Title : 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