Abstract

False positive peptide identifications are a major concern in the field of peptidecentric, mass spectrometry-driven gel-free proteomics. They occur in regions where the score distributions of true positives and true negatives overlap. Removal of these false positive identifications necessarily involves a trade-off between sensitivity and specificity. Existing postprocessing tools typically rely on a fixed or semifixed set of assumptions in their attempts to optimize both the sensitivity and the specificity of peptide and protein identification using MS/MS spectra. Because of the expanding diversity in available proteomics technologies, however, these postprocessing tools often struggle to adapt to emerging technology-specific peculiarity. Here we present a novel tool named Peptizer that solves this adaptability issue by making use of pluggable assumptions. This research-oriented postprocessing tool also includes a graphical user interface to perform efficient manual validation of suspect identifications for optimal sensitivity recovery. Peptizer is open source software under the Apache2 license and is written in Java.

Highlights

  • False positive peptide identifications are a major concern in the field of peptidecentric, mass spectrometry-driven gel-free proteomics

  • The most commonly applied method is based on sequence database searching by database search engines such as SEQUEST [4], Mascot [5], X!Tandem [6], Virtual Expert Mass Spectrometrist [7], or Open Mass Spectrometry Search Algorithm [8]

  • Peptizer was developed as a postprocessing tool aimed at separating true and false positive peptide identifications in a highly configurable manner without relying on any built-in assumptions

Read more

Summary

EXPERIMENTAL PROCEDURES

MS/MS Data—The MS/MS spectra used in this study have been published previously [24]. Full experimental details are provided in the supplemental information. Fractions of 4 min wide were collected and treated with 2,4,6-trinitrobenzenesulfonic acid Such modified primary fractions were loaded for the secondary COFRADIC run wherein the ␣-amino-blocked peptides, which show no altered chromatographic properties, are collected. To estimate the false positive distribution we performed Mascot searches against a concatenated decoy database as described previously [29]. The aggregator used summed all votes together and marked the peptide identification as suspicious if the result was equal to or greater than 2 (or when an agent with veto rights declines)

RESULTS
TABLE I Agent configuration
TABLE II Summary of experimental Peptizer usage
True positives rejected by user
DISCUSSION
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call