HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: T400022-MCP200v1 4/6/835    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nielsen, M. L. Articles by Zubarev, R. A. Search for Related Content PubMed PubMed Citation Articles by Nielsen, M. L. Articles by Zubarev, R. A. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 4:835-845, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Technology

Improving Protein Identification Using Complementary Fragmentation Techniques in Fourier Transform Mass Spectrometry^*

Michael L. Nielsen, Mikhail M. Savitski and Roman A. Zubarev

From the Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, S-75123 Uppsala, Sweden

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 (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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Falth, K. Skold, M. Svensson, A. Nilsson, D. Fenyo, and P. E. Andren Neuropeptidomics Strategies for Specific and Sensitive Identification of Endogenous Peptides Mol. Cell. Proteomics, July 1, 2007; 6(7): 1188 - 1197. [Abstract] [Full Text] [PDF]

				M. Lerner, M. Corcoran, D. Cepeda, M. L. Nielsen, R. Zubarev, F. Ponten, M. Uhlen, S. Hober, D. Grander, and O. Sangfelt The RBCC Gene RFP2 (Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD Mol. Biol. Cell, May 1, 2007; 18(5): 1670 - 1682. [Abstract] [Full Text] [PDF]

				M. L. Nielsen, M. M. Savitski, and R. A. Zubarev Extent of Modifications in Human Proteome Samples and Their Effect on Dynamic Range of Analysis in Shotgun Proteomics Mol. Cell. Proteomics, December 1, 2006; 5(12): 2384 - 2391. [Abstract] [Full Text] [PDF]

				W. Haas, B. K. Faherty, S. A. Gerber, J. E. Elias, S. A. Beausoleil, C. E. Bakalarski, X. Li, J. Villen, and S. P. Gygi Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics Mol. Cell. Proteomics, July 1, 2006; 5(7): 1326 - 1337. [Abstract] [Full Text] [PDF]

				M. M. Savitski, M. L. Nielsen, and R. A. Zubarev ModifiComb, a New Proteomic Tool for Mapping Substoichiometric Post-translational Modifications, Finding Novel Types of Modifications, and Fingerprinting Complex Protein Mixtures Mol. Cell. Proteomics, May 1, 2006; 5(5): 935 - 948. [Abstract] [Full Text] [PDF]

				J. V. Olsen, L. M. F. de Godoy, G. Li, B. Macek, P. Mortensen, R. Pesch, A. Makarov, O. Lange, S. Horning, and M. Mann Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap Mol. Cell. Proteomics, December 1, 2005; 4(12): 2010 - 2021. [Abstract] [Full Text] [PDF]

				M. M. Savitski, M. L. Nielsen, and R. A. Zubarev New Data Base-independent, Sequence Tag-based Scoring of Peptide MS/MS Data Validates Mowse Scores, Recovers Below Threshold Data, Singles Out Modified Peptides, and Assesses the Quality of MS/MS Techniques Mol. Cell. Proteomics, August 1, 2005; 4(8): 1180 - 1188. [Abstract] [Full Text] [PDF]

				R. J. Chalkley, P. R. Baker, L. Huang, K. C. Hansen, N. P. Allen, M. Rexach, and A. L. Burlingame Comprehensive Analysis of a Multidimensional Liquid Chromatography Mass Spectrometry Dataset Acquired on a Quadrupole Selecting, Quadrupole Collision Cell, Time-of-flight Mass Spectrometer: II. New Developments in Protein Prospector Allow for Reliable and Comprehensive Automatic Analysis of Large Datasets Mol. Cell. Proteomics, August 1, 2005; 4(8): 1194 - 1204. [Abstract] [Full Text] [PDF]