The mass range of the instrument has to be estimated from the range of precursors found in the data set.It isn’t practical to filter by retention time, because this information is usually unavailable.The error of doing this is negligibleĬompared with other sources of uncertainty: The database, and the enzyme specificity. The number of observable peptides based on the protein mass, the average amino acid composition of Scan range and the observed nano-LC retention time range.įor reasons of speed, we prefer to make a calculated estimate of Was then filtered to exclude peptides outside the mass spectrometer In silico digests of the protein sequences. To estimate the number of observable peptides, Ishihama et. The difference from the original rule for singly and doubly charged data is negligible compared to the other sources of certainty, as described below. Mascot 2.5 and later count unique parent ions only once, regardless of charge state. However, if peptide matches exist in a number of charge states, such as 2+, 3+, 4+, 5+ and 6+, the rule causes emPAI to be overestimated. Mascot 2.4 and earlier followed this same rule, which works well for singly and doubly charged data. Including different charge states from the same peptide sequence. obtained best proportionalityįor a standard protein mixture by counting unique parent ions, Or the identity threshold, if there is no homology threshold. The count of observed peptides only includes peptide matches with scores at or above the homology threshold, What to include and what to exclude in these two counts. Peptides and N observable is the calculated number of observable peptides for each protein. Where N observed is the number of experimentally observed Present in a search result, and there are no parameter settings, soĮmPAI is 'always on', as long as the MS/MS search contains at least 100 spectra. Unlike the other quantitation protocols, the information required for emPAI is always Sequenced peptides per protein, Molecular & Cellular Proteomics 4 1265-1272 (2005) Y., et al., Exponentially modified protein abundance index (emPAI)įor estimation of absolute protein amount in proteomics by the number of Developed by Ishihama and colleagues, the key publication is Proteins in a mixture based on protein coverage by the peptide matches in a database Index (emPAI) offers approximate, label-free, relative quantitation of the The Exponentially Modified Protein Abundance

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