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Helicobacter pylori proteomics by 2‐DE/MS, 1‐DE‐LC/MS and functional data mining
Author(s) -
Jungblut Peter R.,
Schiele Franziska,
ZimnyArndt Ursula,
Ackermann Renate,
Schmid Monika,
Lange Sabine,
Stein Robert,
Pleissner KlausPeter
Publication year - 2009
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200900361
Subject(s) - proteome , chemistry , proteomics , urea , chromatography , sephadex , helicobacter pylori , biochemistry , biology , gene , enzyme , genetics
With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea-solubilized proteins by 2-DE/MS (data set 2-DE) and by 1-DE-LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1-DE-LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research (http://www.mpiib-berlin.mpg.de/2D-PAGE/), which gives access to raw mass spectra (MALDI-TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT (http://webclu.bio.wzw.tum.de/prompt/) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2-DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2-DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea-insoluble proteins and makes them accessible for separation by SDS-PAGE and LC. The 2-DE/MS analysis with urea-solubilized proteins and the 1-DE-LC/MS analysis with the urea-insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT-generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.