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The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.
Pubmed ID: 23943586 RIS Download
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emPAI (exponentially modified protein abundance index), developed by Ishihama et al., is a measure to describe the protein composition in sample solutions. When the total protein amount in the sample is available, emPAI can be converted to the absolute amount of each protein in the sample. emPAI is derived from PAI, which is defined as the number of the observed peptides divided by the number of the observable peptides per protein. We recently found that log (PAI) had linear relationship to the protein amounts, and that emPAI, 10^(PAI)-1, was proportional to the protein amounts for whole cell lysate digested by trypsin. The accuracy of this method was within factor 5, similar or better than determination of abundance by protein staining
View all literature mentionsA quantitative proteomics software package for analyzing large-scale mass-spectrometric data sets. It is a set of algorithms that include peak detection and scoring of peptides, mass calibration, database searches for protein identification, protein quantification, and provides summary statistics.
View all literature mentionsCell line HeLa is a Cancer cell line with a species of origin Homo sapiens
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