Chapter title |
Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides.
|
---|---|
Chapter number | 12 |
Book title |
Proteomics in Systems Biology
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3341-9_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3339-6, 978-1-4939-3341-9
|
Authors |
Gottfried Pohlentz, Kristina Marx, Michael Mormann |
Editors |
Jörg Reinders |
Abstract |
Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Netherlands | 1 | 10% |
Unknown | 9 | 90% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 4 | 40% |
Researcher | 2 | 20% |
Professor | 1 | 10% |
Librarian | 1 | 10% |
Other | 1 | 10% |
Other | 1 | 10% |
Readers by discipline | Count | As % |
---|---|---|
Agricultural and Biological Sciences | 3 | 30% |
Biochemistry, Genetics and Molecular Biology | 2 | 20% |
Medicine and Dentistry | 2 | 20% |
Neuroscience | 1 | 10% |
Chemistry | 1 | 10% |
Other | 0 | 0% |
Unknown | 1 | 10% |