big variety of doubly or triply glycosylated peptides suggests that the glycopeptide capture-and-release approach made use of within this study offers excellent coverage for abundant N-glycopeptides that originate from plasma proteins, while in situ protein digestion may be sterically hindered by the presence of significant, covalently-bound carbohydrate moieties. In LC-MS/MS evaluation, the assignment of the glycosylation internet sites by SEQUEST was performed by looking the protein database using deamidation of asparagine as a dynamic modification (a monoisotopic mass increment of 0.9840 Da). Such a smaller mass difference may well make the precise assignment of glycosylation web sites complicated due to the restricted mass measurement accuracy of ion-trap instrumentation. This difficulty in website assignment is particularly correct when the peptide has more than a single NXS/T motif, given that it really is not necessarily constantly a one particular motif-one website situation (e.g., 1 peptide which has two NXS/T motifs might have just one particular N-glycosylation web site). Hence, to assess the LC-MS/MS glycosylation site identifications, the identical deglycosylated peptide sample (with no SCX fractionation) was measured working with a single LC-FTICR evaluation,NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Proteome Res. Author manuscript; accessible in PMC 2007 April 10.Liu et al.Pageand the results are summarized in Table 3. A total of 246 distinct peptides covering 95 proteins have been identified using the accurate mass CD119 Proteins Source measurements offered by LC-FTICR; the facts of those site-confirmed glycopeptide identifications are accessible on the internet in Supplementary Table 3. An AMT tag database was generated that contained the calculated masses (based around the unmodified peptide sequences) and NETs of all peptide identifications with at least one particular NXS/ T motif in the LC-MS/MS analyses. Dynamic modification, corresponding to various numbers of deamidation of asparagine residues (i.e., monoisotopic mass increment of n.9840 Da, n=1 to 3), was applied when characteristics were matched to this AMT tag database. Note that peptides that contain the NPS/T motif (which can’t be N-glycosylated) had been also included within the AMT tag database to test the accuracy of this process. Amongst the 229 peptides containing 1 NXS/T motif, 225 peptides had been determined to possess only 1 glycosylation web-site, and four peptides have been determined not to be glycosylated (1.three , excluding one NPS/T Nectin-3/CD113 Proteins Biological Activity motif-containing peptide integrated for test purposes). For the 225 one-site peptides confirmed by LC-FTICR, 169 websites had been annotated as known N-glycosylation web-sites in SWISS-PROT and 49 web sites had been annotated as prospective web-sites (Supplementary table three).
