Identification of cod trypsin isoform
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Bruker Daltonics flex series, instrument model
ORGANISM(S): Gadus Morhua (atlantic Cod)
SUBMITTER: 
Bjarki Stefansson
PROVIDER: PRD000134 | Pride | 2010-12-17
REPOSITORIES: pride
Dataset's files
Items per page: 5 1 - 5 of 6 |
Similar Datasets
Project description:Vacuoles and lysosomes are single-membrane-bound organelles involved in diverse functions such as intracellular digestion and storage or secretion of metabolites. To understand their origin, evolution and fundamental features, the identification of proteins comprising these compartments in missing links would be invaluable. So, we isolated the vacuoles from Cyanidioschyzon merolae, which is considered to be one of the most primitive photosynthetic eukaryotes, and identified 46 proteins by matrix-assisted laser desorption/ionization time of flight-mass spectrometry. Keywords: peptide mass fingerprinting, MALDI-TOF
2009-09-01 | GSE12042 | GEO
Project description:Gel based approach : Concanavalin A bound fiber glycoproteins were resolved through SDS PAGE followed by identification of trypsinised gel pieces through Nano-LC MALDI TOF/TOF. Solution based approach: Concanavalin A bound fiber glycoproteins were solution phase digested and fractionated through SCX followed by Nano-LC MALDI TOF/TOF based identification
2014-03-18 | PXD000178 | Pride
Project description:Proteins were extracted from different regions of HT29 colon carcinoma cells grown as multicell tumour spheroids. Changes in protein expression across the regions were determined by trypsin digestion iTRAQ 4-plex labelling, 2D separation using OffGel (24 fractions) and RP nanoHPLC, MALDI TOF-TOF MS/MS analysis and Mascot database searching.
2014-04-08 | PRD000369 | Pride
Project description:Identification of different Trichuris spp. using MALDI-TOF MS
| PRJEB50468 | ENA
Project description:Clinical Flavobacterium columnare ATCC 49512 was grown on Flavobacterium columnare growth medium (FCGM). Bacteria from four colonies at mid-exponential phase were harvested, total proteins were isolated, and identified using 2-DE MALDI TOF/TOF MS and 2-D LC ESI MS/MS analyses. The MS/MS spectra for all peptides were analyzed using sequest algorithm
2016-03-11 | PRD000810 | Pride
Project description:Bovine brain capillary endothelial cells (BBCEC) are cocultured in presence of rat astrocytes to reinduce blood brain barrier properties. After checking of BBB propereties reinduction, BBCEC are harvested by collagenase treatment, lysed with Triton X-100 based lysis buffer. Triton X-100 soluble proteins are subjected to organic fractionation step and the 5 generated fractions were submitted to 1D-LC-MS/MS analysisi on MALDI-TOF-TOF-MS.
2010-12-03 | PRD000250 | Pride
Project description:Preliminary study of identification of Trichuris suis by MALDI-TOF MS
| PRJEB47832 | ENA
Project description:The present study was initiated to evaluate the quantitative proteomic profiling of Protothecazopfiigenotypes. The cells (P.zopfii genotype 1 –noninfectious and genotype 2 -infectious) were cultured in triplicates until the mid-logarithmic growth phase; the proteins were extracted after sonication on ice, followed by 2D DIGE separation as recommended by the manufacturer (GE Healthcare). The differentially expressed proteins spots were identified using Decodon software analysis (Delta 2D version 4.0 software). Protein identification was carried out by MALDI TOF MS/MS (Ultraflex III TOF/TOF, Bruker Daltonics, Bremen, Germany). The spectra was acquired using the automated option (AutoXecute ) of the Flex Analysis software version 3.3 (Bruker Daltonics, Leipzig, Germany), processed using Flex analysis software version 3.3 (Bruker Daltonics, Leipzig, Germany) and the database search was conducted using the MS/MS ion search (MASCOT, http://www.matrixscience.com) against all entries of NCBInr (GenBank)/Swissprot with subsequent parameters: trypsin digestion, up to one missed cleavage, fixed modifications: carbamidomethyl and with the following variable modifications: oxidation (M), peptide tol.: +- 1.2 Da, MS/MS tol.: +- 0.6 Da, peptide charge: +1. The results were assessed using MOWSE score, p- and E values and those possess positive hits with cRAP database were eliminated from the list.
