Average peptide score: a useful parameter for identification of proteins derived from database searches of liquid chromatography/tandem mass spectrometry data

Abstract

The quantity and variable quality of data that can be generated from liquid chromatography (LC)/mass spectrometry (MS)-based proteomics analyses creates many challenges in interpreting the spectra in terms of the actual proteins in a complex sample. In spite of improvements in algorithms that match putative peptide sequences to MS/MS spectra, the assembly of these lists of possible or probable peptides into a 'correct' set of proteins is still problematic. We have observed a trend in a simple relationship, derived from standard database search outputs, which can be useful in assessing the quality of a MS/MS-based protein identification. Specifically, the ratio of the protein score and number of non-redundant peptides, or average peptide score (APS), can facilitate initial filtering of database search results in addition to providing a useful measure of confidence for the proteins identified. This parameter has been applied to results from the analysis of multi-protein complexes derived from pull-down experiments analyzed using a two-dimensional LC/MS/MS workflow. In particular, the complex list of protein identifications derived from a drug affinity pull-down with immobilized ampicillin and an E. coli lysate was greatly simplified by applying the APS as a filter, allowing for facile identification of the penicillin-binding proteins known to interact with ampicillin. Furthermore, an APS threshold can be used for any data sets derived from electrospray ionization (ESI)- or matrix-assisted laser desorption/ionization (MALDI)-MS/MS experiments and is also not specific to any database search program.

Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

“…Peptide charges of +1, +2, and +3 were selected. Individual ions with MASCOT scores higher than 20 were used, making sure the “average peptide scores” of all identified proteins exceeded 20, a threshold commonly used for confident protein identification from tandem MS data (36). Only bold red peptides were also considered, effectively removed duplicate homologous proteins from the results.…”

Differential Proteomic Analysis of Human Erythroblasts Undergoing Apoptosis Induced by Epo-Withdrawal

“…The raw data files were processed using Proteome Discoverer software v1.2 (Thermo Scientific) with searches performed against the SwissProt Human database (54523 entries) using the Mascot search engine v1.9 (Matrix Science) with the following criteria; peptide tolerance  = 10 ppm, trypsin as the enzyme, carbamidomethylation of cysteine as a fixed modification and oxidation of methionine and phosphorylation of serine, threonine and tyrosine as variable modifications. Individual ions with Mascot scores higher than 20 were used, making sure the average peptide scores of all identified proteins exceeded 20, a threshold commonly used for confident protein identification from tandem MS data [24]. The reverse database search option was enabled and all data was filtered to satisfy false discovery rate (FDR) of less than 5%.…”

The minor salivary gland proteome in Sjögren’s syndrome

“…The average peptide score was designated as protein score to show how well the protein was described by the peptides (Chepanoske et al , 2005). The results from the automated LC‐ESI‐MS/MS runs were validated by manual inspection of two MS/MS spectra of one protein from each peptide fraction.…”