Differential Glycosite Profiling-A Versatile Method to Compare Membrane Glycoproteomes.

Abstract

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose-a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.

• Authors: Malwina Michalak, Martin Simon Kalteis, Aysel Ahadova, Matthias Kloor, Mark Kriegsmann, Katharina Kriegsmann, Uwe Warnken, Dominic Helm, Jürgen Kopitz
• Journal: Molecules (Basel, Switzerland) (Molecules) Vol. 26 Issue 12 (Jun 10 2021) ISSN: 1420-3049 [Electronic] Switzerland
• PMID: 34200965 (Publication Type: Journal Article)

Chemical References

• Glycopeptides
• Glycoproteins
• Membrane Proteins
• Polysaccharides
• Glucose

Topics

• Cell Line, Tumor
• Glucose (metabolism)
• Glycopeptides (metabolism)
• Glycoproteins (metabolism)
• Glycosylation
• Humans
• Membrane Proteins (metabolism)
• Polysaccharides (metabolism)
• Proteomics (methods)