Ion mobility free high throughput peptide collisional cross-section measurements in fourier transform mass spectrometer on the proteome wide scale

The integration of Ion Mobility (IM) devices with mass spectrometers has facilitated the precise determination of peptide collisional cross sections (CCS), introducing an additional dimension to Mass Spectrometry (MS)-based investigations. While the utilization of transient decay rate in Fourier Transform Mass Spectrometry (FTMS) for assessing CCS values has been reported for Orbitrap™ and Ion Cyclotron Resonance (ICR) MS systems, this study presents a novel method that enables real time highly reproducible and accurate measurement of CCS using FTMS in high-throughput LC-MS studies on entire proteome scale.

The key innovation of this approach lies in its ability to infer peptide CCS values directly from the FTMS spectra, without the need for a dedicated IM device, by relating the observed spectral peak resolution to the rate of collisions experienced by ions during the detection determined by their CCS.

Experiments were conducted on the research grade Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer using HeLa cell tryptic and Lys-C lysates, as well as chemically modified peptides (methylation, acetylation, and phosphorylation). The inferred CCS values showed strong correlation with the IM-MS values reported in the literature. The reproducibility of the CCS measurements in the shotgun proteomics experiments was on par with the values obtained using dedicated IM devices.

Our findings illustrate the utility of IM-free FTMS based CCS in the analysis of peptide secondary structures and Post Translational Modifications (PTMs), revealing that the conformation of peptides is likely determined by the balance between hydrophobic interactions and charge repulsion. Modifications such as phosphorylation, methylation, and acetylation were found to impact peptide CCS values, likely due to increased hydrophobicity and loss of charge.

This novel FTMS-based method for CCS determination offers a high-throughput and widely accessible solution for integrating CCS information into standard proteomics workflows without relying on the dedicated IM devices.