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LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.

Journal article
Authors Adnan Halim
Ulla Rüetschi
Göran Larson
Jonas Nilsson
Published in Journal of Proteome Research
Volume 12
Issue 2
Pages 573-584
ISSN 1535-3893
Publication year 2013
Published at Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
Pages 573-584
Language en
Links dx.doi.org/10.1021/pr300963h
Keywords glycoproteomics; glycopeptide; tandem mass spectrometry; PNGase F; hydrazide chemistry
Subject categories Clinical Medicine, Clinical Laboratory Medicine

Abstract

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 nanoLC-ESI-MS/MS with collision-induced dissociation (CID) for glycopeptide characterization. Here, we have introduced peptide N-glycosidase F (PNGase F) pre-treatment of CSF samples to remove the N-glycans facilitating the selective characterization of O-glycopeptides and enabling the use of an automated CID-MS2/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- glycans attached to 1-4 Ser/Thr residues. We characterized 108 O-glycosylations and found Pro residues preferentially in the n-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.

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