Helicobacter pylori proteomics by 2-DE/MS, 1-DE-LC/MS and functional data mining.
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.
Jungblut
Peter R
PR
Core Facility Protein Analysis, Max Planck Institute for Infection Biology, 10117 Berlin, Germany. jungblut@mpiib-berlin.mpg.de
Schiele
Franziska
F
Zimny-Arndt
Ursula
U
Ackermann
Renate
R
Schmid
Monika
M
Lange
Sabine
S
Stein
Robert
R
Pleissner
Klaus-Peter
KP
D001426:Bacterial Proteins / Q000032:analysis*; D002853:Chromatography, Liquid; D057225:Data Mining; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D016480:Helicobacter pylori / Q000737:chemistry*; D020407:Internet; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D020543:Proteome