Abstract
In this study, we investigated the use of metabolic oligosaccharide engineering and bio-orthogonal ligation reactions combined with lectin microarray and mass spectrometry to analyze sialoglycoproteins in the SW1990 human pancreatic cancer line. Specifically, cells were treated with the azido N-acetylmannosamine analog, 1,3,4-Bu3ManNAz, to label sialoglycoproteins with azide-modified sialic acids. The metabolically labeled sialoglyproteins were then biotinylated via the Staudinger ligation, and sialoglycopeptides containing azido-sialic acid glycans were immobilized to a solid support. The peptides linked to metabolically labeled sialylated glycans were then released from sialoglycopeptides and analyzed by mass spectrometry; in parallel, the glycans from azido-sialoglycoproteins were characterized by lectin microarrays. This method identified 75 unique N-glycosite-containing peptides from 55 different metabolically labeled sialoglycoproteins of which 42 were previously linked to cancer in the literature. A comparison of two of these glycoproteins, LAMP1 and ORP150, in histological tumor samples showed overexpression of these proteins in the cancerous tissue demonstrating that our approach constitutes a viable strategy to identify and discover sialoglycoproteins associated with cancer, which can serve as biomarkers for cancer diagnosis or targets for therapy.
Original language | English (US) |
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Article number | 11 |
Journal | Clinical Proteomics |
Volume | 12 |
Issue number | 1 |
DOIs | |
State | Published - 2015 |
Bibliographical note
Funding Information:This work was supported by federal funds from the National Cancer Institute, National Institutes of Health, grants 2R01CA112315, U01CA152813, R01CA112314, and P01HL107153-01. We also gratefully acknowledge the support of Drs. Robert Cole and Robert O’Meally from Johns Hopkins University for their assistance in mass spectrometry analysis and data processing.
Publisher Copyright:
© 2015 Tian et al.
Keywords
- Metabolic oligosaccharide engineering
- Pancreatic cancer cells
- Sialylated glycoproteins