“Molecular” mass spectrometry (MS) emerged with important analytical applications during World War II, particularly for quantitative analysis of light hydrocarbon mixtures. But extension to molecular structure elucidation was slow, highly dependent on improvements in both instrumentation and the understanding of relevant ion chemistry (e.g., “CH5+ is impossible”). Nearly three decades ago the introduction of electrospray ionization (ESI) and MALDI made possible the extension of MS analysis to critical large molecules such as proteins. Sequencing, including MS/MS “top down”, is now a key methodology,1 and “native” MS provides valuable conformational information on larger molecules by minimizing unfolding after ESI.2 Folding is a unique, critically important characteristic of proteins in solution, but there is little agreement on that of their gaseous ion counterparts. Here we present a new mechanism for the folding of gaseous ubiquitin ions, the most studied of gaseous proteins, based on charge site (CS) mass spectra3 measured after H/D exchange with AcOD. Within 0.2s after ESI, 13+ - 10+ ubiquitin ions unfold due to their high charge. Then (1) they equilibrate their 13 - 10 protons into stable distributions on the 14 basic residues, and (2) each protonated basic residue folds by forming H+-bonds to carbonyl groups (C=O---H+-N) of ~8 neighboring residues. Electron capture dissociation occurs at these new charge sites, with the c, z ion yields vs. cleavage site providing the corresponding CS spectra. For H/D exchange, D---O-Ac binds noncovalently to C=O---H+-N, with a decrease in the latter’s H+- charge density increasing the HDX rate. Thus the 12+ to 10+ ions show an additional second, far faster (20D vs. 8D) rate vs. the 13+ ions. Although many molecular ion conformers are possible from this variety of local conformers, their corresponding CS spectra are found for many of the 7+ to 13+ ubiquitin ions. Electrostatics exert strong control on gaseous protein folding, in sharp contrast to that in solution. The previous Cornell postulation of α-helical intermediates disagrees with this new evidence.

Award Address (Nakanishi Prize sponsored by the Nakanishi Prize Endowment). Analytical chemistry of gaseous ubiquitin ions

249th National Meeting (2015)

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