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

Abstract

Assays 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 (MS/MS) 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 MS/MS 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 applied with great success to the detection of trace compounds in complex mixtures involves MS/MS (7) by using targeted compound analysis (reviewed in ref. 8). Here, we present a method building on these previous approaches for the rapid, unambiguous detection of trace amounts of peptides secreted from microorganisms, by using multistage MS (MS ^{and MS) analysis of crude supernatant mixtures with a novel matrix-assisted laser desorption/ionization (MALDI)-quadrupole ion trap mass spectrometer (}9). In this procedure, secreted peptide ions that are hypothesized to be present in the supernatant, but that are not sufficiently abundant to be observed in the regular 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 determination of the mating type of colonies of Saccharomyces cerevisiae and the detection and structural characterization of autoinducing peptides (AIPs) of Staphylococcus aureus that play a crucial role in quorum sensing and bacterial interference.

Haploid yeast cells secrete one of two types of peptide pheromones, i.e., mating factors a or α (10–12). Their expression is controlled (but not encoded) by the alleles at the mating type loci MATa or MATα. MATa cells secrete mating factor a, which binds to a receptor on the surface of MATα cells, and MATα cells secrete mating factor α, which binds to a receptor on the surface of MATa cells. Receptor binding of a mating factor triggers the mating response, i.e., activation of proteins required for cell fusion (13–15). Knowledge about the presence of a specific mating factor is an indicator of a haploid strain, information that is essential when designing mating experiments for genetic studies. Current mating type assays use methods that challenge the strain in question with tester-strains (16) or that analyze it by PCR (17). These assays require overnight incubation (18) and, so, are relatively slow (16, 19). Here, we demonstrate a rapid method for determining the mating type in yeast.

Virulent S. aureus is an invasive pathogen that can infect almost any human tissue. It has the potential to provoke several forms of human disease, including food poisoning (20), skin infections (21), endocarditis (22), and toxic shock syndrome (23). Secreted proteins like the Staphylococcal enterotoxins belong to a family of so-called superantigens that share the ability to trigger excessive and aberrant activation of T cells manifested in various symptoms from atopic dermatitis (24) to toxic shock syndromes (23). Cell density-dependent peptide quorum sensing (25–28) controls the secretion of these toxins, as well as a variety of other virulence factors and surface proteins. The agr locus of S. aureus (and the staphylococci in general) encodes the components agrB, D, C, A, and rnaIII for such a peptide quorum-sensing system (29). AgrB is presumed to process the propeptide precursor AgrD to form the mature autoinducing peptides (AIPs) (30), which are characterized by a thiolactone structure comprised of a pentapeptide ring formed through condensation of the sulfhydryl group of a cysteine with the α-carboxyl group of the C-terminal amino acid. These lariat-like structures are called autoinducing peptides because they stimulate gene expression of several exported proteins as well themselves when they bind to the receptor-histidine kinase, AgrC (30, 31). The agr locus is hypervariable leading to variations in agrD, B, and C that classify S. aureus into four different agr groups (32) [and >20 other putative agr groups in other staphylococcal species (33)]. AIPs from one group usually cross-inhibit virulence specific gene expression in S. aureus strains from a different group, and supernatants of other staphylococcal strains usually inhibit activation in S. aureus (34). This form of bacterial interference (34, 35) is thought to be of clinical relevance in infection and colonization. Thus, a rapid and reliable method to detect and distinguish between different staphylococcal strains based on which AIPs they secrete is potentially of great diagnostic utility.

Acknowledgments

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