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

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 analysis.

Biomaterial characterization of off-the-shelf decellularized porcine pericardial tissue for use in prosthetic valvular applications

“…Our results show that the decellularization process lowers the GAGs compared with the native scaffolds (Figure ), likely due to the caustic nature of SDS. Current research shows that many ECM proteins are glycosylated, which in turn affects protein folding, solubility, binding, and degradation (Barallobre‐Barreiro, Baig, Fava, Yin, & Mayr, ). Potentially reducing the concentration of SDS would allow the GAG content to be more representative of native cusps.…”

Decellularized Splenic Matrix as a Scaffold for Spleen Bioengineering

“…Cellular proteins present in native tissues can make it difficult to identify the components of the ECM. But it is known that the use of SDS can promote enrichment of matrix proteins, facilitating the identification, by removing cellular proteins and improving the solubilization of matrix proteins ( Krasny et al, 2016 ; Barallobre-Barreiro et al, 2017 ). The same is not true for proteoglycans, ECM regulators, secreted factors and proteins associated with the matrix with reduced intensity after decellularization.…”

“…For proteomic analysis, lyophilized samples of native and decellularized tissues ( n = 4) were processed according to the modified protocol of Barallobre-Barreiro et al (2017) . First, cellular contaminants in native tissues were removed.…”

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…Several approaches have been developed to address these methodological blind spots, and they should benefit glycoproteomic methods, too. Specifically, sample preparation protocols that permit use of detergents during protein isolation should improve characterization of membrane bound glycoproteins, i.e., a substantial portion of the glycoproteome (453)(454)(455)(456)(457)(458)(459)(460). Automated sample preparation protocols also promise to streamline glycoproteomics in concert with other "omics", including glycomics and de-glycoproteomics (461).…”