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Using LC-MS with de novo software to fully characterize the multiple methylations of lysine residues in a recombinant fragment of an outer membrane protein from a virulent strain of Rickettsia prowazekii.

Abstract
The outer membrane protein B (OmpB) of the typhus group rickettsiae is an immunodominant antigen and has been shown to provide protection against typhus in animal models. Consequently, OmpB is currently being considered as a potential rickettsiae vaccine candidate to be used in humans. The OmpB from virulent strains are heavily methylated while the attenuated strains are hypomethylated. Western blot analysis of partially digested OmpB revealed that one of the reactive fragments was located at the N-terminus (fragment A, aa 33-272). Recently, we have over expressed, purified, and chemically methylated the recombinant fragment A from Rickettsia prowazekii (Ap). The methylated Ap was thoroughly characterized by LC/MS/MS on the ProteomeX workstation. The protein sequence of Ap with and without methylation was 87.7% and 100% identified, respectively. This high sequence coverage enabled us to determine the sites and extent of methylation on the lysine residues in Ap. All the lysine residues except the C-terminus lysine were either mono-, di- or tri-methylated. In addition, carbamylation on the N-terminus glycine was identified using a combination of denovo sequencing (DeNovoX) and the pattern recognition (SALSA) program with accurate mass measurement. We demonstrated that the use of peptide identification (SEQUEST) in combination with SALSA and denovo sequencing provided a useful means to characterize the sequence and posttranslational modifications of given proteins.
AuthorsChien-Chung Chao, Shiaw-Lin Wu, Wei-Mei Ching
JournalBiochimica et biophysica acta (Biochim Biophys Acta) Vol. 1702 Issue 2 Pg. 145-52 (Nov 01 2004) ISSN: 0006-3002 [Print] Netherlands
PMID15488766 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Bacterial Outer Membrane Proteins
  • Peptide Fragments
  • Recombinant Proteins
  • Lysine
Topics
  • Amino Acid Sequence
  • Animals
  • Bacterial Outer Membrane Proteins (chemistry, genetics, metabolism)
  • Humans
  • Lysine (chemistry)
  • Mass Spectrometry (instrumentation, methods)
  • Methylation
  • Molecular Sequence Data
  • Peptide Fragments (chemistry, genetics, metabolism)
  • Protein Processing, Post-Translational
  • Recombinant Proteins (genetics, metabolism)
  • Rickettsia prowazekii (chemistry, pathogenicity)
  • Sequence Analysis, Protein
  • Software

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