2013-07-22 | PXD000126 | Pride
Project description:The purpose of this study was to generate a basis for the decision of what protein quantities are reliable and find a way for accurate and precise protein quantification. To investigate this we have used thousands of peptide measurements to estimate variance and bias for quantification by iTRAQ (isobaric tags for relative and absolute quantification) mass spectrometry in complex human samples. A549 cell lysate was mixed in the proportions 2:2:1:1:2:2:1:1, fractionated by high resolution isoelectric focusing and liquid chromatography and analyzed by three mass spectrometry platforms; LTQ Orbitrap Velos, 4800 MALDI-TOF/TOF and 6530 Q-TOF. We have investigated how variance and bias in the iTRAQ reporter ions data are affected by common experimental variables such as sample amount, sample fractionation, fragmentation energy, and instrument platform. Based on this, we have suggested a concept for experimental design and a methodology for protein quantification. By using duplicate samples in each run, each experiment is validated based on its internal experimental variation. The duplicates are used for calculating peptide weights, unique to the experiment, which is used in the protein quantification. By weighting the peptides depending on reporter ion intensity, we can decrease the relative error in quantification at the protein level and assign a total weight to each protein that reflects the protein quantitation confidence. We also demonstrate the usability of this methodology in a cancer cell line experiment as well as in a clinical data set of lung cancer tissue samples. In conclusion, we have in this study developed a methodology for improved protein quantification in shotgun proteomics and introduced a way to assess quantification for proteins with few peptides. The experimental design and developed algorithms decreased the relative protein quantification error in the analysis of complex biological samples. Data analysis: LTQ Orbitrap Velos Proteome discoverer 1.1 with Mascot 2.2 (Matrix Science) was used for protein identification. Precursor mass tolerance was set to 10 ppm and for fragments 0.8 Da and 0.015 Da were used for detection in the linear iontrap and the orbitrap, respectively. Oxidized methionine was set as dynamic modification and carbamidomethylation, N-terminal 8plex iTRAQ, and lysyl 8plex iTRAQ as fixed modifications. 4800 MALDI TOF/TOF Peptide identification from the Maldi-TOF/TOF data was carried out using the Paragon algorithm in the ProteinPilot 2.0 software package (Applied Biosystems). Default settings for a 4800 instrument were used (i.e. no manual settings for mass tolerance was given). The following parameters were selected in the analysis method: iTRAQ 8plex peptide labeled as sample type, IAA as alkylating agent of cysteine, trypsin as digesting enzyme, 4800 as instrument, gel based ID and Urea denaturation as special factors, biological modifications as ID focus, and thorough ID as search effort. 6530 QTOF Peptide identification from the QTOF data was carried out using the Spectrum Mill Protein Identification software (Agilent). Data was extracted between MH+ 600 and 4000 Da (Agilent’s definition). Trypsin was used as digesting enzyme, and parent and daughter ion tolerance was set to 25 and 50 ppm, respectively. IAA for cysteine and iTRAQ partial-mix (N-term, K) were set as fixed modifications while oxidized methionine was set as variable modification. Database and peptide cut-off for all searches Searches were performed against the IPI database (build 3.64) limited to human sequences allowing 2 missed cleavages. False discovery rate (FDR) was estimated by searching the data against a database consisting of both forward and reversed sequences and set to < 1 % at the protein level using MAYU. Peptides corresponding to a <1% protein FDR rate was used in the calculations. Peptide and protein identification using Mascot for comparison between instruments Peptide identifications were performed using Mascot Daemon 2.3.2 with Mascot 2.4 for fractions 32 to 36 from IPG-IEF with 400 ug loaded peptides. Carbamidomethylation (CAM) for cysteine was set as fixed modification, oxidized methionine as variable modification and iTRAQ 8plex was set as quantification for all searches. MALDI-TOF/TOF search settings: Parent and daughter ion tolerance was set to 150 ppm and 0.2 Da, respectively. LTQ Orbitrap search settings: Precursor mass tolerance was set to 10 ppm and for fragments 0.8 Da and 0.015 Da were used for data generated in the linear ion trap and the orbitrap, respectively. QTOF search settings: Parent and daughter ion tolerance was set to 25 and 50 ppm, respectively.
2013-08-16 | PXD000418 | Pride
Project description:MALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1,400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. This work presents a generic method and near complete list of MALDI IMS biomarkers that will become a valuable resource for the IMS community. Detailed description of the bioinformatics pipeline can be found in pipeline.txt. Briefly, different Mascot Distiller, Mascot and Scaffold versions have been used for the bottom-up and top-down data.
2013-07-03 | PXD000125 | Pride