FOCUS :M OBILE PROTON MODEL SHORT COMMUNICATION The Mobile Proton Hypothesis in Fragmentation of Protonated Peptides: A Perspective

The Distinguished Contribution Award of the American Society for Mass Spectrometry “—recognizes a focused, singular achievement in or contribution to fundamental or applied mass spectrometry, in contrast to awards that recognize lifetime achievement—a contribution that has had a significant impact on the fundamental understanding and/or practice of mass spectrometry.” The hypothesis that has come to be known as themobile proton model clearly satisfies both the fundamental and practical criteria. It is a pleasure to have been asked to contribute a brief introductory article to this special issue that honors Vicki Wysocki and Simon Gaskell, the principal originators and proponents of the model and the 2009 recipients of this prestigious award. Two excellent reviews cover the relevant literature up to about 2005 [1, 2]. The purpose of this short article is to attempt to view the mobile proton model relative to a wider perspective. Application of mass spectrometry to determination of molecular structure relies on interpretation of fragment ion spectra, however obtained, using a set of rules that are the result of years of experience in extending concepts of classical physical-organic chemistry. Most of the fragmentation rules were derived from experience with positive ion mass spectra obtained using electron ionization. Probably the best-known guide to these interpretative rules is the book authored by McLafferty and Turecek [3]. The underlying theme of these “classical” rules is that the electron rearrangements involved in decomposition of an activated ion into two or more fragments are triggered by localization of charge (and/or unpaired electron spin in the case of radical ions) on specific sites within the molecular structure of the decomposing ion. It is true that these rules are almost entirely empirical, but their continuing practical success indicates that they must correspond to real phenomena in some sense. The introduction of chemical ionization, and later the powerful fast atom bombardment (FAB) [4], electrospray (ESI) [5], and MALDI [6] ionization techniques led to extension of the rules for fragmentations of molecular radical cations to even-electron molecular species formed by adduction of simple ions, protons in the great majority of cases. Indeed, the mobile proton model can be regarded as an extension of these classical rules that permits their appropriate application to protonated peptides or other organic molecules. The main exceptions to interpretations based on triggering by localized charge sites are the charge-remote fragmentations [7] investigated by Gross and his colleagues starting in the late 1980s. These “pseudo-thermal” reactions are known [8, 9] to contribute to fragmentation of protonated peptides at high levels of internal activation, where they compete with the mechanisms subsumed by the mobile proton theory, particularly for singly-protonated peptides containing an Arg residue. As emphasized previously [2], the mobile proton model is not a complete theory that can predict a fragment ion spectrum for any given protonated peptide, but rather a qualitative framework that permits appropriate application of interpretative rules based on charge-site localization. Essentially, the model assumes that for protonated peptides formed by soft ionization methods such as electrospray (ESI), the protons are initially localized on the most basic sites in the molecule. These sites are the N-terminus and the side chains of basic amino acid residues, particularly Arg, Lys, and His. After ion activation the ionizing proton(s) can be transferred from the less-basic of these initially occupied sites to the various peptide linkages, thus triggering charge-site-initiated mechanisms of various kinds that provide the desired sequence ions. In other words, the mobile proton model also implies that heterogeneous populations of protonated forms can be generated upon ion activation and some of these forms are “fragmenting” structures, while others remain intact during the time frame of the mass spectrometer. This framework has now been extensively reviewed and its practical usefulness amply demonstrated [1, 2]. Of course all useful new ideas appear to be simple and obvious once someone else has described them. To illustrate that the mobile proton model was not always regarded as “obvious” and was “linked” only later to the idea of ion populations generated by an “activated”, i.e., “transferable” proton, one can cite some early work published in 1992 by one of the recipients of the Distinguished Contributions Award [10]. This paper was mainly concerned with demonstrating intraionic interactions in peptides containing cysteic acid plus Address reprint requests to Dr. R. K. Boyd, National Research Council, Bldg. M12, 1200 Montreal Road, Ottawa K1A0R6, Canada. E-mail: robert. boyd@nrc.ca