PRIDE Assigned Tags:Biomedical Dataset
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Human bladder,colon,kidney,liver cancer LC MS/MS
We propose that enrichment of low-abundance proteins benefits MPs finding. ProteoMinerTM is an equalizing technique by reducing high-abundance proteins and enriching low-abundance proteins in biological liquids. With triton X-100/TBS buffer extraction, ProteoMinerTM enrichment and peptide fractionation, 25 MPs with 73 unique peptides were identified from four human tissues, including 10 membrane proteins and 6 nucleus proteins. Then 18 MPs were confirmed with at least 2 unique peptide identification and 6 MPs were confirmed with 1 unique peptide identification by PRM assay. Hence these results demonstrated ProteoMinerTM as a powerful means in discovery of MPs.
Sample Processing Protocol
Tissues were ground in a tissue lyser and further lysed by probe sonication in the extraction buffer of PBS (pH 7.5), HEPES (20 mM, pH 8.0), NH4HCO3 (50 mM, pH 8.0) or triton buffer (1% TritonX-100, 20 mM Tris, 150 mM NaCl, pH 7.4) with protease inhibitor cocktail at 0-4 �? Bradford assay was adopted to measure the concentrations of extracted proteins with bovine serum albumin as a standard. Lower abundance proteins were enriched with ProteoMinerTM Protein Enrichment Kits according to manufacturer’s protocol. In brief, the columns were conditioned by the extraction buffer and then the extracted tissue proteins were loaded onto them under gravity speed. The nonspecific binding proteins were washed out by the same extraction buffer, and then the specific binding proteins were eluted by the elution buffer provided in the kits. The amount of input proteins and the volume of ProteoMinerTM beads used were calculated according to the kit manual suggestions. The effects of higher abundance protein depletion were evaluated by protein recovery ratio and the images of SDS-PAGE gels to show the protein pattern changes with ProteoMinerTM treatment.
Data Processing Protocol
Acquired MS data were converted to MGF files by Proteome Discoverer 1.4 (Thermo Scientific, Waltham, MA) and the exported MGF files were searched using Mascot version2.3.02 against the Swiss-prot human database ( released on 2017_04 with 20183 protein sequences). The false discovery rate (FDR) was set to less than 1% at both PSM and protein level. Trypsin was selected as the specific enzyme with a maximum of two missed cleavages permitted per peptide. Parameters included fixed modification: Carbamidomethylation (C); variable modification: Oxidation (M), Deamidatioin (N, Q). Data were searched with a peptide mass tolerance of 20ppm and a fragment mass tolerance of 0.05Da.
Li S, He Y, Lin Z, Xu S, Zhou R, Liang F, Wang J, Yang H, Liu S, Ren Y. Digging more missing proteins using an enrichment approach with ProteoMiner™. J Proteome Res. 2017 PubMed: 28960076
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