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Physicochemical properties determining the detection probability of tryptic peptides in Fourier transform mass spectrometry. A correlation study
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)
2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 19, 5872-5877 p.Article in journal (Refereed) Published
Abstract [en]

Sequence verification and mapping of posttranslational modifications require nearly 100% sequence coverage in the "bottom-up" protein analysis. Even in favorable cases, routine liquid chromatography-mass spectrometry detects from protein digests peptides covering 50-90% of the sequence. Here we investigated the reasons for limited peptide detection, considering various physicochemical aspects of peptide behavior in liquid chromatography-Fourier transform mass spectrometry (LC-FTMS). No overall correlation was found between the detection probability and peptide mass. In agreement with literature data, the signal increased with peptide hydrophobicity. Surprisingly, the pI values exhibited an opposite trend, with more acidic tryptic peptides detected with higher probability. A mixture of synthesized peptides of similar masses confirmed the hydrophobicity dependence but showed strong positive correlation between pI and signal response. An explanation of this paradoxal behavior was found through the observation that more acidic tryptic peptide lengths tend to be longer. Longer peptides tend to acquire higher average charge state in positive mode electrospray ionization than more basic but shorter counterparts. The induced-current detection in FTMS favors ions in higher charge states, thus providing the observed pI-FTMS signal anticorrelation.

Place, publisher, year, edition, pages
2004. Vol. 76, no 19, 5872-5877 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:uu:diva-95262DOI: 10.1021/ac049571qPubMedID: 15456309OAI: oai:DiVA.org:uu-95262DiVA: diva2:169411
Available from: 2006-12-20 Created: 2006-12-20 Last updated: 2013-09-03Bibliographically approved
In thesis
1. 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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