pFind Studio: a computational solution for mass spectrometry-based proteomics
2018
Journal of Biological Chemistry2018. Sugita, S et al.
Konan Univ, Fac Sci & Engn, Dept Biol, Okamoto 8-9-1, Kobe, Hyogo 6588501, Japan.
ABSTRACT:ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA(+)), ClpB forms a hexameric ring structure, with each protomer containing two AAA(+) modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.
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Journal of Biological Chemistry2018. Dickmanns, A et al.
Georg August Univ Gottingen, Dept Mol Struct Biol, Justus von Liebig Weg 11, D-37077 Gottingen, Germany.
ABSTRACT:Utilization of energy-rich carbon sources such as glucose is fundamental to the evolutionary success of bacteria. Glucose can be catabolized via glycolysis for feeding the intermediary metabolism. The methylglyoxal synthase MgsA produces methylglyoxal from the glycolytic intermediate dihydroxyacetone phosphate. Methylglyoxal is toxic, requiring stringent regulation of MgsA activity. In the Gram-positive bacterium Bacillus subtilis, an interaction with the phosphoprotein Crh controls MgsA activity. In the absence of preferred carbon sources, Crh is present in the nonphosphorylated state and binds to and thereby inhibits MgsA. To better understand the mechanism of regulation of MgsA, here we performed biochemical and structural analyses of B. subtilis MgsA and of its interaction with Crh. Our results indicated thatMgsAforms a hexamer (i.e. a trimer of dimers) in the crystal structure, whereas it seems to exist in an equilibrium between a dimer and hexamer in solution. In the hexamer, two alternative dimers could be distinguished, but only one appeared to prevail in solution. Further analysis strongly suggested that the hexamer is the biologically active form. In vitro cross-linking studies revealed that Crh interacts with the N-terminal helices of MgsA and that the Crh-MgsA binding inactivates MgsA by distorting and thereby blocking its active site. In summary, our results indicate that dimeric and hexameric MgsA species exist in an equilibrium in solution, that the hexameric species is the active form, and that binding to Crh deforms and blocks the active site in MgsA.
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Journal of Food Processing and Preservation2018. Wu, ZH et al.
Nanchang Univ, State Key Lab Food Sci & Technol, Nanchang 330047, Jiangxi, Peoples R China.
ABSTRACT:Peanut (Arachis hypogaea) is listed among the eight major food allergens, in which Ara h 2 is the major allergen. The microbial transglutaminase (MTGase)-catalyzed cross-linking reaction reduces the allergenicity of Ara h 2. However, deamidation might occur and influence the cross-linking reaction. In this work, native and reduced Ara h 2 were catalyzed by MTGase. In addition to intermolecular cross-linking, intramolecular cross-linking and deamidation were proven to occur simultaneously. Moreover, intramolecular cross-linking sites were identified using mass spectrometry and the PLINK software. The reactions moved toward different directions because of different reduction conditions and different protein concentrations. Practical applicationsCross-linking is a common processing method used to improve the textural and functional properties of protein. Deamidation reaction might occur during processing and influence the cross-linking reaction. The two types of reactions have a competitive relation, because both of them occur on glutamine residue. In Ara h 2 and MTGase system, protein concentration and reduction content influence the direction of reaction. The results can be applied to modify the reaction system of Ara h 2 catalyzed by MTGase. In addition, the relationship between structure and properties changes on Ara h 2 was discussed based on analysis of cross-linking sites.
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Nature Communications2018. Kolesnikova, O et al.
Inst Genet & Biol Mol & Cellulaire, Equipe Labellisee Ligue Canc, Dept Integrated Struct Biol, F-67404 Illkirch Graffenstaden, France.
ABSTRACT:Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. Here we present the cryo-electron microscopy structure of promoter-bound yeast TFIID at a resolution better than 5 A, except for a flexible domain. We position the crystal structures of several subunits and, in combination with cross-linking studies, describe the quaternary organization of TFIID. The compact tri lobed architecture is stabilized by a topologically closed Taf5-Taf6 tetramer. We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a histone fold domain in the Twin lobe.
