pFind Studio: a computational solution for mass spectrometry-based proteomics
2014
PLOS ONE2014. Poll, G et al.
Univ Regensburg, Lehrstuhl Biochem 3, D-93053 Regensburg, Germany.
ABSTRACT:Eukaryotic ribosome biogenesis is a multistep process involving more than 150 biogenesis factors, which interact transiently with pre-ribosomal particles to promote their maturation. Some of these auxiliary proteins have been isolated in complexes found separate from the ribosomal environment. Among them, are 3 large UTP subcomplexes containing 6 or 7 protein subunits which are involved in the early steps of ribosome biogenesis. The composition of the UTP subcomplexes and the network of binary interactions between protein subunits have been analyzed previously. To obtain further insights into the structural and biochemical properties of UTP subcomplexes, we established a heterologous expression system to allow reconstitution of the yeast tUTP/UTP A and UTP B subcomplexes from their candidate subunits. The results of a series of reconstitution experiments involving different combinations of protein subunits are in good agreement with most of the previously observed binary interactions. Moreover, in combination with additional biochemical analyses, several stable building blocks of the UTP subcomplexes were identified. Based on these findings, we present a refined model of the tUTP/UTP A and UTP B architecture.
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PLOS Biology2014. Zhang, JM et al.
Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT:Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16(XPF)-Swi10(ERCC1) and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 39 endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 59 endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases.
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PLOS ONE2014. Plocinski, P et al.
Polish Acad Sci, Inst Biochem & Biophys, Warsaw, Poland.
ABSTRACT:Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium-and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semiquantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners.
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Molecular & Cellular Proteomics2014. Wang, J et al.
Univ Calif San Diego, Ctr Computat Mass Spectrometry, Dept Comp Sci & Engn, 9500 Gilman Dr,Mail Code 0404, La Jolla, CA 92093 USA.
ABSTRACT:The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein-protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides.
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Journal of Biological chemistry2014. Coffman, K et al.
Natl Inst Biol Sci, Beijing 102206, Peoples R China.
ABSTRACT:Background: mTORC1 recruits its substrate 4E-BP1 via Raptor/4E-BP1 interaction. Chemical cross-linking/mass spectrometry permits characterization of protein-protein interactions. Results: Cross-linked peptides between Raptor and 4E-BP1 were identified. Raptor intramolecular cross-links were also identified. Conclusion: Raptor N-terminal region containing RNC1 is implicated in the interaction with the central region of 4E-BP1. Significance: Our study provides novel insight into how mTORC1 recognizes 4E-BP1. mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.
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Journal of Proteome Research2014. Sriswasdi, S et al.
Wistar Inst Anat & Biol, Ctr Syst & Computat Biol, 3601 Spruce St, Philadelphia, PA 19104 USA.
ABSTRACT:Chemical cross-linking coupled to mass spectrometry provides structural information that is useful for probing protein conformations and providing experimental support for molecular models. "Zero-length" cross-links have greater value for these applications than longer cross-links because they provide more stringent distance constraints. However, this method is less commonly utilized because it cannot take advantage of isotopic labels, MS-labile bonds, or enrichment tags to facilitate identification. In this study, we combined label-free precursor ion quantitation and targeted tandem mass spectrometry with a new software tool, Zero-length Cross-link Miner (ZXMiner), to form a multitiered analysis strategy. A major, critical objective was to simultaneously achieve very high accuracy with essentially no false-positive cross-link identifications while maintaining a good depth of analysis. Our strategy was optimized on several proteins with known crystal structures. Comparison of ZXMiner to several existing cross-link analysis software showed that other algorithms detected less true positive cross-links and were far less accurate. Although prior use of zero-length cross-linking was typically restricted to small proteins, ZXMiner and the associated strategy enable facile analysis of very large protein complexes. This was demonstrated by identification of zero-length cross-links using purified 526 kDa spectrin heterodimers and intact red cell membranes and membrane skeletons.
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Journal of Proteomics2014. Tiroli-Cepeda, AO et al.
Univ Estadual Campinas, UNICAMP, Inst Chem, POB 6154, BR-13083970 Campinas, SP, Brazil.
ABSTRACT:Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. Biological significance Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics. (C) 2014 Elsevier B.V. All rights reserved.
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Biochmical and Biophysical Research Communications2014. Shapiro, AB et al.
AstraZeneca R&D Boston, 35 Gatehouse Dr, Waltham, MA 02451 USA.
ABSTRACT:LptA is a soluble periplasmic component of the lipopolysaccharide (LPS) transport system of Gram-negative bacteria that transports newly synthesized LPS from the inner membrane to the outer leaflet of the outer membrane. LptA links the inner membrane components (LptBFGC) to the outer membrane components (LptDE), but it is uncertain whether LptA is a freely moving LPS shuttle or part of a stable trans-periplasm structure. Escherichia coli LptA forms highly polymerized head-to-tail oligomers in solution, but dimers in vivo. We studied the oligomerization of purified Pseudomonas aeruginosa LptA. Size-exclusion chromatography showed that P. aeruginosa LptA, unlike E. coli LptA, is a dimer over a wide range of concentrations. Chemical crosslinking with bis(sulfosuccinimidyl) suberate confirmed that dimers were the predominant species even at sub-micromolar LptA concentrations, which was unaffected by LPS binding. Mass spectrometry of crosslinked dimers showed that crosslinks occurred between the N-terminal alpha-amino group and either Lys-172 or Lys-173 near the C-terminus. These results support a hypothetical structure for the dimer of isolated P. aeruginosa LptA in which the N-terminus of one monomer is in close proximity to the C-terminus of the other, and the same surface of each monomer forms the interface between them, preventing further oligomerization. (C) 2014 Elsevier Inc. All rights reserved.
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PLOS ONE2014. Plocinski, P et al.
Polish Acad Sci, Inst Biochem & Biophys, Warsaw, Poland.
ABSTRACT:Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium-and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semiquantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners.
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Journal of Proteomics2014. Tiroli-Cepeda, AO et al.
Univ Estadual Campinas, UNICAMP, Inst Chem, POB 6154, BR-13083970 Campinas, SP, Brazil.
ABSTRACT:Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. Biological significance Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics. (C) 2014 Elsevier B.V. All rights reserved.
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