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Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable... Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Biotechnology Springer Journals

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry

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References (43)

Publisher
Springer Journals
Copyright
Copyright © 2003 by Nature Publishing Group
Subject
Life Sciences; Life Sciences, general; Biotechnology; Biomedicine, general; Agriculture; Biomedical Engineering/Biotechnology; Bioinformatics
ISSN
1087-0156
eISSN
1546-1696
DOI
10.1038/nbt827
Publisher site
See Article on Publisher Site

Abstract

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.

Journal

Nature BiotechnologySpringer Journals

Published: May 18, 2003

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