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PNAS2018. Yang, B et al.
Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA.
ABSTRACT:Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues "planting" the reagent at fixed locations on protein. The pendant aryl sulfonyl fluoride is then "cast" across a limited range of the protein surface, where it can react with multiple weakly nucleophilic amino acid sidechains in a proximityenhanced sulfur-fluoride exchange (SuFEx) reaction. Using proteins of known structures, we demonstrated that the heterobifunctional agent formed cross-links between Lys residues and His, Ser, Thr, Tyr, and Lys sidechains. This geometric specificity contrasts with current bis-succinimide esters, which often generate nonspecific cross-links between lysines brought into proximity by rare thermal fluctuations. Thus, the current method can provide diverse and robust distance restraints to guide integrative modeling. This work provides a chemical cross-linker targeting unactivated Ser, Thr, His, and Tyr residues using sulfonyl fluorides. In addition, this methodology yielded a variety of cross-links when applied to the complex Escherichia coli cell lysate. Finally, in combination with genetically encoded chemical crosslinking, cross-linking using this reagent markedly increased the identification of weak and transient enzyme-substrate interactions in live cells. Proximity-dependent cross-linking will dramatically expand the scope and power of CXMS for defining the identities and structures of protein complexes.
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Nature2018. Vos, SM et al.
Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT:Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Honto sapiens DSIF, PAF and SPT6 was determined at 3.1 A resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminaldomain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.
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Journal of Proteome Research2018. Yuan, ZF et al.
Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, Epigenet Inst, Philadelphia, PA 19104 USA.
ABSTRACT:Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extracted ion chromatography to accurately determine the chromatographic profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as aryl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, we anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biology and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family.
Use: pXtract
IEEE Latin America Transactions2018. Damaso, JCG et al.
Univ Fed Rio de Janeiro, Rio De Janeiro, Brazil.
ABSTRACT:This work presents a heuristic to the peptide sequencing in multiplex mass spectra. We developed a methodology that deconvolutes the multiplex mass spectrum, generating a modified specific spectrum for each peptide target mass. A directed spectrum graph is created from each deconvoluted spectrum and a cost function is defined to guide the depth first search algorithm through the sequence paths. The heuristic considers that the peptide with higher intensity would be correctly sequenced from the intact multiplex spectrum. A computational system called DNBuilder was developed. Mass spectra from a thyroid sample were used to test the methodology and compared with three well-known programs from the literature, as Peaks, pNovoPlus and Novor. The results show that our methodology enhances the correct amino acid sequencing amount in co-fragmented peptides spectra.
Use: pNovo
Journal of proteome research2018. Dorfer, V et al.
Univ Appl Sci Upper Austria, Bioinformat Res Grp, Softwarepk 11, A-4232 Hagenberg, Austria.
ABSTRACT:Coeluting peptides are still a major challenge for the identification and validation of MS/MS spectra, but carry great potential. To tackle these problems, we have developed the here presented CharmeRT workflow, combining a chimeric spectra identification strategy implemented as part of the MS Amanda algorithm with the validation system Elutator, which incorporates a highly accurate retention time prediction algorithm. For high resolution data sets this workflow identifies 38-64% chimeric spectra, which results in up to 63% more unique peptides compared to a conventional single search strategy.
Use: pParse
Journal of proteome research2018. Dorfer, V et al.
Univ Appl Sci Upper Austria, Bioinformat Res Grp, Softwarepk 11, A-4232 Hagenberg, Austria.
ABSTRACT:Coeluting peptides are still a major challenge for the identification and validation of MS/MS spectra, but carry great potential. To tackle these problems, we have developed the here presented CharmeRT workflow, combining a chimeric spectra identification strategy implemented as part of the MS Amanda algorithm with the validation system Elutator, which incorporates a highly accurate retention time prediction algorithm. For high resolution data sets this workflow identifies 38-64% chimeric spectra, which results in up to 63% more unique peptides compared to a conventional single search strategy.
Use: pParse