Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated. Top‐down mass spectrometry using infrared multiphoton dissociation (IRMPD) of native membrane proteins such as G protein‐coupled receptors (GPCRs) provides good sequence coverage from native protein ions. We observe successive cleavage of adjacent amino acids, specifically within transmembrane helices, enabling unambiguous identification of membrane proteins and their complexes.

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© 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.

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C.A.L, T.J.E. J.L.B, N.V.K, A.D., C.K, K.G, L.U., and K.P. declare no competing interests. I.L., P.K., and H.‐Y.Y. are employees of OMass Therapeutics. C.V.R. is a consultant of OMass Therapeutics. J.D.H., C.M, and J.E.P.S. are employees of Thermo Fisher Scientific.