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Molecular & Cellular Proteomics 7:1489-1500, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Targeted Quantitative Analysis of Streptococcus pyogenes Virulence Factors by Multiple Reaction Monitoring^*,S

From the ^a Institute of Molecular Systems Biology, ETH Zurich, Zurich 8093, Switzerland, ^e Institute for Systems Biology, Seattle, Washington 98103, ^f Department of Clinical Sciences, Lund University, Lund 22184, Sweden, ^h Seminar für Statistik, ETH Zurich, Zurich 8092, Switzerland, ^j Bioinformatics Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore 138671, Singapore, ^b Competence Center for Systems Physiology and Metabolic Diseases, Zurich 8093, Switzerland, and ^k Faculty of Science, University of Zurich, Zurich 8092, Switzerland

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

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