TY - JOUR
T1 - Glycoproteomics of the extracellular matrix
T2 - A method for intact glycopeptide analysis using mass spectrometry
AU - Barallobre-Barreiro, Javier
AU - Baig, Ferheen
AU - Fava, Marika
AU - Yin, Xiaoke
AU - Mayr, Manuel
PY - 2017/4/21
Y1 - 2017/4/21
N2 - 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.
AB - 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.
KW - Biochemistry
KW - Cardiovascular
KW - Extracellular matrix
KW - Fibrosis
KW - Glycoprotein
KW - Issue 122
KW - Proteomics
KW - Systems biology
UR - http://www.scopus.com/inward/record.url?scp=85017619555&partnerID=8YFLogxK
U2 - 10.3791/55674
DO - 10.3791/55674
M3 - Article
AN - SCOPUS:85017619555
SN - 1940-087X
VL - 2017
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 122
M1 - e55674
ER -