Native and compactly folded in-solution conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of gas-phase pepstatin A - pepsin complex binding strength differences

• Published in: European journal of mass spectrometry (Chichester, England) p. 14690667231178999
• Main Authors: Koy, Cornelia, Glocker, Ursula M, Danquah, Bright D, Glocker, Michael O
• Format: Journal Article
• Language: English
• Published: England 01.12.2023
• Subjects: ITEM-TWO; Pepstatin A – pepsin complex; in-solution protein conformers; intermediate analysis; gas phase complex dissociation; complex binding strength; protein folding; CID; nanoESI-MS
• ISSN: 1751-6838
• DOI: 10.1177/14690667231178999

Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded pepsin conformers (C and C ) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.