This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A new method for the rapid detection and characterization of trace amounts of peptides secreted from microorganisms, including pheromones, virulence factors, and quorum sensing peptides was developed. The procedure, based on targeted multi-stage mass spectrometry (MS), utilizes a novel MALDI-ion trap mass spectrometer to overcome limitations of current MS methods (limited dynamic range, signal suppression effects, chemical noise) that impair observation of low abundance peptides from complex biological matrixes. As a proof of concept, secreted peptides that are hypothesized to be present in the supernatant, but that are not be sufficiently abundant to be observed in single-stage mass spectra, are subjected to multi-stage MS. Highly specific fragmentation signatures enable unambiguous identification of the peptides of interest and differentiation of the signals from the background. As examples, we demonstrate the rapid (1 min) determination of the mating type of cells in colonies of Saccharomyces cerevisiae and the elucidation of autoinducing peptides (AIPs) from supernatants of pathogenic Staphylococci. We confirm the primary structures of the agrD encoded cyclic AIPs of Staphylococcus aureus for group I, II, IV and provide direct evidence that the native group-III AIP is a heptapeptide (INCDFLL). We also show that the homologous peptide from Staphylococcus intermedius is a nonapeptide (RIPTSTGFF) with a lactone ring formed through condensation of the serine side chain with the peptide's C-terminus. This is the first demonstration of cyclization in a staphylococcal AIP that occurs via lactone formation. These examples demonstrate the analytical power of the present procedure for characterizing secreted peptides and its potential utility for identifying microorganisms.A manuscript describing this work has been published:M. Kalkum, G.J. Lyon and B.T. Chait Detection of Secreted Peptides using Hypothesis-driven Multistage Mass Spectrometry Proc Natl Acad. Sci. USA 100 (2003) 2795-2800.
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