ABSTRACT: The raw spectrum data of the proteomic study of Tetragenococcus halophilus under hypo-osmotics stress condition and hyper-osmotic stress condition, the data was generated by combining isobaric laveling regents TMT and reverse phase HPLC-MS/MS.
INSTRUMENT(S): Isobaric labeling reagents Tandem Mass Tags, Reverse Phase HPLC-MS/MS
Project description:Isobaric labelling technique coupled with high resolution mass spectrometry has been widely employed in the proteomics workflows requiring relative quantification. For each high resolution tandem mass spectrum (MS/MS), it can be used not only to quantify the peptide from different samples by reporter ions，but also to identify the peptide it derived from. Since isobaric related ions act as noises in database searching, the MS/MS spectrum should be preprocessed before peptide/protein identification. In this paper, we demonstrated that there were a lot of high frequency, high abundance isobaric related ions in MS/MS spectrum. By combining removing isobaric related ions with deisotoping and deconvolution in MS/MS preprocessing procedure, the peptide/protein identification sensitivity improved significantly. A user-friendly software TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files which can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools.
Project description:Identification of targets of the protein disulfide reductase thioredoxin using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and thiol specific differential labeling with isotope-coded affinity tags (ICAT). Reduction of specific target disulfides is quantified by measuring ratios of cysteine residues labeled with the ?heavy? (13C) and ?light? (12C) ICAT reagents in peptides derived from tryptic digests of Trx-treated and non-treated samples. Keywords: protein, LC-MS/MS, ICAT Overall design: Biological Material: Dissected embryos from seeds from the malting barley (Hordeum vulgare) cultivar Barke (2002 harvest) germinated for 48 h as previously described (Bønsager et al., 2007). Sample preparation: Sample A. Barley embryo protein extract, treated with iodoacetamide (IAM) during extraction, was incubated ± thioredoxin and after 1 h incubation at RT, IAM was again added (final concentration 10 mM) to irreversibly block cysteines reduced by thioredoxin. Excess IAM was removed and remaining cysteine residues were completely reduced by tris(2-carboxyethyl)phosphine (TCEP) under denaturing conditions and ICAT-labeled (+Trx, ICATL; -Trx, ICATH), essentially as recommended by the manufacturer (Applied Biosystems). The ICATL and ICATH labeled samples were mixed 1:1 and subjected to trypsin digestion followed by isolated of ICAT labeled peptides by avidin affinity chromatography and LC-MS/MS analysis on an Agilent 1100 nanoflow HPLC (Agilent, Palo Alto CA) coupled to a QSTAR quadrupole time-of-flight mass spectrometer (Applied Biosystems). Data were acquired in information dependent mode with a cycle of a survey mass spectrum followed by tandem mass spectra of the three most intense multiply charged ions (1 s each) after which the selected ions were placed on an exclusion list for 45 s. Sample B (control). Same as for sample A except IAM addition after thioredoxin incubation was omitted. Data processing and peptide quantification: The raw data .wiff files were processed with the Mascot script for Analyst (QS 2.0) version 1.6b20 (Matrix Science, London, UK). The charge state was determined from the survey spectrum with a default charge setting of multiply charged precursors. The MS/MS data was centroided and de-isotoped without MS/MS averaging to generate the Mascot Generic Format (MGF) peak list. Spectra with less than 10 peaks were rejected. Database searches of the centroided data were performed using the Mascot search engine (Matrix Science) against the Viridiplantae (Green Plants) subset of Swiss-Prot 54.6, nrNCBI (downloaded on 20071206) and the Barley EST database (barley gene index [HvGI] release 9.0). The following search criteria were used: MS mass tolerance 0.2 Da; MS/MS tolerance 0.2 Da; no trypsin miss-cleavage allowed. Variable modifications: ICATH (cysteine), ICATL (cysteine), carbamidomethyl (cysteine) and oxidation (methionine). Tentative consensus sequences (TC sequences) identified in HvGI release 9.0 were blasted (http://www.ncbi.nlm.nih.gov/blast) against the nrNCBI database. Quantification of ICAT labeled peptide pairs was performed using the software MS quant version 1.4.3a12 (http://www.msquant.sourceforge.net). Spectra were manually validated and peptides with a sequence tag of at least three amino acids and a Mascot score of 20 were accepted. References: Bønsager BC, Finnie C, Roepstorff P, Svensson B (2007) Spatio-temporal changes in germination and radical elongation of barley seeds tracked by proteome analysis of dissected embryo, aleurone layer, and endosperm tissues. Proteomics 7:4528-4540.
