title: Potential of liquid-isoelectric-focusing protein fractionation to improve phosphoprotein characterization of Pseudomonas aeruginosa PA14.
abstract: Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes and signal transduction in bacteria. Bacterial-proteome analysis is required to determine which proteins have been conditionally expressed and whether any post-translational modifications are present. One of the greatest challenges of proteome analysis is the fractionation of these complex protein mixtures to detect low-abundance phosphoproteins. Liquid-phase isoelectric focusing (IEF) is a promising analytical tool in proteomics, but as far as we are aware no work has studied the reproducibility of this approach. In this study, we investigated the phosphoproteome of Pseudomonas aeruginosa strain PA14. We first tested in-solution IEF protein fractionation, and then used this technique to fractionate the proteins in the complex mixture. Next, phosphopeptides were enriched with titanium dioxide and analyzed by high-resolution, high-accuracy liquid chromatography-mass spectrometry. With this approach, we succeeded in characterizing 73 unique phosphorylated peptides belonging to 63 proteins. Interestingly, we observed a higher percentage of modified tyrosine, revealing the importance of this phosphorylated residue in bacteria.
authors.affiliation: UMR 6270 CNRS, Laboratoire Polymères, Biopolymères, Surfaces, 76820, Mont-Saint-Aignan, France.
authors.forename: Tassadit
authors.initials: T
authors.lastname: Ouidir
authors.forename: Frédérique
authors.initials: F
authors.lastname: Jarnier
authors.forename: Pascal
authors.initials: P
authors.lastname: Cosette
authors.forename: Thierry
authors.initials: T
authors.lastname: Jouenne
authors.forename: Julie
authors.initials: J
authors.lastname: Hardouin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D010750:Phosphoproteins