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Six chlorinated bispyridinium mono-oximes, analogous to potent charged reactivators K027, K048, and K203, were synthesized with the aim of improving lipophilicity and reducing the pKa value of the oxime group, thus resulting in a higher oximate concentration at pH 7.4 compared to non-chlorinated analogues. The nucleophilicity was examined and the pKa was found to be lower than that of analogous non- chlorinated oximes. All the new compounds efficiently reactivated human AChE inhibited by nerve agents cyclosarin, sarin, and VX. The most potent was the bis-chlorinated analogue of oxime K027 with significantly improved ability to reactivate the conjugated enzyme due to improved binding affinity and molecular recognition. Its overall reactivation of sarin-, VX-, and cyclosarin-inhibited AChE was respectively, three-, seven-, and eight-fold higher than by K027. Its universality, favorable acid dissociation constant coupled with its negligible cytotoxic effect and successful ex vivo scavenging of nerve agents in whole human blood warrant further analysis of this compound as an organophosphorus antidote.

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-μL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC-MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC-MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC-MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 μg of HeLa cells tryptic digest separated during a 60 min gradient at 68 μL/min on a 1.0 mm × 250 mm column held at 55 °C and using an aqua-acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC-MS is an attractive alternative for bottom-up exploratory proteomics.