Dataset Information

Proteogenomic analysis of Helicobacter pylori strain 26695

ABSTRACT: Dear Sir or Madam, we report an in-depth proteogenomics study of Helicobacter pylori strain 26695 and provide the supporting MS data via ProteomExchange. The study includes 2 biological replicates with 6 different datasets: G1: in-gel digestion with trypsin, replicate 1 G2: in-gel digestion with trypsin, replicate 2 T1: SEC fractionation of low molecular weight (LMW) proteins and subsequent trypsin digestion, replicate 1 T2: SEC fractionation of LMW proteins and subsequent trypsin digestion, replicate 2 A1: SEC fractionation of LMW proteins and subsequent AspN digestion, replicate 1 A2: SEC fractionation of LMW proteins and subsequent AspN digestion, replicate 2 L1: SEC fractionation of LMW proteins and subsequent LysC digestion, replicate 1 L2: SEC fractionation of LMW proteins and subsequent LysC digestion, replicate 2 In our proteogenomics approach, we could identify four previously missing protein annotations and were able to correct sequences of six protein coding regions. Furthermore we identified signal peptidase cleavage sites for 72 different proteins. MGFs were generated by Maxquant 1.1 [1] using recalibration of peptide parent masses. For PRIDE (http://www.ebi.ac.uk/pride) submission, we made an additional database search with Mascot and X!Tandem using the SearchGUI [2]. Therefore we searched against a NCBI database of H. pylori strain 26695 complemented with the sequence corrections, signal peptide cleavage sites and missing annotations with the same configurations as described in materials and methods. For pride xml export we used the software PeptideShaker (http://code.google.com/p/peptide-shaker/). The complemented database has entries which will be submitted to the UniProtKB via SPIN. The entries have the according SPIN number as accession number. The NCBI accession numbers for the shortened sequences due to signal peptide cleavage are extended with “_1”. The fasta database is added to the submission. For additional information, please contact me: stephan.mueller@ufz.de Yours sincerely, Stephan Mueller References: [1] Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment. Journal of proteome research. 2011;10:1794-805. [2] Vaudel M, Barsnes H, Berven FS, Sickmann A, Martens L. SearchGUI: An open-source graphical user interface for simultaneous OMSSA and X!Tandem searches. Proteomics. 2011;11:996-9.

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Helicobacter Pylori 26695

SUBMITTER: Stephan MÃ¼ller

PROVIDER: PXD000054 | Pride | 2013-08-01

REPOSITORIES: Pride

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Publications

Identification of new protein coding sequences and signal peptidase cleavage sites of Helicobacter pylori strain 26695 by proteogenomics.

Müller Stephan A SA Findeiß Sven S Pernitzsch Sandy R SR Wissenbach Dirk K DK Stadler Peter F PF Hofacker Ivo L IL von Bergen Martin M Kalkhof Stefan S

Journal of proteomics 20130509

Correct annotation of protein coding genes is the basis of conventional data analysis in proteomic studies. Nevertheless, most protein sequence databases almost exclusively rely on gene finding software and inevitably also miss protein annotations or possess errors. Proteogenomics tries to overcome these issues by matching MS data directly against a genome sequence database. Here we report an in-depth proteogenomics study of Helicobacter pylori strain 26695. MS data was searched against a combin ...[more]

PMID: 23665149

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Project description:Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p < 0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The PT-G affected genes in the microarray analysis were validated by qRT-PCR, however these genes were also affected by PT treated negative controls suggesting that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study demonstrates novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation.

Dataset Information

Proteogenomic analysis of Helicobacter pylori strain 26695

Publications

Identification of new protein coding sequences and signal peptidase cleavage sites of Helicobacter pylori strain 26695 by proteogenomics.

Similar Datasets

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