Expanding Orbitrap collision cross section measurements to native protein applications through kinetic energy and signal decay analysis

Abstract

The measurement of collision cross sections (CCS) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the soft sphere collision model, thus deviating from prior FT-MS CCS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision inducing unfolding and MS/MS experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.

Determining Collisional Cross Sections from Ion Decay with Individual Ion Mass Spectrometry

“…Isotopic resolution is readily achieved for all proteins besides alcohol dehydrogenase and pyruvate kinase in Figure 3 and could be achieved for larger protein complexes by extending the acquisition time to increase the number of ions collected. 23 The Ecm correction factor determined herein showed stability over the course of the study (∼8 months), which is consistent with the correction factor developed by James et al 20 This approach may also be applied to previously collected data using DMTmode if sufficient ion decay was observed. Determining CCS by I 2 MS provides an orthogonal measurement that may be extracted from I 2 MS data sets without increasing the experimental time required, allowing for more detailed characterization of proteins and protein complexes with Orbitrap mass spectrometers.…”

“…Native CCS were determined using the collision energy correction in eq 2 as previously described by James et al 20 The center of mass collision energy (Ecm) for a protein charge state is used to estimate the percent difference between the Orbitrap CCS measurements and IMS CCS measurements to correct values from eq 1. Native CCS were ultimately After sampling, decay constants are extracted from the population decay plots.…”

“…This results in underestimated CCS by eq 1, and the extracted CCS values for native proteins require additional correction by eq 2. 20 After calculating raw CCS values for native proteins, the percent deviation between the Orbitrap CCS and IMS CCS values is plotted in Supplemental Figure S4 to determine the Ecm correction factor in eq 2. Charge state CCS values were then weighted by the number of ions collected for each charge state and averaged together as prior literature suggests native protein CCS does not change significantly across the native charge state distribution.…”