Identification and validation of specific markers of Bacillus anthracis spores by proteomics and genomics approaches.

Bacillus anthracis is the causative bacteria of anthrax, an acute and often fatal disease in humans. The infectious agent, the spore, represents a real bioterrorism threat and its specific identification is crucial. However, because of the high genomic relatedness within the Bacillus cereus group, it is still a real challenge to identify B. anthracis spores confidently. Mass spectrometry-based tools represent a powerful approach to the efficient discovery and identification of such protein markers. Here we undertook comparative proteomics analyses of Bacillus anthracis, cereus and thuringiensis spores to identify proteoforms unique to B. anthracis. The marker discovery pipeline developed combined peptide- and protein-centric approaches using liquid chromatography coupled to tandem mass spectrometry experiments using a high resolution/high mass accuracy LTQ-Orbitrap instrument. By combining these data with those from complementary bioinformatics approaches, we were able to highlight a dozen novel proteins consistently observed across all the investigated B. anthracis spores while being absent in B. cereus/thuringiensis spores. To further demonstrate the relevance of these markers and their strict specificity to B. anthracis, the number of strains studied was extended to 55, by including closely related strains such as B. thuringiensis 9727, and above all the B. cereus biovar anthracis CI, CA strains that possess pXO1- and pXO2-like plasmids. Under these conditions, the combination of proteomics and genomics approaches confirms the pertinence of 11 markers. Genes encoding these 11 markers are located on the chromosome, which provides additional targets complementary to the commonly used plasmid-encoded markers. Last but not least, we also report the development of a targeted liquid chromatography coupled to tandem mass spectrometry method involving the selection reaction monitoring mode for the monitoring of the 4 most suitable protein markers. Within a proof-of-concept study, we demonstrate the value of this approach for the further high throughput and specific detection of B. anthracis spores within complex samples.

Detection of Yersinia pestis in environmental and food samples by intact cell immunocapture and liquid chromatography-tandem mass spectrometry.

Yersinia pestis is the causative agent of bubonic and pneumonic plague, an acute and often fatal disease in humans. In addition to the risk of natural exposure to plague, there is also the threat of a bioterrorist act, leading to the deliberate spread of the bacteria in the environment or food. We report here an immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) method for the direct (i.e., without prior culture), sensitive, and specific detection of Y. pestis in such complex samples. In the first step, a bottom-up proteomics approach highlighted three relevant protein markers encoded by the Y. pestis-specific plasmids pFra (murine toxin) and pPla (plasminogen activator and pesticin). Suitable proteotypic peptides were thoroughly selected to monitor the three protein markers by targeted MS using the selected reaction monitoring (SRM) mode. Immunocapture conditions were optimized for the isolation and concentration of intact bacterial cells from complex samples. The immuno-LC-SRM assay has a limit of detection of 2 × 10(4) CFU/mL in milk or tap water, which compares well with those of state-of-the-art immunoassays. Moreover, we report the first direct detection of Y. pestis in soil, which could be extremely useful in confirming Y. pestis persistence in the ground.

The Yersinia pseudotuberculosis complex: characterization and delineation of a new species, Yersinia wautersii.

The genus Yersinia contains three species pathogenic for humans, one of which is the enteropathogen Yersinia pseudotuberculosis. A recent analysis by Multi Locus Sequence Typing (MLST) of the 'Y. pseudotuberculosis complex' revealed that this complex comprises three distinct populations: the Y. pestis/Y. pseudotuberculosis group, the recently described species Yersinia similis, and a third not yet characterized population designated 'Korean Group', because most strains were isolated in Korea. The aim of this study was to perform an in depth phenotypic and genetic characterization of the three populations composing the Y. pseudotuberculosis complex (excluding Y. pestis, which belonged to the Y. pseudotuberculosis cluster in the MLST analysis). Using a set of strains representative of each group, we found that the three populations had close metabolic properties, but were nonetheless distinguishable based on D-raffinose and D-melibiose fermentation, and on pyrazinamidase activity. Moreover, high-resolution electrospray mass spectrometry highlighted protein peaks characteristic of each population. Their 16S rRNA gene sequences shared high identity (≥99.5%), but specific nucleotide signatures for each group were identified. Multi-Locus Sequence Analysis also identified three genetically closely related but distinct populations. Finally, an Average Nucleotide Identity (ANI) analysis performed after sequencing the genomes of a subset of strains of each group also showed that intragroup identity (average for each group ≥99%) was higher than intergroup diversity (94.6-97.4%). Therefore, all phenotypic and genotypic traits studied concurred with the initial MLST data indicating that the Y. pseudotuberculosis complex comprises a third and clearly distinct population of strains forming a novel Yersinia species that we propose to designate Yersinia wautersii sp. nov. The isolation of some strains from humans, the detection of virulence genes (on the pYV and pVM82 plasmids, or encoding the superantigen ypmA) in some isolates, and the absence of pyrazinamidase activity (a hallmark of pathogenicity in the genus Yersinia) argue for the pathogenic potential of Y. wautersii.

