In this paper a novel and general procedure is presented for detection of organophosphate-inhibited human butyrylcholinesterase (HuBuChE), which is based on electrospray tandem mass spectrometric analysis of phosphylated nonapeptides obtained after pepsin digestion of the enzyme. The utility of this method is exemplified by the positive analysis of serum samples from Japanese victims of the terrorist attack with sarin in the Tokyo subway in 1995.

To develop a mass spectrometric assay for the detection of sulfur mustard adducts with human serum albumin, the following steps were performed: quantitation of the binding of the agent to the protein by using [(14)C]sulfur mustard and analysis of acidic and tryptic digests of albumin from blood after exposure to sulfur mustard for identification of alkylation sites in the protein. The T5 fragment containing an alkylated cysteine could be detected in the tryptic digest with micro-LC/tandem MS analysis. Attempts to decrease the detection limit for in vitro exposure of human blood by analysis of the alkylated T5 fragment were not successful. After Pronase treatment of albumin, S-[2-[(hydroxyethyl)thio]ethyl]Cys-Pro-Phe was analyzed by means of micro-LC/tandem MS, allowing a detection limit for in vitro exposure of human blood of 10 nM, which is 1 order of magnitude lower than that obtained by means of modified Edman degradation. The analytical procedure could be successfully applied to the analysis of albumin samples from Iranian victims of the Iran-Iraq war.

The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach ensures constant growth conditions, both in the presence and absence of toluene. A considerable negative effect of toluene on the cell yield was found. The need for energy in the defence against toluene was reflected by differentially expressed proteins for cell energy management. In toluene-stressed cells the balance between proton motive force (PMF) enforcing and dissipating systems was shifted. NAD(P)H generating systems were upregulated whereas the major proton-driven system, ATP synthase, was downregulated. Other differentially expressed proteins were identified: outer membrane proteins, transport proteins, stress-related proteins and translation-related proteins. In addition, a protein with no assigned function was found. This study yielded a more detailed view of the effect of toluene on the intracellular energy management of P. putida S12 and several novel leads have been obtained for further targeted investigations.

The protein toxin ricin, which originates from the seeds of Ricinus communis plants, has been the subject of increased interest, due to its potential terrorist use. Exceptionally, this toxin is also subject to the Chemical Weapons Convention. In this paper, it is shown that mass spectrometry can be used to unambiguously verify the presence of ricin in crude toxin preparations. It is demonstrated that MALDI MS can be used for screening, either by direct analysis or by trypsin digestion and peptide mapping. Purified ricin from several varieties of R. communis was characterized by LC-ES MS(/MS). A crude ricin preparation from a single bean was similarly characterized. An LC method was set up with product ion MS/MS detection of selected marker peptides specific for ricin: T5, T7, T11, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. This method was then used to unambiguously identify ricin in a crude preparation of ricin. The MALDI MS molecular weight analysis and the marker peptides LC-ES MS/MS analysis give a forensic level of identification of ricin when combined with activity testing.

A convenient and rapid micro-anion exchange liquid chromatography (LC) tandem electrospray mass spectrometry (MS) procedure was developed for quantitative analysis in serum of O-isopropyl methylphosphonic acid (IMPA), the hydrolysis product of the nerve agent sarin. The mass spectrometric procedure involves negative or positive ion electrospray ionization and multiple reaction monitoring (MRM) detection. The method could be successfully applied to the analysis of serum samples from victims of the Tokyo subway attack and of an earlier incident at Matsumoto, Japan. IMPA levels ranging from 2 to 135 ng/ml were found. High levels of IMPA appear to correlate with low levels of residual butyrylcholinesterase activity in the samples and vice versa. Based on our analyses, the internal and exposure doses of the victims were estimated. In several cases, the doses appeared to be substantially higher than the assumed lethal doses in man.