PRIDE Assigned Tags:Biological Dataset
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Normal human mitral valve proteome: a preliminary investigation by gel-based and gel-free proteomic approaches
The mitral valve is a highly complex structure which regulates blood flow from the left atrium to the left ventricle (LV) avoiding a significant forward gradient during diastole or regurgitation during systole. The integrity of the mitral valve is also essential for the maintenance of normal LV size, geometry, and function. Significant advances in the comprehension of the biological, functional, and mechanical behavior of the mitral valve have recently been made. However, current knowledge of protein components in the normal human mitral valve is still limited and complicated by the low cellularity of this tissue and the presence of high abundant proteins from the extracellular matrix. We employed here an integrated proteomic approach to analyse the protein composition of the normal human mitral valve and reported confident identification of 422 proteins, some of which have not been previously described in this tissue. In particular, we described the ability of pre-MS separation technique based on liquid-phase IEF and SDS-PAGE to identify the largest number of proteins. These initial results provide a valuable basis for future studies aimed at analysing in depth the mitral valve protein composition and at investigating potential pathogenetic molecular mechanisms.
Sample Processing Protocol
Gel pieces from 1-DE or 2-DE gels were destained and proteins were digested with trypsin. For gel slices derived from 1-DE gels cysteine residues were reduced with DTT (10 mmol/L) and derivatised by treatment with iodoacetamide (25 mmol/L) prior to addition of trypsin. Peptides mixtures were analysed using a nanoACQUITY UPLC system coupled to a Synapt-MS G1 (Waters Corporation, Milford, MA, USA) for DDA LC-MS/MS analysis. Proteins extracted from mitral valve tissue or valvular interstitial cells (VICs, dissolved in 25 mmol/L NH4HCO3 containing 0.1% RapiGest, were reduced with DTT (5 mmol/L) and derivatised by treatment with iodoacetamide (10 mmol/L) prior to digestion with trypsin. Additionally, peptide mixtures from mitral valve tissue were off-line fractionated by high pH reverse phase separation using ZipTipC18. Nanoscale LC separations of fractionated or unfractionated peptides mixtures obtained by low pH reverse phase separation were performed using a nanoACQUITY UPLC system coupled to a Synapt-MS G1 (Waters Corporation, Milford, MA, USA) for LC/MSE analysis.
Data Processing Protocol
Data were processed and searched using PLGS v2.5 (Waters Corporation, Milford, MA, USA) with a human species-specific UniProt database (release 2011-12; number of human sequence entries, 20249). For LC-MS/MS analysis of in gel digestions the following criteria were used: methionine oxidation and phosphorylation (Thr, Tyr and Ser) were considered as variable modifications, one missed cleavage per peptide was allowed, and the mass tolerance window was set to 20 ppm for peptide precursors and 0.05 Da for fragments. In parallel, the spectra were also searched against Uniprot database using Mascot (Matrix Science, London, UK). Valid identification required two or more peptides independently matching the same protein sequence, with a significant peptide score (higher than the identity score from Mascot). The following search criteria were used for protein identification from LC/MSE data: default search parameters included the “automatic” setting for mass accuracy (approximately 10 ppm for precursor ions and 25 ppm for product ions); a minimum of one peptide match per protein, a minimum of three consecutive product ion matches per peptide, and a minimum of seven total product ion matches per protein; up to one missed cleavage site allowed; carbamidomethyl-cysteine as fixed modification; and methionine oxidation as variable modification. Additionally, for LC/MSE, a randomized version of the databank was created in order to check the real false discovery rate, and only proteins identified in two replicates were considered as valid identifications.
Brioschi M, Baetta R, Ghilardi S, Gianazza E, Guarino A, Parolari A, Polvani G, Tremoli E, Banfi C. Normal human mitral valve proteome: A preliminary investigation by gel-based and gel-free proteomic approaches. Electrophoresis. 2016 Jul 23 PubMed: 27450324
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||65510||MItral valve proteome||180||2355||1734||28074||1878||
|2||65511||MItral valve proteome||171||2151||1665||26804||1792||
|3||65512||MItral valve proteome||173||2176||1673||25375||1781||
|4||65513||Mitral valve proteome||224||2747||2320||19643||2504||
|5||65514||Mitral valve proteome||212||2247||1842||13432||2003||
|6||65515||Mitral valve proteome||210||2020||1637||13145||1777||
|7||65516||mitral valve proteome||143||2204||1807||63253||1976||
|8||65517||mitral valve proteome||128||1796||1403||85280||1537||
|9||65518||mitral valve proteome||188||2685||2091||86818||2304||
|10||65519||mitral valve proteome||150||2099||1672||77097||1854||