Monsterscript_reverse_transcriptase_comparison
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Geobacillus Stearothermophilus (ncbitaxon:1422)
SUBMITTER: 
Wei Yan
PROVIDER: MSV000083126 | MassIVE | Fri Nov 09 13:37:00 GMT 2018
SECONDARY ACCESSION(S): PXD011649
REPOSITORIES: MassIVE
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Project description:S-nitrosoproteome of human unterine smooth muscle in different states of pregnancy (Labor, non labor, and preterm labor). Samples were isolated by the biotin switch and streptavidin pulldown before being analyzed by MS/MS on an Orbitrap instrument. Database Searching and bioinformatics: Tandem mass spectra were extracted; charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, version v.27, rev. 11). Sequest was initiated to search the database containing ipi.HUMAN.v3.87, the Global Proteome Machine cRAP v.2012.01.01, and random decoy sequences (183158 entries) assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine, iodoacetamide derivative of cysteine, and n-ethylmaleimide on cysteine were specified in Sequest as variable modifications. Criteria for Protein Identification-PROTEOIQ (V2.6, www.nusep.com) was used to validate MS/MS based peptide and protein identifications. Peptides were parsed before analysis with a minimum Xcorr value of 1.5 and a minimum length of 6 amino acids. There were no matches to the concatenated decoy database and therefore a false discovery value is not applicable or "0". Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides with 5 spectra per peptide. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
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2013-04-08 | PXD000141 | Pride
Project description:Gram-positive bacteria are known to export many proteins to the cell wall and growth medium and, accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed at determining the fate of native exported proteins in the model organism Bacillus subtilis. Specifically, we employed a GeLC-MS approach to distinguish the roles of the membrane-associated quality control proteases HtrA and HtrB from those of eight other proteases that are present in the cell wall and/or growth medium of B. subtilis. Notably, HtrA and HtrB were previously shown to counteract potentially detrimental 'protein export stresses' upon overproduction of membrane- or secreted proteins. Our results show that many secreted proteins, lipoproteins and membrane proteins of B. subtilis are potential substrates of extracytoplasmic proteases. Moreover, potentially important roles of HtrA and HtrB in the folding of native secreted proteins into a protease-resistant conformation, the liberation of lipoproteins from the membrane-cell wall interface, and the degradation of membrane proteins are uncovered. Altogether, our observations show that HtrA and HtrB are crucial for maintaining the integrity of the B. subtilis cell even under non-stress conditions. Bionformatics pipeline and data processing: MS and MS/MS data were acquired with the LTQ-Orbitrap mass spectrometer. After a survey scan in the Orbitrap (r= 30,000), MS/MS data were recorded for the five most intensive precursor ions in the linear ion trap. Singly charged ions were not taken into account for MS/MS analysis. The lock mass option was enabled throughout the analysis. The mass spectrometric data was then subjected to database searching via Sorcerer-Sequest. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version v.27, rev. 11). Sequest was set up to search a B. subtilis _target-decoy protein sequence database that included the complete proteome set of _B. subtilis _extracted from UniprotKB release 12.7, and a set of common laboratory contaminants compiled with Bioworks Browser (Thermo Fisher Scientific), assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a search tolerance of 10 ppm for the overview scans. Oxidation of methionine was specified in Sequest as a variable modification. Scaffold (version Scaffold_3_00_04, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they exceeded Xcorr values of 2.2, 3.5, 3.75 for doubly, triply and quadruply charged ions. A protein was regarded as significantly identified when at least two peptides per protein were identified in at least three of four biological replicates. These criteria resulted in no false positive identifications.
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2013-02-19 | PXD000128 | Pride
Project description:Tandem mass tag spectrometry was performed on eluted peptides from 8 single cell clones derived from a DNA mismatch repair deficient (MMRd) lung cancer cell line, which was derived ex vivo from tumors induced autochthonously in the lungs of mice in the MMRd models described in Westcott, PMK, et al., Nature Genetics, 2023 (in press). Whole exome sequencing and neoantigen prediction was performed on all single cell clones, and predicted neoantigens associated with somatic mutations were provided in the inclusion list for searching spectra. Tandem mass spectra were searched with Sequest (Thermo Fisher Scientific; version IseNode in Proteome Discoverer 2.5.0.400). Sequest was set up to search a mouse uniprot database (v.July 3, 2020; 55650 entries containing common contaminants and the proteins GFP, Cas9, Puromycin, and P2A (present in the cell lines) assuming no digestion enzyme (unspecific). Sequest was searched with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 10.0 ppm. TMTpro was added as a fixed modification on the K and N terminus of peptides. Oxidation of methionine was specified in Sequest as a variable modification. Resulting peptides were filtered to exclude peptides with an isolation interference of greater than 30 percent and ppm error greater than plus/minus 3 ppm of the median ppm error of all peptide-spectrum matches (PSMs). Peptides were further prioritized based on concordance of relative abundance across the single cell clones with presence/absence of the associated somatic mutation.
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2014-02-03 | PXD000410 | Pride
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2013-04-09 | PXD000113 | Pride