Global Analysis of the Membrane Subproteome of Pseudomonas aeruginosa Using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer. Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P. aeruginosa and for prokaryotes in general.

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

“…Both methods utilized the same sample preparation steps, except one used Brij-58, a nonionic detergent, to solubilize the proteins obtained from microsomal fractions and the other used methanol, an organic solvent that is directly compatible with µrpLC-MS/MS. [21][22][23] As determined by SCXC and µrpLC-MS/MS analysis, a total of 1109 peptides were identified with the Brij-58 method and 673 peptides were identified using the methanol approach. To remove any ambiguities regarding protein identification, each peptide was searched using BLASTP to match it with a unique protein.…”

“…[16][17][18] The use of MS-compatible organic solvents such as methanol for membrane protein solubilization, followed by direct insolution tryptic digestion and LC-MS/MS, proved effective in proteomic studies of bacterial and human membranes. 17,[19][20][21][22][23] The application of this protocol to plant membrane proteomics has not been reported, although differential solubilization of membrane proteins in chloroform/methanol mixtures has been widely used in the extraction of highly hydrophobic proteins of the chloroplast envelope and mitochondrial membrane. 3,7,24 This procedure is biased toward hydrophobic membrane proteins and is often combined with extraction by basic and saline solutions to increase the diversity of membrane proteins obtained.…”

Cysteine tagging for MS‐based proteomics

“…Several members of important membrane protein families were identified, and in particular the six ATP-binding cassette (ABC) transporters (Goshe, Blonder, & Smith, 2003). A similar strategy for the global analysis of the membrane sub-proteome of Pseudomonas aerugonisa was also reported (Blonder et al, 2004). In another study, von Haller et al carried out parallel analyses with the same cysteine biotin-affinity tag and a standard gel-based approach on membrane associated lipid rafts from Jurkat T-cells.…”

“LANESPECTOR”, a tool for membrane proteome profiling based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/liquid chromatography - tandem mass spectrometry analysis: Application toListeria monocytogenes membrane proteins

“…The progress in all these areas has now raised increased interest in exploiting LC-MS/MS for comparative investigations of integral membrane proteins not confined to those containing only b-barrel strands [18,30]. However, proposals addressing the questions of how to display and explore these kinds of complex data are overdue.…”