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PhosTShunter: A fast and reliable tool to detect phosphorylated peptides in liquid chromatography Fourier transform tandem mass spectrometry data sets
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics. (Laboratory for Biological and Medical Mass Spectrometry)
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics. (Laboratory for Biological and Medical Mass Spectrometry)
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics. (Laboratory for Biological and Medical Mass Spectrometry)
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics. (Laboratory for Biological and Medical Mass Spectrometry)
2006 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 3, 659-668 p.Article in journal (Refereed) Published
Abstract [en]

A database independent search algorithm for the detection of phosphopeptides is described. The program interrogates the tandem mass spectra of LC-MS/MS data sets regarding the presence of phosphorylation specific signatures. To achieve maximum informational content, the complementary fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD) are used independently for peptide fragmentation. Several criteria characteristic for peptides phosphorylated on either serine or threonine residues were evaluated. The final algorithm searches for product ions generated by either the neutral loss of phosphoric acid or the combined neutral loss of phosphoric acid and water. Various peptide mixtures were used to evaluate the program. False positive results were not observed because the program utilizes the parts-per-million mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Additionally, false negative results were not generated owing to the high sensitivity of the chosen criteria. The limitations of database dependent data interpretation tools are discussed and the potential of the novel algorithm to overcome these limitations is illustrated.

Place, publisher, year, edition, pages
2006. Vol. 5, no 3, 659-668 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:uu:diva-95354DOI: 10.1021/pr0503836PubMedID: 16512682OAI: oai:DiVA.org:uu-95354DiVA: diva2:169531
Available from: 2007-01-17 Created: 2007-01-17 Last updated: 2017-12-14Bibliographically approved
In thesis
1. New Proteomics Methods and Fundamental Aspects of Peptide Fragmentation
Open this publication in new window or tab >>New Proteomics Methods and Fundamental Aspects of Peptide Fragmentation
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Nya Proteomik Metoder och Fundamentala Aspekter av Peptid Fragmentering
Abstract [en]

The combination of collision-activated dissociation, (CAD) and electron capture dissociation, (ECD) yielded a 125% increase in protein identification. The S-score was developed for measuring the information content in MS/MS spectra. This measure made it possible to single out good quality spectra that were not identified by a search engine. Poor quality MS/MS data was filtered out, streamlining the identification process.

A proteomics grade de novo sequencing approach was developed enabling to almost completely sequence 19% of all MS/MS data with 95% reliability in a typical proteomics experiment.

A new tool, Modificomb, for identifying all types of modifications in a fast, reliable way was developed. New types of modifications have been discovered and the extent of modifications in gel based proteomics turned out to be greater than expected.

PhosTShunter was developed for sensitive identification of all phosphorylated peptides in an MS/MS dataset.

Application of these programs to human milk samples led to identification of a previously unreported and potentially biologically important phosphorylation site.

Peptide fragmentation has been studied. It was shown emphatically on a dataset of 15.000 MS/MS spectra that CAD and ECD have different cleavage preferences with respect to the amino acid context.

Hydrogen rearrangement involving z• species has been investigated. Clear trends have been unveiled. This information elucidated the mechanism of hydrogen transfer.

Partial side-chain losses in ECD have been studied. The potential of these ions for reliably distinguishing Leu/Iso residues was shown. Partial sidechain losses occurring far away from the cleavage site have been detected.

A strong correlation was found between the propensities of amino acids towards peptide bond cleavage employing CAD and the propensity of amino acids to accept in solution backbone-backbone H-bonds and form stable motifs. This indicated that the same parameter governs formation of secondary structures in solution and directs fragmentation in peptide ions by CAD.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 264
Keyword
Bioinformatics, Proteomics, Peptide fragmentation, Bioinformatik
Identifiers
urn:nbn:se:uu:diva-7438 (URN)978-91-554-6775-X (ISBN)
Public defence
2007-02-08, B21, BMC, Husargatan 3, Uppsala, 14:15
Opponent
Supervisors
Available from: 2007-01-17 Created: 2007-01-17 Last updated: 2013-09-04Bibliographically approved

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