Article

The Standard Protein Mix Database: A Diverse Data Set To Assist in the Production of Improved Peptide and Protein Identification Software Tools

Institute for Systems Biology, Seattle, Washington 98103, USA.
Journal of Proteome Research (Impact Factor: 5). 02/2008; 7(1):96-103. DOI: 10.1021/pr070244j
Source: PubMed

ABSTRACT Tandem mass spectrometry (MS/MS) is frequently used in the identification of peptides and proteins. Typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptides in a database. The probabilities that these spectrum-to-sequence assignments are correct can be determined by statistical software such as PeptideProphet or through estimations based on reverse or decoy databases. However, many of the software applications that assign probabilities for MS/MS spectra to sequence matches were developed using training data sets from 3D ion-trap mass spectrometers. Given the variety of types of mass spectrometers that have become commercially available over the last 5 years, we sought to generate a data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools. We analyzed the proteolytic peptides in a mixture of tryptic digests of 18 proteins, named the "ISB standard protein mix", using 8 different mass spectrometers. These include linear and 3D ion traps, two quadrupole time-of-flight platforms (qq-TOF), and two MALDI-TOF-TOF platforms. The resulting data set, which has been named the Standard Protein Mix Database, consists of over 1.1 million spectra in 150+ replicate runs on the mass spectrometers. The data were inspected for quality of separation and searched using SEQUEST. All data, including the native raw instrument and mzXML formats and the PeptideProphet validated peptide assignments, are available at http://regis-web.systemsbiology.net/PublicDatasets/.

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    ABSTRACT: Background Mass spectrometry-based protein identification is a very challenging task. The main identification approaches include de novo sequencing and database searching. Both approaches have shortcomings, so an integrative approach has been developed. The integrative approach firstly infers partial peptide sequences, known as tags, directly from tandem spectra through de novo sequencing, and then puts these sequences into a database search to see if a close peptide match can be found. However the current implementation of this integrative approach has several limitations. Firstly, simplistic de novo sequencing is applied and only very short sequence tags are used. Secondly, most integrative methods apply an algorithm similar to BLAST to search for exact sequence matches and do not accommodate sequence errors well. Thirdly, by applying these methods the integrated de novo sequencing makes a limited contribution to the scoring model which is still largely based on database searching. Results We have developed a new integrative protein identification method which can integrate de novo sequencing more efficiently into database searching. Evaluated on large real datasets, our method outperforms popular identification methods.
    BMC Bioinformatics 01/2013; 14(2). DOI:10.1186/1471-2105-14-S2-S24 · 2.67 Impact Factor
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    • "Database search for peptide identification was done with Sequest [13], and the statistical analysis of identification results was done with PeptideProphet [18]. Notice that some possible contaminants are considered in the datasets [20], and the summary of the two datasets is given in Table 1. "
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    • "This dataset uses spectra generated from a linear ion trap Fourier transform instrument that was published in [8]. In particular the spectra from Mixture 3 was used, where 16 known trypsin-digested proteins from different mammals were analyzed. "
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    ABSTRACT: PeptideProphet is a post-processing algorithm designed to evaluate the confidence in identifications of MS/MS spectra returned by a database search. In this manuscript we describe the "what and how" of PeptideProphet in a manner aimed at statisticians and life scientists who would like to gain a more in-depth understanding of the underlying statistical modeling. The theory and rationale behind the mixture-modeling approach taken by PeptideProphet is discussed from a statistical model-building perspective followed by a description of how a model can be used to express confidence in the identification of individual peptides or sets of peptides. We also demonstrate how to evaluate the quality of model fit and select an appropriate model from several available alternatives. We illustrate the use of PeptideProphet in association with the Trans-Proteomic Pipeline, a free suite of software used for protein identification.
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