Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Journal of Visualized Experiments : JoVE
J Barallobre-BarreiroM Mayr

Abstract

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS an...Continue Reading

Citations

Jan 16, 2020·Applied Microbiology and Biotechnology·Ziyan WangHuili Lu
Sep 1, 2019·Molecular & Cellular Proteomics : MCP·Rekha RaghunathanJoseph Zaia
Jun 30, 2019·Laboratory Investigation; a Journal of Technical Methods and Pathology·Chiaki Nagai-OkataniAtsushi Kuno
Sep 18, 2020·Molecular & Cellular Proteomics : MCP·Nicholas M RileySharon J Pitteri
Nov 1, 2019·Molecular & Cellular Proteomics : MCP·Rekha RaghunathanJoseph Zaia
Feb 16, 2021·Molecular & Cellular Proteomics : MCP·Nicholas M RileySharon J Pitteri

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Methods Mentioned

BETA
glycosylation
Precipitation
electrophoresis
deamidation
assess

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