Characterization and of Gas-Phase Native(-Like) Proteins Using Structures for Lossless Ion Manipulations

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.