A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors

Effects of Different Waveforms on the Performance of Active Capillary Dielectric Barrier Discharge Ionization Mass Spectrometry

Active capillary dielectric barrier discharge ionization (DBDI) is emerging as a compact, low-cost, and robust method to form intact ions of small molecules for detection in near real time by portable mass spectrometers. Here, we demonstrate that by using a 10 kHz, ~2.5 kV p-p high-voltage square-wave alternating current plasma, active capillary DBDI can consume less than 1 μW of power. In contrast, the power consumed using a sine and triangle alternating current waveform is more than two orders of magnitude higher than that for the square waveform to obtain a similar voltage for plasma generation. Moreover, the plasma obtained using a square waveform can be significantly more homogenous than that obtained using sine and triangle waveforms. Protonated dimethyl methylphosphonate (DMMP) and deprotonated perfluorooctanoic acid (PFOA) can be detected at about the same or higher abundances using square-wave DBDI mass spectrometry compared with the use of sine and triangle waveforms. By use of benzylammonium thermometer ions, the extent of internal energy deposition using square, sine, or triangle waveform excited plasmas are essentially the same at the optimum voltages for ion detection. Using an H-bridge circuit driving a transformer optimized to reduce losses, square-wave active capillary DBDI can be continuously powered for ~50 h by common 9 V-battery (PP3). [Figure not available: see fulltext.