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High resolution mobility-based ion separations in Structures for Lossless Ion Manipulations (SLIM) have been useful for ion mobility separations for a variety of molecular classes in the gas phase. Here, we present multi-pass SLIM separations for gas-phase proteins in their near-native state exhibiting charge state dependent arrival time distributions using carbonic anhydrase (29 kDa), alcohol dehydrogenase (148 kDa), and apo-transferrin (79 kDa). For the selected charge states of each protein species, we investigate the conformational space using molecular dynamic simulations and calculated the collision cross section (CCS) values using IMoS. The measured CCS values obtained from the SLIM arrival time distributions (ATDs) agreed within ~6% difference when compared to the calculated CCS values. The experimental CCS values were obtained from calibration curves for the arrival times of Agilent Tune Mix ions. For multi-pass separations, the ATDs were converted to CCS values by deconvoluting the multi-pass arrival times into accurate single-pass values amenable to the single-pass calibration curves. Mass spectra of carbonic anhydrase (CA) showed three different charge states (z = 9+ to 11+). Their corresponding mobility peaks were baseline-separated using 8-m single-pass separations. Single-pass analysis of alcohol dehydrogenase (ADH) exhibit three predominant charge states (z = 23+ to 25+) with mobility overlap between adjacent charge states. The mobility peak resolution for ADH improved with multi-pass separations (up to 24-m path length). In addition, CCS distributions obtained for charge states z = 16+ to 18+ of apo-transferrin reveal a transition from a compact unimodal form (z = 18+ and 19+) to broader multi-modal CCS distributions for z = 16+. For apo-transferrin, 40-m multi-pass separations were performed allowing for complete isolation of the selected mobility range corresponding to z = 17+ leading to selective isolation of a narrow arrival time window. The extended mobility separations provided minimal alterations to the structure of the proteins, and the experimentally derived CCS values showed minimal change as a function of separation time or number of passes. Mobility-based ion separations for native-like proteins, using SLIM, open opportunities for native-IMS applications as well as other manipulations enabled by SLIM like mobility selective isolation and collection. 

Insights into the effect of temperature (T) and relative humidity (RH) as well as structure and polarisation on ion mobility help the comparison and interpretation of mobility and mass-based data. We measured alkylammonium ions in air under different T (14 °C, 24 °C, 34 °C and 41 °C) and RH (0 %, 20 %, 40 %) conditions using two individual setups (in both cases a planar differential mobility analyser coupled with a time-of-flight mass spectrometer) and the results are in excellent agreement. Mobility increases with rising T and decreases with water vapour loading. When separating the measurement mobility by structures, clear mass dependence was observed. The measured mobilities exhibited large deviations from theoretically calculated results in dry conditions, which are possibly caused by adduct formation on the monomer ions via clustering (or reactions). This phenomenon seems to be unavoidably associated with light ions under atmospheric pressures, which is worth further exploration and bearing in mind when comparing measurements to calculations. Both methanol and oxygen (occasionally nitrogen or alkyl chain elongation) are possible candidates of the adduct. Under spherical assumption, we used the modified Mason–Schamp's approximation to link the measured mobility to the mobility equivalent diameter. The drag enhancement factor and the effective gas-molecule collision diameter derived from our measurement data are comparable to literature values. Our data also exposed a non-linear dependence on the polarisation parameter . Polarisation, and were parameterised using linear models against ion structures, T, and RH for primary, secondary and tertiary alkylammonium ions with identical alkyl groups. Our model parametrisations predict mobilities within ±10 % deviation from the measured data. The model also has satisfying predicting power for alkylammonium ions with unidentical alkyl structures. 

This study introduces a high-field calculation method for ion mobility, focusing on structural changes in ions resulting from heating due to high fields. This approach notably improves ion mobility prediction in arbitrary field systems.