A method to correlate the uninterpreted tandem mass spectra of peptides produced under low energy (lo-50 eV) collision conditions with amino acid sequences in the Genpept database has been developed. In this method the protein database is searched to identify linear amino acid sequences within a mass tolerance of * 1 u of the precursor ion molecular weight. A cross-correlation function is then used to provide a measurement of similarity between the mass-to-charge ratios for the fragment ions predicted from amino acid sequences obtained from the database and the fragment ions observed in the tandem mass spectrum. In general, a difference greater than 0.1 between the normalized cross-correlation functions of the first-and second-ranked search results indicates a successfol match between sequence and spectrum. Searches of species-specific protein databases with tandem mass spectra acquired from peptides obtained from the enzymatically digested total proteins of E. coli and S. cerevisiae cells allowed matchmg of the spectra to amino acid sequences within proteins of these organisms. The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database, fJ Am Sot Mass Spectrom 1994, 5, 976-989) A mino acid sequence analysis is often the initial step in characterizing a newly isolated protein.Conventional sequencing strategies employ chemical reagents to remove one amino acid at a time from the amino terminus followed by isolation and analysis of the released amino acid derivative [l, 21. Limitations in the chemical efficiency of the process prevents determination of the complete sequence of a protein from small quantities of sample. Partial sequence information, however, can be used to search a protein or nucleotide database to discover relationships to previously identified proteins or to determine if the protein sequence is novel 13, 41. Although sequence information may have been determined previously, the context in which the protein is identified may be relevant to the biological process under study [51. Another method to identify known protein sequences employs site-specific proteolysis followed by measurement of the mass-to-charge ratios of the pep tides by mass spectrometry. The set of observed peptide mass-to-charge ratios is then used to search a protein database to find a set of peptide masses predicted from enzymatic digestion of each protein in the database [6-101. Both chemical degradation and peptide mapping approaches require the use of fairly homogeneous samples to avoid ambiguity in assigning Address reprint requests to John R.

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.

We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, and databases of known physical interactions. Using this approach, we identify 997 messenger RNAs responding to 20 systematic perturbations of the yeast galactose-utilization pathway, provide evidence that approximately 15 of 289 detected proteins are regulated posttranscriptionally, and identify explicit physical interactions governing the cellular response to each perturbation. We refine the model through further iterations of perturbation and global measurements, suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and a variety of other metabolic pathways.