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Improving protein identification using complementary fragmentation techniques in Fourier transform mass spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences. (Laboratory for Biological and Medical Mass Spectrometry)
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences. (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)
2005 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 6, 835-845 p.Article in journal (Refereed) Published
Abstract [en]

Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.

Place, publisher, year, edition, pages
2005. Vol. 4, no 6, 835-845 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:uu:diva-95349PubMedID: 15772112OAI: oai:DiVA.org:uu-95349DiVA: diva2:169526
Available from: 2007-01-17 Created: 2007-01-17 Last updated: 2013-09-03Bibliographically 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
2. Characterization of Polypeptides by Tandem Mass Spectrometry Using Complementary Fragmentation Techniques
Open this publication in new window or tab >>Characterization of Polypeptides by Tandem Mass Spectrometry Using Complementary Fragmentation Techniques
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the growing field of proteomics identification of proteins by tandem mass spectrometry (MS/MS) is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein database. One problem with this approach is the false-positive identifications. MS-based proteomics experiments are further affected by a rather poor efficiency typical in the range of 10-15%, implicating that only a low percentage of acquired mass spectrometric data is significantly identified and assigned a peptide sequence.

In this thesis improvement in spectrum specificity is accomplished by using a combination of high-accuracy mass spectrometry and techniques that will yield complementary sequence information. Performing collision-activated dissociation (CAD) and electron capture dissociation (ECD) upon the same peptide ion will yield such complementary sequence information. Implementing this into a proteomics approach and showing the advantages of using complementary fragmentation techniques for improving peptide identification is shown. Furthermore, a novel database-independent score is introduced (S-score) based upon the maximum length of the peptide sequence tag derived from complementary use of CAD and ECD. The S-score can be used to separate poor quality spectra from good quality spectra. An-other aspect of the S-score is the development of the ‘reliable sequence tag’ which can be used to recover below threshold identifications and for a reliable backbone for de novo sequencing of peptides.

A novel proteomics-grade de novo sequencing algorithm has also been developed based upon the RST, which can retrieve peptide identification with the highest reliability (>95%). Furthermore, a novel software tool for unbiased identifications of any post-translational modifications present in a peptide sample is introduced (ModifiComb). Combining all the tools described in this thesis increases the identification specificity (>30 times), recovers false-negative identifications and increases the overall efficiency of proteomics experiements to above 40%. Currently one of the highest achieved in large-scale proteomics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 252
Keyword
Analytical chemistry, Mass Spectrometry, Electron capture dissociation (ECD), Collision-activated dissociation (CAD), Proteomics, Post-translational modifications, De Novo sequencing, Bioinformatics, Analytisk kemi
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-7409 (URN)91-554-6755-5 (ISBN)
Public defence
2007-01-11, B21, BMC, Husargatan 3, Uppsala, 14:15
Opponent
Supervisors
Available from: 2006-12-20 Created: 2006-12-20 Last updated: 2013-09-04Bibliographically approved

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