Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

Abstract

A method is presented for the rapid detection and characterization of trace amounts of peptides secreted from microorganisms, including pheromones, virulence factors, and quorum-sensing peptides. The procedure, based on targeted multistage MS, uses a novel matrix-assisted laser desorption͞ionization-ion trap mass spectrometer to overcome limitations of current MS methods (limited dynamic range, signal suppression effects, and chemical noise) that impair observation of low abundance peptides from complex biological matrixes. Here, secreted peptides that are hypothesized to be present in the supernatant, but that may not be sufficiently abundant to be observed in single-stage mass spectra, are subjected to multistage 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 groups I, II, and 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 C terminus of the peptide. 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 ssays that provide information on specific peptides that are secreted by living cells (e.g., toxins, pheromones, virulence factors, and quorum-sensing peptides) can be valuable as diagnostic aids. For example, the presence and identity of a specific microorganism may be inferred by detection of characteristic secreted peptides, which may also be used to distinguish between subtypes of a given strain. Several authors have described the use of MS to identify microorganisms based on fingerprint masses of protein constituents as well as secreted peptides and proteins (1). Recently, electrospray ionization-Fourier transform tandem MS (MS͞MS) of intact proteins has been applied for the characterization of biomarkers from Bacillus cereus T spores (2), and a combined approach of single-stage MS and MS͞MS was used to identify proteins secreted from adipocytes (3). Because these methods rely on the initial detection by single-stage MS of intact peptide ions from a highly complex milieu, it may be difficult to detect trace amounts of the secreted peptides. In addition to limitations imposed by dynamic range and signal suppression effects, low-level signals of interest tend to disappear into the ''chemical noise '' (4-6). An approach that has been widely...

Inactivation of a bacterial virulence pheromone by phagocyte-derived oxidants: New role for the NADPH oxidase in host defense

“…A 10-m i.d. PicoTip nanospray emitter (New Objective, Woburn, MA) was used with a spray voltage set between 1,800-2,100 V. AIP tandem MS fragmentation (30) sequencing of ions 961 and 977 was performed by using the QSTAR oMALDI source with a laser power of 5%, multichannel panel of 2,225, and a collision active dissociation gas of 55. Peptide samples for oMALDI analysis were further purified and plated by using C 18 ZipTips (Millipore) and eluted with 4.0 l of 50% 0.1% trifluoroacetic acid͞50% acetonitrile solution saturated with recrystallized ␣-cyano-4-hydroxycinnamic acid (10 mg͞ml).…”

Stepping Transfer Messenger RNA through the Ribosome

“…5). We collected and fragmented ions from all four of the samples in a hypothesis-driven multistage MS experiment (28) and noticed that the y 6 ion, indicative of the presence of SmpB, was obtained only from the complexes with the mutant tmRNA and not from the TP30 and 50 control samples (Fig. 4, spectra below A and B).…”

“…The fragmentation spectrum of the tryptic SmpB peptide ion at m/z 1250.67 (Fig. 5) was utilized as a characteristic signature to detect SmpB in hypothesis-driven multistage MS experiments in a fashion similar to that described for the detection of low-abundant peptides (28). These experiments were necessary to test for the presence of SmpB in cases in which peptide mass fingerprints alone did not permit a conclusive identification.…”

Why the move to microfluidics for protein analysis?

“…This kind of micro-or nanospray microfabricated emitter is appearing at a time when exciting developments in mass spectrometry are starting to emerge, such as new methodologies based on the capabilities of MS for the structural elucidation of protein complexes or complex mixture analysis. For example, Kalkum et al [18] recently introduced a methodology called 'hypothesis-driven multistage mass spectrometry' to assess the presence of particular compounds in a mixture: peptides of an expected mass are very selectively isolated by tandem mass spectrometry (MS/MS) and their identification further confirmed by MS/MS/MS (where the third mass stage is used to fragment the isolated molecules and further confirm their identity). No previous separation is required.…”