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

Abstract

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 in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution 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 in-solution pepsin conformers (Ca and Cb) 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 in-solution pepsin conformers by interrogating quantitative traits of pepstatin A – pepsin complexes in the gas phase.

Mass Spectrometric ITEM-ONE and ITEM-TWO Analyses Confirm and Refine an Assembled Epitope of an Anti-Pertuzumab Affimer

“…The specific binding of an antigen's epitope peptide(s) to an antibody in orthodox fashion [24,25] is unequivocally determined by mass spectrometric analyses of the mass and/or the collision-induced fragment ions of the complex-released epitope peptides' ions. In parallel, upon stepwise increasing of the collision energy, the gas-phase binding strengths of the non-covalent immune complexes' ions are determined [26,27], providing information about the immune complex stability.…”