Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G |
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Authors: | Moo-Jin Suh David J ClarkPrashanth P Parmer Robert D FleischmannScott N Peterson Rembert Pieper |
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Institution: | Pathogen Functional Genomics Resource Center, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA |
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Abstract: | The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A51 and the C-terminal cell wall anchor site at residue T1086. The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572 Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A51, were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties. |
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Keywords: | PTMs post-translational modifications PMF peptide mass fingerprinting TMPP-Ac-OSu N-succinimidyloxycarbonyl-methyl-tris(2 4 6-trimethoxyphenyl) phosphonium bromide SasG Staphylococcus aureus surface protein G ESI-MS electrospray ionization mass spectrometry MALDI-TOF matrix-assisted laser desorption ionization-time of flight TFA trifluoroacetic acid 2DE two dimensional gel electrophoresis CID collision-induced dissociation PBS phosphate buffered saline EDTA ethylenediaminetetraacetic acid BAM benzamidine-HCl PMSF phenylmethylsulphonyl fluoride |
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