title: Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G.
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 A(51) and the C-terminal cell wall anchor site at residue T(1086). 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 A(51), 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.
authors.affiliation: Pathogen Functional Genomics Resource Center, J. Craig Venter Institute, Rockville, MD 20850, USA. msuh@jcvi.org
authors.forename: Moo-Jin
authors.initials: MJ
authors.lastname: Suh
authors.forename: David J
authors.initials: DJ
authors.lastname: Clark
authors.forename: Prashanth P
authors.initials: PP
authors.lastname: Parmer
authors.forename: Robert D
authors.initials: RD
authors.lastname: Fleischmann
authors.forename: Scott N
authors.initials: SN
authors.lastname: Peterson
authors.forename: Rembert
authors.initials: R
authors.lastname: Pieper
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018441:Biofilms; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021382:Protein Sorting Signals / Q000502:physiology*; D013211:Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; C524374:SasG protein, Staphylococcus aureus; D037521:Virulence Factors