Abstract
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.
Highlights
Two-dimensional LC combined with whole protein and peptide mass spectrometry is used to characterize proteins secreted by methicillin-resistant Staphylococcus aureus COL
Protein Identification Strategy—Sibbald et al [29] used a rigorous approach utilizing a combination of computational tools and an optimized type I signal peptidase (SpsB) recognition search pattern to estimate that 71 extracellular proteins are produced by S. aureus COL
To identify proteins present in S. aureus COL extracellular medium, proteins were extracted by TCA precipitation from cultures at stationary growth phase, a phase during which extracellular proteins are preferentially expressed [27, 41]
Summary
Two-dimensional LC combined with whole protein and peptide mass spectrometry is used to characterize proteins secreted by methicillin-resistant Staphylococcus aureus COL. LC and MS Characterization of S. aureus COL Secretome fore, a comprehensive description of secretory proteins (the secretome) of different S. aureus strains is vital to gain insights into its pathogenesis This information will be valuable in identifying novel virulence factors and should help in the development of new diagnostic tools and vaccines. To this end, S. aureus secretory proteins have been identified using a variety of classical techniques including Western blot, ELISA, and one- and two-dimensional gel electrophoresis (1DE/2DE) with N-terminal sequencing (18 –20). They were able to tentatively identify three and four secreted proteins in NCTC 8325 and MRSA 3543 strains, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
