LC鈥揗S/MS Characterization of O-Glycosylation Sites and Glycan Structures of Human Cerebrospinal Fluid Glycoproteins

详细信息    查看全文

• 作者：Adnan Halim ; Ulla R眉etschi ; G枚ran Larson ; Jonas Nilsson
• 刊名：Journal of Proteome Research
• 出版年：2013
• 出版时间：February 1, 2013
• 年：2013
• 卷：12
• 期：2
• 页码：573-584
• 全文大小：653K
• 年卷期：v.12,no.2(February 1, 2013)
• ISSN：1535-3907

文摘

The GalNAc O-glycosylation on Ser/Thr residues of extracellular proteins has not been well characterized from a proteomics perspective. We previously reported a sialic acid capture-and-release protocol to enrich tryptic N- and O-glycopeptides from human cerebrospinal fluid glycoproteins using nano-LC鈥揈SI鈥揗S/MS with collision-induced dissociation (CID) for glycopeptide characterization. Here, we have introduced peptide N-glycosidase F (PNGase F) pretreatment of CSF samples to remove the N-glycans facilitating the selective characterization of O-glycopeptides and enabling the use of an automated CID鈥揗S²/MS³ search protocol for glycopeptide identification. We used electron-capture and -transfer dissociation (ECD/ETD) to pinpoint the glycosylation site(s) of the glycopeptides, identified as predominantly core-1-like HexHexNAc-O- structure attached to one to four Ser/Thr residues. We characterized 106 O-glycosylations and found Pro residues preferentially in the n 鈥?1, n + 1, and/or n + 3 positions in relation to the Ser/Thr attachment site (n). The characterization of glycans and glycosylation sites in glycoproteins from human clinical samples provides a basis for future studies addressing the biological and diagnostic importance of specific protein glycosylations in relation to human disease.r>

Keywords:

glycoproteomics; glycopeptide; tandem mass spectrometry; PNGase F; hydrazide chemistry