Development and validation of a spectral library searching method for peptide identification from MS/MS

Abstract

A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptides by sequence database searching methods, which often are time-consuming and error-prone. A more precise and efficient method, in which previously observed and identified peptide MS/MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching, is seen as a promising alternative. To that end, an open-source, functionally complete, high-throughput and readily extensible MS/MS spectral searching tool, SpectraST, was developed. A high-quality spectral library was constructed by combining the high-confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines. The resulting library consists of over 30 000 spectra for Saccharomyces cerevisiae. Using this library, SpectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits. A unique advantage of SpectraST is its full integration into the popular Trans Proteomic Pipeline suite of software, which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment, quantification, and data visualization. This method of spectral library searching is especially suited for targeted proteomics applications, offering superior performance to traditional sequence searching.

Bifurcating fragmentation behavior of gas-phase tryptic peptide dications in collisional activation

“…At the same time, sequences X 1 Y 2 Z 3 and X 1 Z 2 Y 3 gave strongly anti-correlating frequencies for aa 2 and aa 3 (r ϭ Ϫ0.86) in y mϪ3 ϩ formation ( Figure 5, right), as expected for an asymmetric oxazolone intermediate. In y mϪ4 ϩ formation, the two N-terminal to the cleavage site amino acids aa 3 and aa 4 also behave in an anti-symmetric way.…”

“…Two alternative approaches were pursued in implementation of this idea. One approach was to collect a massive database of tandem mass spectra of all possible peptides and to make direct comparison between the standard MS/MS datasets and the experimental ones [3]. Although this approach has many merits, it faces a huge challenge in the form of an astronomical number of peptide sequences existing in nature.…”

Exploring the potential of public proteomics data

“…The creation of spectral libraries most strongly benefits from the growing amount of shared data 51, 52. Several repositories, including PeptideAtlas, GPMDB, and PRIDE, and research groups such as the one at NIST (National Institute of Standards and Technology), provide spectral libraries for different species, which can in turn be used to perform spectral searches.…”

Evaluating reproducibility and similarity of mass and intensity data in complex spectra—applications to tubulin

“…While several papers [1][2][3][4] have used the spectral dot product for evaluating spectral similarity, particularly the similarity of a query spectrum against library references [1, 3, 4], only one paper [4] has addressed the critical issue of how an algorithm or investigator attributes significance to a spectral dot product-a nontrivial detail because the dot product alone can yield equivocal information. Dot product ambiguity arises both from the number of peaks, as has been described [4], and from the different sampling distribution of the dot product at different values. We introduce the 95% confidence intervals on the spectral dot product to address both these sources of ambiguity.…”

“…The spectral dot product has an established use as the means by which mass spectra are similar to spectra of known substances in a reference library [1,3,4,7]. Monigiatti and Berndt took a different approach and used consensus spectra for the purpose of database searching with peptide mass fingerprints by using a Spearman's rank correlation coefficient [8].…”