Comparing Selected-Ion Collision Induced Unfolding with All Ion Unfolding Methods for Comprehensive Protein Conformational Characterization

Abstract

Structural analysis by native ion mobility−mass spectrometry provides a direct means to characterize protein interactions, stability, and other biophysical properties of disease-associated biomolecules. Such information is often extracted from collision-induced unfolding (CIU) experiments, performed by ramping a voltage used to accelerate ions entering a trap cell prior to an ion mobility separator. Traditionally, to simplify data analysis and achieve confident ion identification, precursor ion selection with a quadrupole is performed prior to collisional activation. Only one charge state can be selected at one time, leading to an imbalance between the total time required to survey CIU data across all protein charge states and the resulting structural analysis efficiency. Furthermore, the arbitrary selection of a single charge state can inherently bias CIU analyses. We herein aim to compare two conformation sampling methods for protein gas-phase unfolding: (1) traditional quadrupole selection-based CIU and (2) nontargeted, charge selection-free and shotgun workflow, all ion unfolding (AIU). Additionally, we provide a new data interpretation method that integrates across all charge states to project collisional cross section (CCS) data acquired over a range of activation voltages to produce a single unfolding fingerprint, regardless of charge state distributions. We find that AIU in combination with CCS accumulation across all charges offers an opportunity to maximize protein conformational information with minimal time cost, where additional benefits include (1) an improved signal-to-noise ratios for unfolding fingerprints and (2) a higher tolerance to charge state shifts induced by either operating parameters or other factors that affect protein ionization efficiency.

Native Mass Spectrometry and Collision-Induced Unfolding of Laser-Ablated Proteins

“…A Synapt G2-S traveling wave ion mobility spectrometry quadrupole time-of-flight (TWIMS-q-ToF) IM-MS system (Waters, Milford, MA) was used for native IM-MS and all multiplexed collision-induced unfolding (MPX-CIU) experiments. , MassLynx software V4.1 (Waters) was utilized for instrument control and data acquisition. Instrumental parameters for native IM-MS experiments were optimized (Table S1) to minimize collisional and electrostatic ion heating , and kept consistent for all multiplicate MS and CIU experiments.…”

“…Protein ion unfolding experiments were performed without quadrupole isolation per charge state by applying an “all-ion/charge multiplexed” approach, , allowing simultaneous acquisition of unfolding pathways for all charge states observed; the broad-band CIU approach reduced the time required to probe all unfolding pathways of interest from surface-sampled proteins. Reproducibility of CIU curves can be affected by chemical noise and low signal-to-noise ratio (S/N); hence, to generate average or representative CIU curves, all IM-MS analyses were performed in triplicate.…”

“…Once spray conditions were optimized, the acquisition of each data set was completed in ∼15 min, resulting in total acquisition times of ∼45 min per broad-band triplicate data set. A data acquisition time of 15 min with a broad-band approach was chosen to enhance speed (bypassing precursor ion selection), 64 minimize potential ion heating, 65 and maintain sufficient ion signal for high activation energy ranges where ion loss can be most prevalent. Ion collisional activation was achieved in the pre-IM trap cell by establishing a potential difference from 5 to 100 V in 5 V increments (ΔV step = 5 V and range = 5−100).…”

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

“…The CIU curve is similar to ones measured by ion mobility, in which the increase in CCS during CIU frequently ranges from 30-50%. [56][57][58][59][60] Figure 5. Orbitrap CCS of native-like carbonic anhydrase (9+, 29 kDa monomer, m/z 3233) relative to the collision energy used for in-source CID to cause collisional heating.…”

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

“…The CIU curve is similar to ones measured by ion mobility, in which the increase in CCS during CIU frequently ranges from 30-50%. [58][59][60][61][62] Figure 5. Orbitrap CCS of native-like carbonic anhydrase (9+, 29 kDa monomer, m/z 3233) relative to the collision energy used for in-source collisional activation to cause collisional heating.…”