Project description:We found the bone marrow stromal-derived neural progenitor cells secretome have the neural protection effect. Proteomic analysis was performed nn order to analyze the protection factor in the secretome. Keywords: Neural protection, secretome Overall design: Secretome samples were run first by HPLC. After that, 20 fractions were selected for MS/MS.
Project description:Transcriptional profiling of Arabidopsis transgenic 35S:AtMYB102-SRDX plant under MS medium containing 200mM NaCl for 24h. Transgenic Arabidopsis (background : Col-0) overexpressing AtMYB102 fused to an active transcriptional repression domain (SRDX) showed strong salt tolerance. Overall design: Two-condition experiment, Control condition (3-week old plant was transfered to MS medium for 24h) and Salt stress condition (plant was transferred to MS containg 200mM NaCl for 24h), Biological replicates: 3 control replicates and 3 salt stress condition replicates.
Project description:Quantitative proteomic analysis of human post-mortem substantia nigra from patients with Parkinson’s disease (n=3) versus controls (n=3) based on sixplex TMT (TMT6) isobaric labeling which allowed protein simultaneous identification and quantification. Samples were digested and pooled after TMT6 labeling. Peptides were fractionated by OFFGEL electrophoresis (24 fractions). Each peptidic fraction was analyzed in two technical replicates by LC−MS/MS analysis on a LTQ Orbitrap XL mass spectrometer equipped with a NanoAcquity HPLC system. MS data were processed using EasyProtConv (v. 1.21). Peak lists were generated from raw data combining CID and HCD spectra, and submitted to Easyprot (v2.2) which uses Phenyx (GeneBio, Geneva, Switzerland) for protein identification . Searches were conducted against UniProt Swiss-Prot database (08-Feb-2011, 525’207 entries) specifying Homo sapiens taxonomy. Trypsin was selected as the proteolytic enzyme, one missed cleavage was allowed, cysteines carbamidomethylation, TMT6 amino terminus and TMT6 lysine were set as a fixed modification whereas oxidized methionine as variable. The minimum peptide length was five amino acids and precursor error tolerance was 10 ppm. False positive ratios were estimated using a reverse decoy database . Peptide z-scores were set to maintain a false positive peptide ratio below 1%. Proteins with at least two unique distinct peptide sequences were selected and clustered based on shared peptides indistinguishable by MS  using Isobar (v 1.3.1) package. The protein entry containing the most peptides was selected as the group reporter. For protein quantification, TMT6 reporter ion intensities were extracted, corrected for isotopic impurities as provided by the manufacturer and each channel was normalized imposing equal median intensity. Only spectra from protein specific peptides not eliminated after outlier filtering were used for quantification. Isobar R package (v 1.3.1.) was used to calculate protein ratios and select statistically significant differentially regulated proteins between the two states.
Project description:Investigation of whole genome expression pattern of 60 and 72 hours post fertilization Danio Rerio embryos exposed to TMT and vehicle control Overall design: Embryos were exposed to 10uM TMT or control from 48hpf to 60 or 72 hpf. Three replicates were collected for each time point. 40 embryos were pooled to comprise a replicate.
Project description:Investigation of whole genome expression pattern of 60 and 72 hours post fertilization Danio Rerio embryos exposed to TMT and vehicle control Embryos were exposed to 10uM TMT or control from 48hpf to 60 or 72 hpf. Three replicates were collected for each time point. 40 embryos were pooled to comprise a replicate.
Project description:We used whole mouse genome microarrays to the effects of γ-TmT on global gene expression in the prostate TRAMP and age-matched C57BL/6 mice were administered either 600 mg/kg of γ-TmT or a control vehicle via oral gavage. Twelve hours after dosing, prostate tissues were collected for RNA extraction.
Project description:High throughput “omics technologies” such as transcriptomics and proteomics provide insights into the metabolic potential of an organism and have been used to understand the genetic and the central carbon metabolism mechanisms for the production of desired end products in various cellulolytic clostridia cultured on different substrates In this study, C. termitidis was cultured on lignocellulose derived simple and complex sugars: cellobiose, xylose, xylan and α–cellulose as sole carbon sources. 2D HPLC-MS/MS quantitative Proteomic profiles and RNA seq transcriptome profiles (next generation sequencing to identify and quantify RNA in biological samples) were analyzed to identify the genes involved in substrate degradation, cellodextrin transport and end product synthesis related genes Identification of these genes is important in understanding the metabolic networks of C. termitidis and could be valuable engineering targets for improving biomass to biofuel production. Closridium termitidis was cultured on 2g/L each of α-cellulose, xylan, cellobiose and xylose. Samples were collected from the exponential phase. 2 replicate experiments were conducted under each substrate condition
Project description:A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on isobaric TMT reporter ion quantification.