Tes, and 114 had been unknown either for the reason that the web-sites were not annotated or mainly because the corresponding proteins did not possess a SWISS-PROT entry (Supplementary Table 1). Twenty-six peptides had greater than one putative N-Calcitonin Proteins Recombinant Proteins glycosylation web site. Two peptides have been identified with 3 putative web pages, and all of these internet sites had been annotated in SWISS-PROT as known or ICOS Proteins supplier probable N-glycosylation sites. The peptide R.ETIYPNASLLIQNVTQNDTGFYTLQVIK.S, with all three sites annotated as known glycosylation web sites, was identified from carcinoembryonic antigen-related cell adhesion molecule 1, which has a total of five identified web pages and 15 prospective web pages. The other triply Nglycosylated peptide K.NNMSFVVLVPTHFEWNVSQVLANLSWDTLHPPLVWERPTK.V was identified from -2-antiplasmin, and all 3 on the identified web-sites were annotated as possible internet sites. The potential to recognize a big variety of doubly or triply glycosylated peptides suggests that the glycopeptide capture-and-release technique utilised in this study provides fantastic coverage for abundant N-glycopeptides that originate from plasma proteins, while in situ protein digestion might be sterically hindered by the presence of substantial, covalently-bound carbohydrate moieties. In LC-MS/MS evaluation, the assignment of the glycosylation sites by SEQUEST was performed by browsing the protein database applying deamidation of asparagine as a dynamic modification (a monoisotopic mass increment of 0.9840 Da). Such a tiny mass difference may perhaps make the accurate assignment of glycosylation web sites complicated due to the restricted mass measurement accuracy of ion-trap instrumentation. This difficulty in web site assignment is specifically true when the peptide has greater than one particular NXS/T motif, considering the fact that it’s not necessarily normally a 1 motif-one web page scenario (e.g., 1 peptide that has two NXS/T motifs may have just one particular N-glycosylation site). Thus, to assess the LC-MS/MS glycosylation website identifications, the identical deglycosylated peptide sample (without the need of 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; obtainable in PMC 2007 April 10.Liu et al.Pageand the results are summarized in Table three. A total of 246 diverse peptides covering 95 proteins had been identified using the precise mass measurements offered by LC-FTICR; the facts of these site-confirmed glycopeptide identifications are readily available on line 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 no less than one NXS/ T motif in the LC-MS/MS analyses. Dynamic modification, corresponding to different numbers of deamidation of asparagine residues (i.e., monoisotopic mass increment of n.9840 Da, n=1 to three), was applied when functions have been matched to this AMT tag database. Note that peptides that contain the NPS/T motif (which cannot be N-glycosylated) were also incorporated in the AMT tag database to test the accuracy of this method. Amongst the 229 peptides containing one NXS/T motif, 225 peptides had been determined to have only one glycosylation web-site, and 4 peptides were determined not to be glycosylated (1.three , excluding 1 NPS/T motif-containing peptide integrated for test purposes). For the 225 one-site peptides confirmed by LC-FTICR, 169 websites have been annotated as known N-glycosylation websites in SWISS-PROT and 49 sites were annotated as prospective websites (Supplementary table 3).