Ion Mobility Mass Spectrometry for the Separation and Characterization of Small Molecules

Single‐cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in‐depth characterization of individual cells by mass spectrometry (MS)‐based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow‐rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10‐fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS‐isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single‐cell proteomes to transcriptome data revealed a stable‐core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra‐high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease. A new ultra‐high sensitivity LC‐MS workflow increases sensitivity by up to two orders of magnitude and enables true single‐cell proteome analysis. In‐depth comparison indicates that the single‐cell transcriptome is stochastic while the single‐cell proteome is complete and stable.

Collision cross section (CCS) measurements resulting from ion mobility-mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values (DTCCSN2) for over 120 unique ion species with the lowest measurement uncertainty to date. The reproducibility of these DTCCSN2 values are evaluated across three additional laboratories on a commercially available DTIM-MS instrument. The traditional stepped field CCS method performs with a relative standard deviation (RSD) of 0.29% for all ion species across the three additional laboratories. The calibrated single field CCS method, which is compatible with a wide range of chromatographic inlet systems, performs with an average, absolute bias of 0.54% to the standardized stepped field DTCCSN2 values on the reference system. The low RSD and biases observed in this interlaboratory study illustrate the potential of DTIM-MS for providing a molecular identifier for a broad range of discovery based analyses.

Improvements in the performance and availability of commercial instrumentation have made ion mobility-mass spectrometry (IM-MS) an increasingly popular approach for the structural analysis of ionic species as well as for separation of complex mixtures. Here, a new research instrument is presented which enables complex experiments, extending the current scope of IM technology. The instrument is based on a Waters SYNAPT G2-S i IM-MS platform, with the IM separation region modified to accept a cyclic ion mobility (cIM) device. The cIM region consists of a 98 cm path length, closed-loop traveling wave (TW)-enabled IM separator positioned orthogonally to the main ion optical axis. A key part of this geometry and its flexibility is the interface between the ion optical axis and the cIM, where a planar array of electrodes provides control over the TW direction and subsequent ion motion. On either side of the array, there are ion guides used for injection, ejection, storage, and activation of ions. In addition to single and multipass separations around the cIM, providing selectable mobility resolution, the instrument design and control software enable a range of "multifunction" experiments such as mobility selection, activation, storage, IMS n, and importantly custom combinations of these functions. Here, the design and performance of the cIM-MS instrument is highlighted, with a mobility resolving power of approximately 750 demonstrated for 100 passes around the cIM device using a reverse sequence peptide pair. The multifunction capabilities are demonstrated through analysis of three isomeric pentasaccharide species and the small protein ubiquitin.