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

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.

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Figure 1: Schematic diagram of quantitative analysis of N-linked glycopeptides.
Figure 2: Isolation of glycoproteins from serum.
Figure 3: Quantitative analysis of formerly N-linked glycosylated peptides by MALDI QqTOF MS.
Figure 4: Subcellular location of glycoproteins identified from a crude microsomal fraction of LNCaP prostate epithelial cells.

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Acknowledgements

This work was supported in part by grants from the National Cancer Institute (R33, CA93302) and by a sponsored research agreement from MacroGenics. The Institute for Systems Biology is supported by a generous gift from Merck & Co. D.B.M is supported by a National Institutes of Health career development award (K08CA97282-01).

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Correspondence to Ruedi Aebersold.

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Zhang, H., Li, Xj., Martin, D. et al. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21, 660–666 (2003). https://doi.org/10.1038/nbt827

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