An evaluation of standardized software for processing GC/MS data from different vendors' instruments in a forensic laboratory.

Forensic science laboratories perform analyses on a variety of materials using gas chromatography/mass spectrometry (GC/MS). Instruments from different vendors may be used, requiring analysts to be proficient in the use of multiple proprietary software packages for collecting and processing data. There is no standardized GC/MS software available that can acquire data from different vendors' instruments. However, there are third-party processing software products that can import data files in different formats. The Centre of Forensic Sciences compared the data processing performance of one such product, ACD/MS Manager Suite, with three instrument vendors' software used for casework analysis. This product was tested for its compatibility with the existing software, its capability to load and present data, and to initiate searches of commercial libraries. The study shows that the MS Manager module provides a means for the forensic analyst to view, process, and report on data from different sources in one software package.

Combining targeted and nontargeted data analysis for liquid chromatography/high-resolution mass spectrometric analyses.

Increasing importation of food and the diversity of potential contaminants have necessitated more analytical testing of these foods. Historically, mass spectrometric methods for testing foods were confined to monitoring selected ions (SIM or MRM), achieving sensitivity by focusing on targeted ion signals. A limiting factor in this approach is that any contaminants not included on the target list are not typically identified and retrospective data mining is limited. A potential solution is to utilize high-resolution MS to acquire accurate mass full-scan data. Based on the instrumental resolution, these data can be correlated to the actual mass of a contaminant, which would allow for identification of both target compounds and compounds that are not on a target list (nontargets). The focus of this research was to develop software algorithms to provide rapid and accurate data processing of LC/MS data to identify both targeted and nontargeted analytes. Software from a commercial vendor was developed to process LC/MS data and the results were compared to an alternate, vendor-supplied solution. The commercial software performed well and demonstrated the potential for a fully automated processing solution.

The dissociation chemistry of ionized methyl carbamate and its isomers revisited: theory and experiment in concert.

Early combined computational and experimental studies by J.K. Terlouw c.s. in Refs. 1-3 propose that low-energy methyl carbamate ions, NH(2)COOCH(3)(•+) (MC-1), rearrange into distonic ions NH(2)C(OH)OCH(2)(•+) and hydrogen-bridged radical cations [NH(2)C=O--H--OCH(2)](•+) (MC-5) en route to the observed losses of HCO(•) and CO. In this study, we report on the generation of ions MC-5 by decarbonylation of ionized methyl oxamate NH(2)COCOOCH(3)(•+). Theory and experiment agree that ion MC-5 is a key intermediate in the dissociation of low-energy ions MC-1. The subsequent HCO(•) loss, however, may not proceed via the route proposed in Ref. 2, but rather by an entirely different mechanism involving proton-transport catalysis (PTC) in ion MC-5. This view is further supported by the dissociation behaviour of the MC-5 isotopologue [ND(2)C=O--D--OCH(2)](•+), which is conveniently generated from the d(3)-labelled glycolamide ion DOCH(2)C(=O)ND(2)(•+).

Binding site characterization and resistance to a class of non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase.

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzimidazole 5-carboxamide and related compounds were found at three amino acid positions in the thumb domain: Pro(495) with substitutions to Ser, Leu, Ala, or Thr; Pro(496) substitutions to Ser or Ala; and a V499A substitution. Mutations at each of these positions conferred different levels of resistance to this drug class: the Pro(495) changes provided the greatest shifts in compound potency, followed by moderate changes in potency with the Pro(496) substitutions, and finally only minor shifts in potency with V499A. Combinations that include the benzimidazole 5-carboxamide polymerase inhibitors and compounds that bind other sites or other HCV targets, including HCV protease inhibitors, are complementary in cell culture models of HCV RNA replication at suppressing the emergence of resistant variants. This novel class of compounds and unique binding site expand the diversity of HCV antivirals currently under development and offer the potential to improve the treatment of chronic HCV infection.

Characterization of the binding site for inhibitors of the HPV11 E1-E2 protein interaction on the E2 transactivation domain by photoaffinity labeling and mass spectrometry.

An indandione-containing class of inhibitors abrogates DNA replication of human papillomavirus (HPV) types 6 and 11 by binding reversibly to the transactivation domain (TAD) of the viral E2 protein and inhibiting its interaction with the viral E1 helicase. To locate the binding site of this class of protein-protein interaction inhibitors, a benzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site of covalent modification of the E2-TAD was identified by proteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides by LC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminal methyl of residue Met101. Evidence further pinpointed the site of photoaffinity attachment to the terminal carbon atom, which is significant in providing a definitive example of the ability to locate photoinduced cross-linking to a polypeptide with atomic resolution using solely mass spectrometric detection. The location of the inhibitor binding site vis-à-vis the Glu39 and Glu100 residues sensitive to mutation for HPV 11 E2-TAD is discussed in relation to the crystal structure of the E2-TAD from the related HPV type 16.