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Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
2006 (Engelska)Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, nr 12, s. 2384-2391Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The complexity of the uman proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at ≥1 % level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.

Ort, förlag, år, upplaga, sidor
2006. Vol. 5, nr 12, s. 2384-2391
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-95353DOI: 10.1074/mcp.M600248-MCP200ISI: 000242852000015PubMedID: 17015437OAI: oai:DiVA.org:uu-95353DiVA, id: diva2:169530
Tillgänglig från: 2007-01-17 Skapad: 2007-01-17 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
Ingår i avhandling
1. New Proteomics Methods and Fundamental Aspects of Peptide Fragmentation
Öppna denna publikation i ny flik eller fönster >>New Proteomics Methods and Fundamental Aspects of Peptide Fragmentation
2007 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2007. s. 56
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 264
Nyckelord
Bioinformatics, Proteomics, Peptide fragmentation, Bioinformatik
Identifikatorer
urn:nbn:se:uu:diva-7438 (URN)978-91-554-6775-X (ISBN)
Disputation
2007-02-08, B21, BMC, Husargatan 3, Uppsala, 14:15
Opponent
Handledare
Tillgänglig från: 2007-01-17 Skapad: 2007-01-17 Senast uppdaterad: 2013-09-04Bibliografiskt granskad
2. Characterization of Polypeptides by Tandem Mass Spectrometry Using Complementary Fragmentation Techniques
Öppna denna publikation i ny flik eller fönster >>Characterization of Polypeptides by Tandem Mass Spectrometry Using Complementary Fragmentation Techniques
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2006. s. 65
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 252
Nyckelord
Analytical chemistry, Mass Spectrometry, Electron capture dissociation (ECD), Collision-activated dissociation (CAD), Proteomics, Post-translational modifications, De Novo sequencing, Bioinformatics, Analytisk kemi
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:uu:diva-7409 (URN)91-554-6755-5 (ISBN)
Disputation
2007-01-11, B21, BMC, Husargatan 3, Uppsala, 14:15
Opponent
Handledare
Tillgänglig från: 2006-12-20 Skapad: 2006-12-20 Senast uppdaterad: 2013-09-04Bibliografiskt granskad

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