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

, "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" (2004). Uniformed Services University of the Health Sciences. 4. http://digitalcommons.unl.edu/usuhs/4Using 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 AbstractThe 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. D

Serological Reactivity and Biochemical Characterization of Methylated and Unmethylated Forms of a Recombinant Protein Fragment Derived from Outer Membrane Protein B of Rickettsia typhi

“…Although our conditions were similar to theirs, we used a different reaction temperature for the methylation reaction. The difference in reaction temperature has been attributed to the presence of all mono-, di-, and trimethylated lysines in our previous work (3). Similarly, this may explain our current observation of mono-, di-, and trimethylated lysines in mrAt.…”

Genomic, proteomic, and transcriptomic analysis of virulent and avirulentRickettsia prowazekiireveals its adaptive mutation capabilities

“…Post-translational modifications may also play a role in the host-R. prowazekii relationship as was shown previously in other hostparasite systems (Polevoda and Sherman 2007). The relation between post-translational modifications, especially methylation, and virulence of R. prowazekii strains has been previously suggested (Rodionov et al 1991;Ching et al 1993;Turco and Winkler 1994;Chao et al 2004). Here, we report only subtle differences in methylation of proteins, mainly concerning methyltransferase (RP789) itself, when comparing Rp22 with Erus.…”

Structural characterization of tick cement cones collected from in vivo and artificial membrane blood-fed Lone Star ticks (Amblyomma americanum)

“…The gel slices were washed with HPLC grade water followed by 100% acetonitrile, and then dried in a speedvac as described by Chao et al (2004). The proteins were digested with trypsin (0.5 mg/mL, Promega, Madison, WI, USA) using a 50:1 ratio (protein:trypsin) overnight at 37° C. Hydrophilic peptides were eluted using NANOpure water, followed by the elution of hydrophobic peptides with 50% acetonitrile with 5% trifluoroacetic acid.…”