Current methodologies for protein quantitation include 2-dimensional gel electrophoresis techniques, metabolic labeling, and stable isotope labeling methods to name only a few. The current literature illustrates both pros and cons for each of the previously mentioned methodologies. Keeping with the teachings of William of Ockham, "with all things being equal the simplest solution tends to be correct", a simple LC/MS based methodology is presented that allows relative changes in abundance of proteins in highly complex mixtures to be determined. Utilizing a reproducible chromatographic separations system along with the high mass resolution and mass accuracy of an orthogonal time-of-flight mass spectrometer, the quantitative comparison of tens of thousands of ions emanating from identically prepared control and experimental samples can be made. Using this configuration, we can determine the change in relative abundance of a small number of ions between the two conditions solely by accurate mass and retention time. Employing standard operating procedures for both sample preparation and ESI-mass spectrometry, one typically obtains under 5 ppm mass precision and quantitative variations between 10 and 15%. The principal focus of this paper will demonstrate the quantitative aspects of the methodology and continue with a discussion of the associated, complementary qualitative capabilities.

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-Si 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.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values ( K 0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E / N ; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method‐dependent results) only if the gas nature, temperature or E / N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

We present the use of drug-like molecules as a traveling wave (T-wave) ion mobility (IM) calibration sample set, covering the m/z range of 122.1-609.3, the nitrogen collision cross-section (Ω(N(2))) range of 124.5-254.3 Å(2) and the helium collision cross-section (Ω(He)) range of 63.0-178.8 Å(2). Absolute Ω(N(2)) and Ω(He) values for the drug-like calibrants and two diastereomers were measured using a drift-tube instrument with radio frequency (RF) ion confinement. T-wave drift-times for the protonated diastereomers betamethasone and dexamethasone are reproducibly different. Calibration of these drift-times yields T-wave Ω(N(2)) values of 189.4 and 190.4 Å(2), respectively. These results demonstrate the ability of T-wave IM spectrometry to differentiate diastereomers differing in Ω(N(2)) value by only 1 Å(2), even though the resolution of these IM experiments were ∼40 (Ω/ΔΩ). Demonstrated through density functional theory optimized geometries and ionic electrostatic surface potential analysis, the small but measurable mobility difference between the two diastereomers is mainly due to short-range van der Waals interactions with the neutral buffer gas and not long-range charge-induced dipole interactions. The experimental RF-confining drift-tube and T-wave Ω(N(2)) values were also evaluated using a nitrogen based trajectory method, optimized for T-wave operating temperature and pressures, incorporating additional scaling factors to the Lennard-Jones potentials. Experimental Ω(He) values were also compared to the original and optimized helium based trajectory methods.

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