Research output: Contribution to journal › Article › peer-review
Original language | English |
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Article number | e55674 |
Journal | Journal of Visualized Experiments |
Volume | 2017 |
Issue number | 122 |
DOIs | |
Accepted/In press | 28 Dec 2016 |
Published | 21 Apr 2017 |
Additional links |
Glycoproteomics of the extracellular_BARALLOBRE-BARREIRO_Published21April2017_GOLD VoR
Glycoproteomics_of_the_extracellular_BARALLOBRE_BARREIRO_Published21April2017_GOLD_VoR.pdf, 680 KB, application/pdf
Uploaded date:04 May 2017
Version:Final published version
Licence:CC BY-NC-ND
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.
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