Extracellular Matrix Analysis of Human Varicose Veins
To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix.
Sample Processing Protocol
Vein tissues were subjected to our sequential extraction procedure. In brief, vessels were washed briefly with cold PBS, incubated in 0.5M sodium chloride (NaCl) plus proteinase and phosphatase inhibitors for 4 hours, followed by decellularization using 0.08% SDS and extraction of ECM proteins using 4M guanidine hydrochloride (GuHCl) for 48h before processing for proteomics analysis. The salt and guanidine fractions were deglycosylated in O18-water, reduced, alkylated and digested by trypsin overnight. Tryptic peptides were separated on a nanoflow LC system and directly injected into Q Exactive Plus MS. Spectra were collected using full MS mode over the mass-to-charge (m/z) range 350-1600. MS/MS was performed on the top 15 ions in each MS scan using the data-dependent acquisition mode with dynamic exclusion enabled.
Data Processing Protocol
MS data were searched using Proteome Discoverer (version 18.104.22.1688) using Mascot (version 2.3.01) against UniProt/Swiss-Prot human database (version 2015_02). The following parameters were used: precursor mass tolerance 10ppm, fragment mass tolerance 20mmu, trypsin, 2 missed cleavage, carbamidomethylation of cysteine as fixed modification, oxidation of methionine, proline, lysine and O18-deamidation of asparagine as varible modifications, FDR <1%.
Barallobre-Barreiro J, Oklu R, Lynch M, Fava M, Baig F, Yin X, Barwari T, Potier DN, Albadawi H, Jahangiri M, Porter KE, Watkins MT, Misra S, Stoughton J, Mayr M; Extracellular Matrix Remodeling in Response to Venous Hypertension: Proteomics of Human Varicose Veins., Cardiovasc Res, 2016 Apr 11, PubMed: 27068509