Using Dicationic Ion-Pairing Compounds To Enhance the Single Cell Mass Spectrometry Analysis Using the Single-Probe: A Microscale Sampling and Ionization Device

A unique mass spectrometry (MS) method has been developed to determine the negatively charged species in live single cells using the positive ionization mode. The method utilizes dicationic ion-pairing compounds through the miniaturized multifunctional device, the single-probe, for reactive MS analysis of live single cells under ambient conditions. In this study, two dicationic reagents, 1,5p entanediyl-bis(1-butylpyrrolidinium) difluoride (C-5(bpyr)(2)F-2) and 1,3-propanediyl-bis- I I I (tripropylphosphonium) difluoride (C-3(triprp)(2)F-2), were added in the solvent and introduced into single cells to extract cellular contents for real-time MS analysis. The negatively charged (1 charged) cell metabolites, which form stable ion-pairs (1+ charged) with dicationic compounds (2+ charged), were detected in positive ionization mode with a greatly improved sensitivity. We have tentatively assigned 192 and 70 negatively charged common metabolites as adducts with (C-5(bpyr)(2)F-2) and (C-5(triprp)(2)F-2), respectively, in three separate SCMS experiments in the positive ion mode. The total number of tentatively assigned metabolites is 285 for C-5(bpyr)(2)F-2 and 143 for C-3(triprp)(2)F-2. In addition, the selectivity of dicationic compounds in the complex formation allows for the discrimination of overlapped ion peaks with low abundances. Tandem (MS/MS) analyses at the single cell level were conducted for selected adduct ions for molecular identification. The utilization of the dicationic compounds in the single-probe MS technique provides an effective approach to the detection of a broad range of metabolites at the single cell level.