MassWiz: a novel scoring algorithm with target-decoy based analysis pipeline for tandem mass spectrometry

Mass spectrometry has made rapid advances in the recent past and has become the preferred method for proteomics. Although many open source algorithms for peptide identification exist, such as X!Tandem and OMSSA, it has majorly been a domain of proprietary software. There is a need for better, freely available, and configurable algorithms that can help in identifying the correct peptides while keeping the false positives to a minimum. We have developed MassWiz, a novel empirical scoring function that gives appropriate weights to major ions, continuity of b-y ions, intensities, and the supporting neutral losses based on the instrument type. We tested MassWiz accuracy on 486,882 spectra from a standard mixture of 18 proteins generated on 6 different instruments downloaded from the Seattle Proteome Center public repository. We compared the MassWiz algorithm with Mascot, Sequest, OMSSA, and X!Tandem at 1% FDR. MassWiz outperformed all in the largest data set (AGILENT XCT) and was second only to Mascot in the other data sets. MassWiz showed good performance in the analysis of high confidence peptides, i.e., those identified by at least three algorithms. We also analyzed a yeast data set containing 106,133 spectra downloaded from the NCBI Peptidome repository and got similar results. The results demonstrate that MassWiz is an effective algorithm for high-confidence peptide identification without compromising on the number of assignments. MassWiz is open-source, versatile, and easily configurable.     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Proanthocyanidin exposure to B6C3F1 mice significantly attenuates dimethylnitrosamine-induced liver tumor induction and mortality by differentially modulating programmed and unprogrammed cell deaths

Proanthocyanidins are of current interest as chemopreventive agents. The potential of the pre-, post- and co-exposure of proanthocyanidin-rich grape seed extract (GSPE) in preventing, reducing and/or delaying dimethylnitrosamine (N-nitrosodimethylamine, DMN)-induced liver tumorigenesis, carcinogenesis and mortality in male B6C3F1 mice was determined. Animals were divided into six groups: I—control, II—GSPE alone, III—DMN alone, IV—GSPE + DMN, V—DMN exposure (3 months) followed by GSPE diet (9 months) and VI—GSPE diet (3 months) + DMN (3 months) + control diet (6 months). DMN exposure (0–8 weeks: 5 mg/kg; 8–12 weeks: 10 mg/kg, i.p.) was limited to a total period of 3 months. GSPE was incorporated in laboratory chow (ADI: 100 mg/kg b.w.). Animals were sacrificed at 3 month intervals, and serum chemistry, liver histopathology, integrity of hepatic genomic DNA, antioxidant status, and rates of apoptotic and necrotic cell deaths were determined. DMN-induced liver tumor formation (85%) and animal lethality (38%) were powerfully antagonized by co-administration of GSPE + DMN (tumor positive: 45%; death: 11%). More than 75% of the DMN-treated animals had numerous tumors (five or more), which were significantly reduced in the GSPE + DMN group (35%). GSPE also negatively influenced other protocols specifically designed to test initiation and progression phases. Thus, GSPE was instrumental in modulating metabolic cascades and regulated orchestration of cell death processes involved during the multistage tumorigenic process. These results unraveled that long-term exposure to proanthocyanidin-rich grape seed extract may serve as a potent barrier to all three stages of DMN-induced liver carcinogenesis and tumorigenesis by selectively altering oxidative stress, genomic integrity and cell death patterns in vivo.     

Cytokine release induced by killed bacteria associated with anti-IFN-gamma antibody in Shigella infection

An effort was made to analyze the effect of in vitro stimulation on macrophages using killed Shigella dysenteriae type-1 (KSD1) coupled with anti-Interferon Gamma (anti-IFN-gamma) antibody. The stimulated macrophages were co-cultured with primed or non-primed T-cells from Shigella infected patients. T-cell cultures were also established by co-culturing KSD1 coupled with or without PHA stimulated macrophages. Emulsified KSD1 coupled with anti-IFN-gamma antibody was found to act as a potent immunogen, inducing the release of Th1 cytokine from primed T-cells cultured in acute stage of the disease. It was observed that the levels of IFN-gamma and IL-2 production rather than IL-4 and IL-6 were increased as the disease became more severe. On comparison, the subsequent values of IL-6, IFN-gamma, IL-4 and IL-2 were found to be less significant in healthy primed T-cell cultures. This was also associated with a substantial production of superoxide ions (O2-), which probably inhibits the colonization of intracellular Shigella due to the presence of anti-IFN-gamma antibodies. On the other hand KSD1 with or without PHA failed to induce such responses. The above findings reflect that in the presence of anti-IFN-gamma antibody, KSD1 acts as a potential immunogen for eliciting cellular immunity against shigellosis.     

Cysteine and serine protease-mediated proteolysis in body homogenate of a zooplankter, Moina macrocopa, is inhibited by the toxic cyanobacterium, Microcystis aeruginosa PCC7806

The paper describes the characterization of proteases in the whole body homogenate of Moina macrocopa, which can possibly be inhibited by the extracts of Microcystis aeruginosa PCC7806. With the use of oligopeptide substrates and specific inhibitors, we detected the activities of trypsin, chymotrypsin, elastase and cysteine protease. Cysteine protease, the predominant enzyme behind proteolysis of a natural substrate, casein, was partially purified by gel filtration. The substrate SDS-polyacrylamide gel electrophoresis of body homogenate revealed the presence of nine bands of proteases (17-72 kDa). The apparent molecular mass of an exclusive cysteine protease was 60 kDa, whereas of trypsin, it was 17-24 kDa. An extract of M. aeruginosa PCC7806 significantly inhibited the activities of trypsin, chymotrypsin and cysteine protease in M. macrocopa body homogenate at estimated IC(50) of 6- to 79-microg dry mass mL(-1). Upon fractionation by C-18 solid-phase extraction, 60% methanolic elute contained all the protease inhibitors, and these metabolites could be further separated by reverse-phase liquid chromatography. The metabolites inhibitory to M. macrocopa proteases also inhibited the corresponding class of proteases of mammalian/plant origin. The study suggests that protease inhibition may contribute to chemical interaction of cyanobacteria and crustacean zooplankton.     

Effect of water stress and heavy metals on induction of somatic embryogenesis in wheat leaf base cultures

In vitro cultures of plant tissues are known to mimic the response of field-grown plants when subjected to stress treatments. This investigation on Triticum aestivum explores the effect of drought stress on somatic embryogenesis and endogenous proline content. Leaf bases were cultured on MS medium supplemented with 2,4-D (10 microM) and different concentrations of PEG (2.5, 5, 7.5%) or mannitol (0.25 and 0.5 M) and also subjected to different periods of aerial drying in the laminar flow for one-day and subsequently transferred to MS basal medium. PEG treatment induced a high percentage (up to 50%) of embryoid formation. However, with mannitol and aerial drying, percentage of embryoid formation decreased with increasing concentrations and duration. After ten days, the endogenous proline content of explants treated with different concentrations of PEG, mannitol and different durations of aerial drying increased with increasing concentration and increasing duration of the treatment, thus, corroborating the role of proline as an osmolyte during stress conditions. Similarly, addition of metals such as cadmium and cobalt caused a reduction in percentage explants depicting embryogenesis. However, when cadmium was employed alone, 22% explants displayed somatic embryogenesis as compared to 54% in 2,4-D treated cultures.