Impact of Protonation\nSites on Collision-Induced Dissociation-MS/MS\nUsing CIDMD Quantum Chemistry Modeling

Protonation is the most frequent adduct found in positive\nelectrospray\nionization collision-induced mass spectra (CID-MS/MS). In a parallel\nreport Lee, J. J. Chem.\nInf. Model. 2024, 10.1021/acs.jcim.4c00760, we developed a quantum\nchemistry framework to predict mass spectra by collision-induced dissociation\nmolecular dynamics (CIDMD). As different protonation sites affect\nfragmentation pathways of a given molecule, the accuracy of predicting\ntandem mass spectra by CIDMD ultimately depends on the choice of its\nprotomers. To investigate the impact of molecular\nprotonation sites on MS/MS spectra, we compared CIDMD-predicted spectra\nto all available experimental MS/MS spectra by similarity matching.\nWe probed 10 molecules with a total of 43 protomers, the largest study\nto date, including organic acids (sorbic acid, citramalic acid, itaconic\nacid, mesaconic acid, citraconic acid, and taurine) as well as aromatic\namines including uracil, aniline, bufotenine, and psilocin. We demonstrated\nhow different protomers can converge different fragmentation pathways\nto the same fragment ions but also may explain the presence of different\nfragment ions in experimental MS/MS spectra. For the first time, we\nused in silico MS/MS predictions to test the impact of solvents on\nproton affinities, comparing the gas phase and a mixture of acetonitrile/water\n(1:1). We also extended applications of in silico MS/MS predictions\nto investigate the impact of protonation sites on the energy barriers\nof isomerization between protomers via proton transfer. Despite our\ninitial hypothesis that the thermodynamically most stable protomer\nshould give the best match to the experiment, we found only weak inverse\nrelationships between the calculated proton affinities and corresponding\nentropy similarities of experimental and CIDMD-predicted MS/MS spectra.\nCIDMD-predicted mechanistic details of fragmentation reaction pathways\nrevealed a clear preference for specific protomer forms for several\nmolecules. Overall, however, proton affinity was not a good predictor\ncorresponding to the predicted CIDMD spectra. For example, for uracil,\nonly one protomer predicted all experimental MS/MS fragment ions,\nbut this protomer had neither the highest proton affinity nor the\nbest MS/MS match score. Instead of proton affinity, the transfer of\nprotons during the electrospray process from the initial protonation\nsite (i.e., mobile proton model) better explains the differences between\nthe thermodynamic rationale and experimental data. Protomers that\nundergo fragmentation with lower energy barriers have greater contributions\nto experimental MS/MS spectra than their thermodynamic Boltzmann populations\nwould suggest. Hence, in silico predictions still need to calculate\nMS/MS spectra for multiple protomers, as the extent of distributions\ncannot be readily predicted.