PNNL

Abstract

High resolution mobility-based ion separations in Structures for Lossless Ion Manipulations (SLIM) have been useful for determining conformational distributions 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 collision cross section (CCS) values for selected conformations using IMoS. The measured CCS values obtained from the SLIM arrival time distributions (ATDs) were in agreement within ~6% error. 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 indicating mobility differences between adjacent charge states. 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) revealing two conformers for the lowest charge state z = 23+ that were not resolved via single-pass separation. 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 CCS distributions with multiple conformers (z = 16+). 40-m multi-pass separations were performed allowing for complete isolation of the selected mobility range for z = 17+ leading to selective isolation of targeted conformations of interest. Mobility-based ion separations, combined with high selectivity for intact proteins, show the path to broad utility, including the production of high-purity biomolecular subpopulations from heterogenous samples for high-resolution molecular structure determination.