Experimental Protein Mixture for Validating Tandem Mass Spectral Analysis

Keller, Andrew; Purvine, Samuel O.; Nesvizhskii, Alexey I.; Stolyar, Sergey; Goodlett, David R.; Kolker, Eugene

doi:10.1089/153623102760092805

Cited by 239 publications

(245 citation statements)

References 11 publications

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“…11 and 19-24; (ii) LC coupled to Fourier transform ion cyclotron resonance MS (25,26); (iii) LC coupled to quadruple-TOF MS (27); and (iv) 2D gel electrophoresis followed by LC-MS͞MS (16,23). Common approaches used in different proteome analyses included: LC-MS͞MS implemented by using the LCQ platform (Thermo Electron, San Jose, CA), standard sample processing (Supporting Materials), standard top-down data-dependent ion selection (11,(19)(20)(21)(22)(23)(24), use of the TURBO-SEQUEST (Thermo Electron) search engine, and use of the recently developed standard mixtures for proteome studies (24). These approaches, in turn, allowed implementation of the same stringent criteria (28) for all peptide and protein identifications included in this study (Supporting Materials).…”

Section: Methodsmentioning

confidence: 99%

Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations

Kolker

Picone

Galperin

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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114

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The ␥-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, Ϸ40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized ''hypothetical'' genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.computational biology ͉ expression analysis ͉ microarrays ͉ proteomics ͉ integrative microbiology

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Section: Methodsmentioning

confidence: 99%

Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations

Kolker

Picone

Galperin

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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114

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“…Peptides were submitted for mass spectrometry analysis using either a Micromass Q-TOF Tandem Hybrid Mass Spectrometer (at the Department of Medical Chemistry of the University of Washington), a Thermo Electron LCQ DECA XP or an LTQ Linear Ion Trap (SBRI Proteomics Core). The collision induced dissociation (CID) spectra were compared with a L. major protein database (version 5.2, downloaded from GeneDB) using TURBOSEQUEST software, and protein matches determined using PEPTIDEPROPHET and PROTEINPROPHET (Keller et al, 2002;Nesvizhskii et al, 2003).…”

Section: Mass Spectrometry Analysismentioning

confidence: 99%

Characterization of the RNA polymerase II and III complexes in Leishmania major

Martínez‐Calvillo

Saxena

Green

et al. 2007

International Journal for Parasitology

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Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function.

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“…Small reference sets of tryptic peptides have been made from known proteins [16,17] and larger datasets have been made from the yeast proteome [18,19] and human serum proteins collectively in the HUPO initiative [20]. Although these are useful databases, they are time consuming to generate and are not publicly available as a reference set.…”

mentioning

confidence: 99%

Validated MALDI-TOF/TOF mass spectra for protein standards

Falkner

Kachman

Veine

et al. 2007

J. Am. Soc. Mass Spectrom.

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A current focus of proteomics research is the establishment of acceptable confidence measures in the assignment of protein identifications in an unknown sample. Development of new algorithmic approaches would greatly benefit from a standard reference set of spectra for known proteins for the purpose of testing and training. Here we describe an openly available library of mass spectra generated on an ABI 4700 MALDI TOF/TOF from 246 known, individually purified and trypsin-digested protein samples. The initial full release of the Aurum Dataset includes gel images, peak lists, spectra, search result files, decoy database analysis files, FASTA file of protein sequences, manual curation, and summary pages describing protein coverage and peptides matched by MS/MS followed by decoy database analysis using Mascot, Sequest, and X!Tandem. The data are publicly available for use at

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Experimental Protein Mixture for Validating Tandem Mass Spectral Analysis

Cited by 239 publications

References 11 publications

Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations

Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations

Characterization of the RNA polymerase II and III complexes in Leishmania major

Validated MALDI-TOF/TOF mass spectra for protein standards

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