High-throughput determination of dry mass of single bacterial cells by ultrathin membrane resonators.

How bacteria are able to maintain their size remains an open question. Techniques that can measure the biomass (dry mass) of single cells with high precision and high-throughput are demanded to elucidate this question. Here, we present a technological approach that combines the transport, guiding and focusing of individual bacteria from solution to the surface of an ultrathin silicon nitride membrane resonator in vacuum. The resonance frequencies of the membrane undergo abrupt variations at the instants where single cells land on the membrane surface. The resonator design displays a quasi-symmetric rectangular shape with an extraordinary capture area of 0.14 mm2, while maintaining a high mass resolution of 0.7 fg (1 fg = 10-15 g) to precisely resolve the dry mass of single cells. The small rectangularity of the membrane provides unprecedented frequency density of vibration modes that enables to retrieve the mass of individual cells with high accuracy by specially developed inverse problem theory. We apply this approach for profiling the dry mass distribution in Staphylococcus epidermidis and Escherichia coli cells. The technique allows the determination of the dry mass of single bacterial cells with an accuracy of about 1% at an unparalleled throughput of 20 cells/min. Finally, we revisit Koch & Schaechter model developed during 60 s to assess the intrinsic sources of stochasticity that originate cell size heterogeneity in steady-state populations. The results reveal the importance of mass resolution to correctly describe these mechanisms.

The Omnitrap Platform: A Versatile Segmented Linear Ion Trap for Multidimensional Multiple-Stage Tandem Mass Spectrometry.

Multidimensional multiple-stage tandem processing of ions is demonstrated successfully in a novel segmented linear ion trap. The enhanced performance is enabled by incorporating the entire range of ion activation methods into a single platform in a highly dynamic fashion. The ion activation network comprises external injection of reagent ions, radical neutral species, photons, electrons, and collisions with neutrals. Axial segmentation of the two-dimensional trapping field provides access to a unique functionality landscape through a system of purpose-designed regions for processing ions with maximum flexibility. Design aspects of the segmented linear ion trap, termed the Omnitrap platform, are highlighted, and motion of ions trapped by rectangular waveforms is investigated experimentally by mapping the stability diagram, tracing secular frequencies, and exploring different isolation techniques. All fragmentation methods incorporated in the Omnitrap platform involving radical chemistry are shown to provide complete sequence coverage for partially unfolded ubiquitin. Three-stage (MS3) tandem mass spectrometry experiments combining collision-induced dissociation of radical ions produced by electron meta-ionization and further involving two intermediate steps of ion isolation and accumulation are performed with high efficiency, producing information rich spectra with signal-to-noise levels comparable to those obtained in a two-stage (MS2) experiment. The advanced capabilities of the Omnitrap platform to provide in-depth top-down MSn characterization of proteins are portrayed. Performance is further enhanced by connecting the Omnitrap platform to an Orbitrap mass analyzer, while successful integration with time-of-flight analyzers has already been demonstrated.