Tes, and 114 have been unknown either since the websites were not annotated or for the reason that the corresponding proteins did not have a SWISS-PROT entry (Supplementary Table 1). Twenty-six peptides had greater than a Glycophorin-A/CD235a Proteins web single putative N-glycosylation internet site. Two peptides had been identified with three putative websites, and all of these web-sites had been annotated in SWISS-PROT as recognized or probable N-glycosylation web pages. The peptide R.ETIYPNASLLIQNVTQNDTGFYTLQVIK.S, with all 3 internet sites annotated as identified glycosylation sites, was identified from carcinoembryonic antigen-related cell adhesion molecule 1, which features a total of five recognized websites and 15 prospective 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 potential to determine a sizable quantity of doubly or triply glycosylated peptides suggests that the glycopeptide capture-and-release process applied in this study supplies good coverage for abundant N-glycopeptides that originate from plasma proteins, although in situ protein digestion may very well be sterically hindered by the presence of large, covalently-bound carbohydrate moieties. In LC-MS/MS analysis, the assignment with the glycosylation websites by SEQUEST was performed by browsing the protein CD66c/CEACAM6 Proteins manufacturer database using deamidation of asparagine as a dynamic modification (a monoisotopic mass increment of 0.9840 Da). Such a modest mass difference may make the accurate assignment of glycosylation web-sites difficult due to the limited mass measurement accuracy of ion-trap instrumentation. This difficulty in site assignment is specifically correct when the peptide has greater than one particular NXS/T motif, since it really is not necessarily constantly a a single motif-one website situation (e.g., a single peptide which has two NXS/T motifs might have just one particular N-glycosylation web-site). Therefore, to assess the LC-MS/MS glycosylation internet site identifications, the identical deglycosylated peptide sample (without 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 ten.Liu et al.Pageand the outcomes are summarized in Table 3. A total of 246 different peptides covering 95 proteins have been identified applying the correct mass measurements supplied by LC-FTICR; the details of these site-confirmed glycopeptide identifications are obtainable on line in Supplementary Table 3. An AMT tag database was generated that contained the calculated masses (primarily based on the unmodified peptide sequences) and NETs of all peptide identifications with at the least one NXS/ T motif in the LC-MS/MS analyses. Dynamic modification, corresponding to distinctive numbers of deamidation of asparagine residues (i.e., monoisotopic mass increment of n.9840 Da, n=1 to three), was applied when attributes have been matched to this AMT tag database. Note that peptides that contain the NPS/T motif (which can’t be N-glycosylated) were also included in the AMT tag database to test the accuracy of this approach. Amongst the 229 peptides containing one NXS/T motif, 225 peptides were determined to have only 1 glycosylation web page, and 4 peptides had been determined not to be glycosylated (1.3 , excluding one NPS/T motif-containing peptide integrated for test purposes). For the 225 one-site peptides confirmed by LC-FTICR, 169 web-sites had been annotated as known N-glycosylation web pages in SWISS-PROT and 49 web pages have been annotated as potential web-sites (Supplementary table three).
