Comprehensive characterization of methicillin-resistant Staphylococcus aureus subsp. aureus COL secretome by two-dimensional liquid chromatography and mass spectrometry.
Two-dimensional LC combined with whole protein and peptide mass spectrometry is used to characterize proteins secreted by methicillin-resistant Staphylococcus aureus COL. Protein identifications were accomplished via off-line protein fractionation followed by digestion and subsequent peptide analysis by reverse phase LC-ESI-LTQ-FT-MS/MS. Peptide MS/MS analysis identified 127 proteins comprising 59 secreted proteins, seven cell wall-anchored proteins, four lipoproteins, four membrane proteins, and 53 cytoplasmic proteins. The identified secreted proteins included various virulence factors of known functions (cytotoxins, enterotoxins, proteases, lipolytic enzymes, peptidoglycan hydrolases, etc.). Accurate whole protein mass measurement (+/-1.5 Da) of the secreted proteins combined with peptide analysis enabled identification of signal peptide cleavage sites and various post-translational modifications. In addition, new observations were possible using the present approach. Although signal peptide cleavage is highly specific, signal peptide processing can occur at more than one site. Surprisingly, cleaved signal peptides and their fragments can be observed in the extracellular medium. The prediction accuracies of several signal peptide prediction programs were also evaluated.
Ravipaty
Shobha
S
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Reilly
James P
JP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002853:Chromatography, Liquid / Q000379:methods*; D005583:Fourier Analysis; D055624:Methicillin-Resistant Staphylococcus aureus / Q000378:metabolism*; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins