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.

Mark S. Lowenthal

Kiersta S. Davis

Trina Formolo

Lisa E. Kilpatrick

Karen W. Phinney

Journal of Proteome Research

《蛋白质组研究杂志》出版内容涵盖全球蛋白质分析和功能的各个方面，包括基因组学、时空蛋白质组学、代谢组学和代谢组学、临床和农业蛋白质组学的动态方面，以及包括生物信息学在内的方法学进展。主题和重点是通过不同类型的“组学”之间的协同作用，对生命科学采取多学科方法。

The Journal of Proteome Research covers various aspects of global protein analysis and functionality, including genomics, spatiotemporal proteomics, metabolomics, and dynamic studies in clinical and agricultural proteomics, as well as methodological advancements including bioinformatics. The journal focuses on a multidisciplinary approach to life sciences through the synergy of different types of 'omics'.

《Journal of Proteome Research》涵盖全球蛋白质分析和功能的各个方面，包括基因组学、时空蛋白质组学、代谢组学、临床和农业蛋白质组学的动态研究，以及生物信息学等方法学的进展。该期刊的主题和重点是通过不同类型的“组学”之间的协同作用，采用多学科的方法来研究生命科学。

Identification of Novel N-Glycosylation Sites at Noncanonical Protein Consensus Motifs

Novel glycosylation sites within noncanonical consensus motifs, in the conformation N-X-C, are reported, based on mass spectrometry analysis of partially deglycosylated glycopeptide targets, and A1AG in porcine and canine sources is highly N-glycosylations at a noncanonicals motif (N-Q-C) based on semiquantitative multiple reaction monitoring analysis.

Category	Quartile
生物学	2
生物学, 生化研究方法	2

Identification of Novel N-Glycosylation Sites at Noncanonical Protein Consensus Motifs

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Category	Quartile
BIOCHEMICAL RESEARCH METHODS	1