Unravelling the favorability of radical-directed xn-H2O dissociation at serine and threonine

Among tandem-mass-spectrometry approaches, radical-directed dissociation (RDD) is uniquely sensitive to molecular structure because the location and types of cleavage observed are dictated by radical migration propensities. Although the underlying chemistry for many RDD fragmentation pathways has been previously explained, x_n-H₂O fragment ions that occur exclusively at serine and threonine residues, have not been examined in detail. Creation of this fragment type inherently requires two dissociation events, one to lose water and another to cleave the peptide backbone. Double dissociations are typically disfavored relative to pathways requiring a single cleavage, yet x_n-H₂O fragment ions are abundant in RDD spectra. To understand why this fragmentation pathway is favorable, we used a combination of computational chemistry and experiments on peptides with a variety of covalent modifications. Our results explore the energetics, location, and migration of the radical in each step of the mechanism, revealing that favorability can be attributed to the stability of the required radical intermediates and access to low-energy pathways connecting them. Ultimately, the abundant nature of x_n-H₂O ions and the selectivity associated with their exclusive generation at Ser/Thr provides high value sequence information in RDD experiments.