pFind Studio: a computational solution for mass spectrometry-based proteomics
2015
Nucleic Acids Research2015. Trahan, C et al.
Inst Rech Clin Montreal, Dept Syst Biol, Montreal, PQ H2W 1R7, Canada.
ABSTRACT:Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes.
Use: pLink
Methods2015. Rivera-Santiago, RF et al.
Wistar Inst Anat & Biol, 3601 Spruce St, Philadelphia, PA 19104 USA.
ABSTRACT:Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein-protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of "zero-length" cross-linking. Zero-length cross-linking reagents do not add any atoms to the cross-linked species due to the lack of a spacer arm. This provides a major advantage in the form of providing more precise distance constraints as the cross-linkable groups must be within salt bridge distances in order to react. However, identification of cross-linked peptides using these reagents presents unique challenges. We discuss recent efforts by our group to minimize these challenges by using multiple cycles of LC-MS/MS analysis and software specifically developed and optimized for identification of zero-length cross-linked peptides. Representative data utilizing our current protocol are presented and discussed. (C) 2015 Elsevier Inc. All rights reserved.
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Molecular & Cellular Proteomics2015. Matthew M Makowski et al.
Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands
ABSTRACT:In recent years, cross-linking mass spectrometry has proven to be a robust and effective methodofinterrogating macromolecularproteincomplex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenouscomplexesobtained through time-limiting reconstitution, tandemaffinitypurification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probingchromatin-associatedproteincomplexesusingsmall volumesofmammalian whole cell lysates,singleaffinitypurification, and on-bead cross-linking followed by LC-MS/MSanalysis. We first benchmarkedxIP-MSusingthe structurally well-characterized phosphoribosyl pyrophosphate synthetase complex. We then appliedxIP-MSto thechromatin-associatedcohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCMcomplexes, and we provide novel structural and biological insights into their architectures and molecular function.Ofnote, we usexIP-MSto performtopologicalstudies under cell cycle perturbations, showing that thexIP-MSprotocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates thatxIP-MSis a robust, flexible, and widely applicable methodology for interrogatingchromatin-associatedproteincomplex architectures.
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Science2015. Yan, CY et al.
Tsinghua Univ, Minist Educ, Key Lab Prot Sci,Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci,Ctr Struct Bio, Beijing 100084, Peoples R China.
ABSTRACT:Splicing of precursor messenger RNA (pre-mRNA) in yeast is executed by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs), NTC (nineteen complex), NTC-related proteins (NTR), and a number of associated enzymes and cofactors. Here, we report the three-dimensional structure of a Schizosaccharomyces pombe spliceosome at 3.6-angstrom resolution, revealed by means of single-particle cryogenic electron microscopy. This spliceosome contains U2 and U5 snRNPs, NTC, NTR, U6 small nuclear RNA, and an RNA intron lariat. The atomic model includes 10,574 amino acids from 37 proteins and four RNA molecules, with a combined molecular mass of approximately 1.3 megadaltons. Spp42 (Prp8 in Saccharomyces cerevisiae), the key protein component of the U5 snRNP, forms a central scaffold and anchors the catalytic center. Both the morphology and the placement of protein components appear to have evolved to facilitate the dynamic process of pre-mRNA splicing. Our near-atomic-resolution structure of a central spliceosome provides a molecular framework for mechanistic understanding of pre-mRNA splicing.
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Analytical Chemistry2015. He, L et al.
Scripps Res Inst, Dept Physiol Chem, 10550 North Torrey Pines Rd, La Jolla, CA 92037 USA.
ABSTRACT:Extraction of data from the proprietary RAW files generated by Thermo Fisher mass spectrometers is the primary step for subsequent data analysis. High resolution and high mass accuracy data obtained by state-of-the-art mass spectrometers (e.g., Orbitraps) can significantly improve both peptide/protein identification and quantification. We developed RawConverter, a stand-alone software tool, to improve data extraction on RAW files from high-resolution Thermo Fisher mass spectrometers. RawConverter extracts full scan and MS n data from RAW files like its predecessor RawXtract; most importantly, it associates the accurate precursor mass-tocharge (m/z) value with the tandem mass spectrum. RawConverter accepts RAW data generated by either data-dependent acquisition (DDA) or data-independent acquisition (DIA). It generates output into MS1/MS2/MS3, MGF, or mzXML file formats, which fulfills the format requirements for most data identification and quantification tools. Using the tandem mass spectra extracted by RawConverter with corrected m/z values, 32.8%, 27.1%, and 84.1%, peptide spectra matches (PSMs) produce 17.4% (13.0%), 14.4% (11.5%), and 45.7% (36.2%) more peptide (protein) identifications than ProteoWizard, pXtract, and RawXtract, respectively. RawConverter is implemented in C# and is freely accessible at http://fields.scripps.edu/rawconv.
Use: pXtract; pParse
Molecular & Cellular Proteomics2015. Yuan, ZF et al.
Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, Epigenet Program, Room 9-124,3400 Civ Ctr Blvd,Bldg 421, Philadelphia, PA 19104 USA.
ABSTRACT:Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.
Use: pXtract; pParse
Journal of Proteome Research2015. Barnidge, DR et al.
Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA.
ABSTRACT:In our previous work, we showed that electrospray ionization of intact polydonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.
Use: pNovo
IEEE Transactions on Nanobioscience2015. Yan, Y et al.
Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT:With tandem mass spectrometry (MS/MS), spectra can be generated by various fragmentation techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD) and so on. At the same time, de novo sequencing using multiple spectra from the same peptide generated by different fragmentation techniques is becoming popular in proteomics studies. The focus of this study is the use of paired spectra from CID (or HCD) and ECD (or ETD) fragmentation because of the complementarity between them. We present a de novo peptide sequencing framework for multiple tandem mass spectra, and apply it to paired spectra sequencing problem. The performance of the framework on paired spectra is compared to another successful method named pNono+. The results show that our proposed method outperforms pNono+ in terms of full length peptide sequencing accuracy on three pairs of experimental datasets, with the accuracy increasing up to 13.6% compared to pNono+.
Use: pNovo
Journal of Proteome Research2015. Barnidge, DR et al.
Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA.
ABSTRACT:In our previous work, we showed that electrospray ionization of intact polydonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.
Use: pNovo
IEEE Transactions on Nanobioscience2015. Yan, Y et al.
Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT:With tandem mass spectrometry (MS/MS), spectra can be generated by various fragmentation techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD) and so on. At the same time, de novo sequencing using multiple spectra from the same peptide generated by different fragmentation techniques is becoming popular in proteomics studies. The focus of this study is the use of paired spectra from CID (or HCD) and ECD (or ETD) fragmentation because of the complementarity between them. We present a de novo peptide sequencing framework for multiple tandem mass spectra, and apply it to paired spectra sequencing problem. The performance of the framework on paired spectra is compared to another successful method named pNono+. The results show that our proposed method outperforms pNono+ in terms of full length peptide sequencing accuracy on three pairs of experimental datasets, with the accuracy increasing up to 13.6% compared to pNono+.
Use: pNovo