Determination of hATG8 Binding Selectivity of AIM (Autophagy-Interacting Motif) Peptides Using Native Electrospray Ionization Mass Spectrometry

Brennan, Andrew; Oldham, Neil; Layfield, Robert; Scott, Daniel

Abstract

Establishing the hATG8 binding selectivity of AIM (autophagy-interacting motif) sequences found within autophagy system proteins provides insights into their biological roles, and in the case of disease-associated AIM mutations, potential pathophysiological mechanisms. Given the sometimes small differences in affinity for an individual AIM amongst the six hATG8 proteins, establishing AIM preferences can be experimentally challenging. We describe a native mass spectrometry method that is suitable for detecting such differences, using synthetic AIM peptides and recombinant hATG8 proteins, to probe hATG8-AIM interactions in the gas phase. Binding preferences of a single AIM peptide against multiple hATG8s, or two AIM peptides against a single hATG8 (e.g., wild-type versus mutant AIM), may be determined.

Citation

Brennan, A., Oldham, N., Layfield, R., & Scott, D. (2024). Determination of hATG8 Binding Selectivity of AIM (Autophagy-Interacting Motif) Peptides Using Native Electrospray Ionization Mass Spectrometry. In I. P. Nezis (Ed.), Selective autophagy: methods and protocols (237-246). Springer. https://doi.org/10.1007/978-1-0716-4067-8_19

• Acceptance Date: Aug 9, 2024
• Online Publication Date: Aug 9, 2024
• Publication Date: Aug 9, 2024
• Deposit Date: Aug 19, 2024
• Publicly Available Date: Aug 10, 2025
• Peer Reviewed: Peer Reviewed
• Pages: 237-246
• Series Title: Methods in molecular biology
• Series Number: 2845
• Series ISSN: 1064-3745
• Book Title: Selective autophagy: methods and protocols
• ISBN: 9781071640661
• DOI: https://doi.org/10.1007/978-1-0716-4067-8_19
• Public URL: https://nottingham-repository.worktribe.com/output/38383324
• Publisher URL: https://link.springer.com/protocol/10.1007/978-1-0716-4067-8_19