Dataset Information

Prototheca zopfii genotype proteomics analysis

ABSTRACT: 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.

INSTRUMENT(S): Bruker Daltonics flex series, instrument model

ORGANISM(S): Chlorella-like Algae Prototheca Zopfii

SUBMITTER: Jayaseelan Murugaiyan

PROVIDER: PXD000126 | Pride | 2013-07-22

REPOSITORIES: Pride

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Publications

Two-dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style.

Murugaiyan Jayaseelan J Weise Christoph C von Bergen Martin M Roesler Uwe U

Proteomics 20130901 17

Biochemical, serological, and genetic analyses have identified two genotypes of Prototheca zopfii, a unicellular microalga belonging to the family Chlorellaceae. The P. zopfii genotype 1, abundantly present in cow barns and environment, remains nonpathogenic, while P. zopfii genotype 2 has been isolated from cows with bovine mastitis. The present study was carried out to identify the protein expression level difference between the pathogenic and nonpathogenic strains of P. zopfii. A total of 782 ...[more]

PMID: 23852777

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Project description:Datasets contain protein identification and raw data from MALDI protein identifications. Identified proteins resulted from a comparison of GLC1 and GLC1 SCLC cell lines using 2D DIGE. MALDI-TOF-MS analyses were performed on an UltraFlexTM II (Bruker Daltonics) instrument according to the instructions of the manufacturer. The instrument was equipped with a scoutTM MTP MALDI target. The spectra were acquired in the positive ion mode according to the settings given by the manufacturer. For external calibration, a peptide standard (m/z 757.399, 1296.684, 1619.822, 2093.086 and 3147.471) was used. The MALDI-PMF spectra were processed using the FlexAnalysis™ 2.4 software (Bruker Daltonics) and converted in the .xml format. For peak detection, the spectra were subjected to an internal recalibration using 13 different monoisotopic masses from autolysis products of trypsin and fragments of keratins ranging from m/z 842.509 – 2825.406. Following parameters were applied: snap peak detection algorithm, signal to noise threshold of 6, maximal number of peaks 100, quality factor threshold 50 and baseline subtraction TopHat. The generated mass lists were subsequently sent to ProteinScapeTM 1.3 (Bruker Daltonics, Bremen, Germany), triggering database searches using ProFound (Version 2002.03.01, Proteometrics LLC) and MASCOT (Version 2.3.02, Matrix Science, London, UK). The following search parameters were selected: fixed cysteine modification with propionamide, variable modification due to methionine oxidation, one maximal missed cleavage sites in case of incomplete trypsin hydrolysis and no details about 2-DE derived protein mass and pI. Using the Score booster function of ProteinScapeTM the mass lists were recalibrated and background masses removed using a list containing 44 masses occurring in a minimum of 10% of generated peak lists. The database searches were run with a mass tolerance of 40 ppm using UniProt’s human complete proteome set (downloaded on 26.10.2012) containing 68.109 protein entries. The used database is a composite database consisting of the UniProtKB entries and a duplicate of the same database, in which the amino acid sequence of each protein entry was randomly shuffled. Proteins reaching Profound score > 1.5 or Mascot score > 64 were considered as identified. Using these criteria one decoy database entry was found by the search engines indicating high confidence of protein identifications. If several database entries of homologues proteins matched these criteria only the entry with the highest score was reported.

Project description:Genetic and environmental factors mediate via different physiological and molecular processes a shifted energy balance leading to overweight and obesity. Significant progress in understanding the molecular mechanisms of appetite regulation and control of food consumption was made when the hypothalamus was identified as an important player in regulating energy and glucose homeostasis by sensing circulating hormones and nutrients. To get insights in the underlying processes involved in energy intake and weight gain, we compared hypothalamic tissue of mice kept on a high-fat or control diet for 10 days by a proteomic approach. Using 2D difference gel electrophoresis in combination with LC-MS/MS we observed significant abundance changes in 16 protein spots. One isoform of the protein DJ-1 was elevated in the high fat diet group in the analyzed three different mouse strains SWR/J, C57BL/6N and AKR/J. Large scale validation of DJ-1 isoforms in individual samples and tissues confirmed a shift in the pattern of DJ-1 isoforms towards more acidic isoforms in several brain and peripheral tissues after feeding a high-fat diet for 10 days. The identification of an oxidation of cysteine 106 as well as a 2-succinyl modification of the same residue by mass spectrometry not only explains the isoelectric shift of DJ-1 but also links our results to similar shifts of DJ-1 observed in neurodegenerative disease states under oxidative stress. We hypothesize that DJ-1 is a common physiological sensor involved in both nutrition-induced effects and neurodegenerative disease states. Peak lists of MS/MS spectra acquired on the HCT ion trap (Bruker Daltonics, Bremen, Germany) were generated using the software tool Data Analysis (version 4 SP4 Build 281, Bruker Daltonics) with default parameters. For peptide and protein identification using MASCOT 2.4.1, peak lists were correlated with the Swiss-Prot part of the UniProtKB release 12/2012 including a reverse decoy version of all entries resulting in a total of 33,160 mus musculus sequences. All searches were performed for peptides with charge states 2+, 3+ and 4+ with tryptic specificity allowing one missed cleavage. No fixed modification was set, oxidation of methionine was considered as variable modification. Ion trap MS/MS spectra were accepted with cut off scores of 30 as well as mass tolerance of 0.4 Da was applied for MS and MS/MS experiments. Only proteins with at least 2 identified peptides were considered to be identified. Raw files from the LTQ Orbitrap ELITE were further processed for protein and peptide identification and quantification using the MaxQuant software suite version 1.3.0.5 (Max Planck Institute of Biochemistry, Planegg, Germany) mainly with default parameters. Within the software suite database searches were carried out using 16,615 mouse sequences from the SwissProt part of UniProtKB (release June 2013) applying the following parameters: mass tolerance Fourier transformed mass spectra (Orbitrap) first/second search: 20 ppm / 6 ppm, mass tolerance fragment spectra (linear ion trap): 0.5 Da, no fixed modification was specified. For variable modifications methionine oxidation, acetylation at protein N-termini and carbamidomethylation were considered as well as one of the following modifications per search event: trioxidation of cysteines, 2-succinylation of cysteines. Peptides and proteins were accepted at a false discovery rate of 1 % and proteins identified with a minimum of two peptides.

Dataset Information

Prototheca zopfii genotype proteomics analysis

Publications

Two-dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style.

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