DiagnoMass: A proteomics hub for pinpointing discriminative spectral clusters.

MOTIVATION: There are several well-established paradigms for identifying and pinpointing discriminative peptides/proteins using shotgun proteomic data; examples are peptide-spectrum matching, de novo sequencing, open searches, and even hybrid approaches. Such an arsenal of complementary paradigms can provide deep data coverage, albeit some unidentified discriminative peptides remain. RESULTS: We present DiagnoMass, software tool that groups similar spectra into spectral clusters and then shortlists those clusters that are discriminative for biological conditions. DiagnoMass then communicates with proteomic tools to attempt the identification of such clusters. We demonstrate the effectiveness of DiagnoMass by analyzing proteomic data from Escherichia coli, Salmonella, and Shigella, listing many high-quality discriminative spectral clusters that had thus far remained unidentified by widely adopted proteomic tools. DiagnoMass can also classify proteomic profiles. We anticipate the use of DiagnoMass as a vital tool for pinpointing biomarkers. AVAILABILITY: DiagnoMass and related documentation, including a usage protocol, are available at http://www.diagnomass.com.

RawVegetable - A data assessment tool for proteomics and cross-linking mass spectrometry experiments.

We present RawVegetable, a software for mass spectrometry data assessment and quality control tailored toward shotgun proteomics and cross-linking experiments. RawVegetable provides four main modules with distinct features: (A) The charge state chromatogram that independently displays the ion current for each charge state; useful for optimizing the chromatography for highly charged ions and with lower XIC values such as those typically found in cross-linking experiments. (B) The XL-Artefact determination, which flags possible noncovalently associated peptides. (C) The TopN density estimation, for detecting retention time intervals of under or over-sampling, and (D) The chromatography reproducibility module, which provides pairwise comparisons between multiple experiments. RawVegetable, a tutorial, and the example data are freely available for academic use at: http://patternlabforproteomics.org/rawvegetable. SIGNIFICANCE: Chromatography optimization is a critical step for any shotgun proteomic or cross-linking mass spectrometry experiment. Here, we present a nifty solution with several key features, such as displaying individual charge state chromatograms, highlighting chromatographic regions of under- or over-sampling and checking for reproducibility.

Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins.

Cross-linking coupled with mass spectrometry (XL-MS) has emerged as a powerful strategy for the identification of protein-protein interactions, characterization of interaction regions, and obtainment of structural information on proteins and protein complexes. In XL-MS, proteins or complexes are covalently stabilized with cross-linkers and digested, followed by identification of the cross-linked peptides by tandem mass spectrometry (MS/MS). This provides spatial constraints that enable modeling of protein (complex) structures and regions of interaction. However, most XL-MS approaches are not capable of differentiating intramolecular from intermolecular links in multimeric complexes, and therefore they cannot be used to study homodimer interfaces. We have recently developed an approach that overcomes this limitation by stable isotope-labeling of one of the two monomers, thereby creating a homodimer with one 'light' and one 'heavy' monomer. Here, we describe a step-by-step protocol for stable isotope-labeling, followed by controlled denaturation and refolding in the presence of the wild-type protein. The resulting light-heavy dimers are cross-linked, digested, and analyzed by mass spectrometry. We show how to quantitatively analyze the corresponding data with SIM-XL, an XL-MS software with a module tailored toward the MS/MS data from homodimers. In addition, we provide a video tutorial of the data analysis with this protocol. This protocol can be performed in ∼14 d, and requires basic biochemical and mass spectrometry skills.

How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding.

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Sera of chagasic patients react with antigens from the tomato parasite Phytomonas serpens.

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These flagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identified, resulting in 22 different putative proteins. The identified proteins were classified into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.

Sera of Chagasic patients react with antigens from the tomato parasite Phytomonas serpens

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These fagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twenty-eight polypeptides were identifed, resulting in 22 different putative proteins. The identifed proteins were classifed into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.