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry.

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.

Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases.

OBJECTIVE: We investigated SF and serum proteomic fingerprints of patients suffering from RA, OA and other miscellaneous inflammatory arthritides (MIAs) in order to identify RA-specific biomarkers. METHODS: SF profiles of 65 patients and serum profiles of 31 patients were studied by surface-enhanced laser desorption and ionization-time-of-flight-mass spectrometry technology. The most discriminating RA biomarkers were identified by matrix-assisted laser desorption ionization-time of flight and their overexpression was confirmed by western blotting and ELISA. RESULTS: Three biomarkers of 10 839, 10 445 and 13 338 Da, characterized as S100A8, S100A12 and S100A9 proteins, were the most up-regulated proteins in RA SF. Their expression was about 10-fold higher in RA SF vs OA SF. S100A8 exhibited a sensitivity of 82% and a specificity of 69% in discriminating RA from other MIAs, whereas S100A12 displayed a sensitivity of 79% and a specificity of 64%. Three peptides of 3351, 3423 and 3465 Da, corresponding to the alpha-defensins-1, -2 and -3, were also shown to differentiate RA from other MIAs with weaker sensitivity and specificity. Levels of S100A12, S100A8 and S100A9 were statistically correlated with the neutrophil count in MIA SF but not in the SF of RA patients. S100A8, S100A9, S100A12 and alpha-defensin expression in serum was not different in the three populations. CONCLUSION: The most enhanced proteins in RA SF, the S100A8, S100A9 and S00A12 proteins, distinguished RA from MIA with high accuracy. Possible implication of resident cells in this increase may play a role in RA physiopathology.

The cell line secretome, a suitable tool for investigating proteins released in vivo by tumors: application to the study of p53-modulated proteins secreted in lung cancer cells.

Malignant processes such as metastasis, invasion, or angiogenesis are tightly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. p53, a major tumor suppressor protein very frequently mutated and/or inactivated in cancer cells, is known to modulate the release of proteins by the tumor cells; however, while p53-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. Here, we characterized the p53-dependent secretome of a lung tumor model in vitro (H358 human nonsmall cell lung adenocarcinoma cell line with a homozygous deletion of p53) and demonstrate that the modulation of exported proteins can also be detected in vivo in the plasma of tumor-bearing mice. We used a clone of H358, stably transfected with a tetracycline-inducible wild-type p53-expressing vector. With the use of iTRAQ labeling and LC-MALDI-MS/MS analysis, we identified 909 proteins released in vitro by the cells, among which 91 are p53-modulated. Three proteins (GDF-15, FGF-19, and VEGF) were also investigated in H358/TetOn/p53 xenograft mice. The ELISA dosage on total tumor protein extracts confirmed the influence of p53 on the release of these proteins in vivo. Moreover, the GDF-15 concentration was measured in the plasma and its p53-dependent modulation was confirmed. To our knowledge, this is the first report establishing that the in vitro cell line secretome is reliable and reflects the extracellular release of proteins from tumor cells in vivo and could be used to identify putative tumor markers.

Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry.

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL-1 in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.

Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling.

BACKGROUND: The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes. RESULTS: We evaluated OFFGEL fractionator technology to separate peptides from two complex proteomes, human secretome and human plasma, using a 24-wells device encompassing the pH range 3-10. In combination with reverse phase liquid chromatography, peptides from these two samples were separated and identified by MALDI TOF-TOF. The repartition profiles of the peptides in the different fractions were analyzed and explained by their content in charged amino acids using an algorithmic model based on the possible combinations of amino acids. We also demonstrated for the first time the compatibility of OFFGEL separation technology with the quantitative proteomic labeling technique iTRAQ allowing inclusion of this technique in complex samples comparative proteomic workflow. CONCLUSION: The reported data showed that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic proteomes (plasma and secretome) and is compatible with iTRAQ labeling quantitative studies. We therefore consider peptides OFFGEL fractionation as an effective addition to our strategy and an important system for quantitative proteomics studies.

Detection of Yersinia pestis in Complex Matrices by Intact Cell Immunocapture and Targeted Mass Spectrometry.

We describe an immunoaffinity-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) protocol for the direct (i.e., without prior culture), sensitive and specific detection of Yersinia pestis in complex matrices. Immunoaffinity enables isolation and concentration of intact bacterial cells from food and environmental samples. After protein extraction and digestion, suitable proteotypic peptides corresponding to three Y. pestis-specific protein markers (murine toxine, plasminogen activator and pesticin) are monitored by targeted LC-MS/MS using the selected reaction monitoring (SRM) mode. This immuno-LC-MS/MS assay has a limit of detection of 2 × 104 CFU/mL in milk or tap water, and 4.5 × 105 CFU in 10 mg of soil.