title: Identification of Novel N-Glycosylation Sites at Noncanonical Protein Consensus Motifs.
abstract: N-glycosylation of proteins is well known to occur at asparagine residues that fall within the canonical consensus sequence N-X-S/T but has also been identified at a small number of asparagine residues within N-X-C motifs, including the N491 residue of human serotransferrin. Here we report novel glycosylation sites within noncanonical consensus motifs, in the conformation N-X-C, based on mass spectrometry analysis of partially deglycosylated glycopeptide targets. Alpha-1-acid glycoprotein (A1AG) and serotransferrin (Tf) were observed for the first time to be N-glycosylated on asparagine residues within a total of six unique noncanonical motifs. N-glycosylation was initially predicted in silico based on the evolutionary conservation of the N-X-C motif among related mammalian species and demonstrated experimentally in A1AG from porcine, canine, and feline sources and in human serotransferrin. High-resolution liquid chromatography-tandem mass spectrometry was employed to collect fragmentation data of predicted GlcNAcylated peptides and to assign modification sites within N-X-C motifs. A combination of targeted analytical techniques that includes complementary mass spectrometry platforms, enzymatic digestions, and partial-deglycosylation procedures was developed to confirm the novel observations. Additionally, we found that A1AG in porcine and canine sources is highly N-glycosylated at a noncanonical motif (N-Q-C) based on semiquantitative multiple reaction monitoring analysis-the first report of an N-X-C motif exhibiting substantial N-glycosylation. Although reports of N-X-C motif N-glycosylation are relatively uncommon in the literature, this work adds to a growing list of glycoproteins reported with glycosylation at various forms of noncanonical motifs.
authors.affiliation: Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology , 100 Bureau Drive, Stop 8314, Gaithersburg, Maryland 20899, United States.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Lowenthal
authors.affiliation: Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology , 100 Bureau Drive, Stop 8314, Gaithersburg, Maryland 20899, United States.
authors.forename: Kiersta S
authors.initials: KS
authors.lastname: Davis
authors.affiliation: Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology , 100 Bureau Drive, Stop 8314, Gaithersburg, Maryland 20899, United States.
authors.forename: Trina
authors.initials: T
authors.lastname: Formolo
authors.affiliation: Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology , 100 Bureau Drive, Stop 8314, Gaithersburg, Maryland 20899, United States.
authors.forename: Lisa E
authors.initials: LE
authors.lastname: Kilpatrick
authors.affiliation: Material Measurement Laboratory, Biomolecular Measurement Division, National Institute of Standards and Technology , 100 Bureau Drive, Stop 8314, Gaithersburg, Maryland 20899, United States.
authors.forename: Karen W
authors.initials: KW
authors.lastname: Phinney
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D002415:Cats; D002853:Chromatography, Liquid; D004285:Dogs; D006020:Glycopeptides; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D013552:Swine
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D020543:Proteome; D001216:Asparagine
keywords: A1AG; LC−MS/MS; N-X-C; N-glycosylation; consensus motif; evolutionary conservation; mass spectrometry; noncanonical glycosylation; transferrin