Impact of Protonation Sites on Collision-Induced Dissociation-MS/MS Using CIDMD Quantum Chemistry Modeling

Abstract

Protonation is the most frequent adduct found in positive electrospray ionization collision-induced mass spectra (CID-MS/MS). In a parallel report Lee, J. J. Chem. Inf. Model. 2024, 10.1021/acs.jcim.4c00760, we developed a quantum chemistry framework to predict mass spectra by collision-induced dissociation molecular dynamics (CIDMD). As different protonation sites affect fragmentation pathways of a given molecule, the accuracy of predicting tandem mass spectra by CIDMD ultimately depends on the choice of its protomers. To investigate the impact of molecular protonation sites on MS/MS spectra, we compared CIDMD-predicted spectra to all available experimental MS/MS spectra by similarity matching. We probed 10 molecules with a total of 43 protomers, the largest study to date, including organic acids (sorbic acid, citramalic acid, itaconic acid, mesaconic acid, citraconic acid, and taurine) as well as aromatic amines including uracil, aniline, bufotenine, and psilocin. We demonstrated how different protomers can converge different fragmentation pathways to the same fragment ions but also may explain the presence of different fragment ions in experimental MS/MS spectra. For the first time, we used in silico MS/MS predictions to test the impact of solvents on proton affinities, comparing the gas phase and a mixture of acetonitrile/water (1:1). We also extended applications of in silico MS/MS predictions to investigate the impact of protonation sites on the energy barriers of isomerization between protomers via proton transfer. Despite our initial hypothesis that the thermodynamically most stable protomer should give the best match to the experiment, we found only weak inverse relationships between the calculated proton affinities and corresponding entropy similarities of experimental and CIDMD-predicted MS/MS spectra. CIDMD-predicted mechanistic details of fragmentation reaction pathways revealed a clear preference for specific protomer forms for several molecules. Overall, however, proton affinity was not a good predictor corresponding to the predicted CIDMD spectra. For example, for uracil, only one protomer predicted all experimental MS/MS fragment ions, but this protomer had neither the highest proton affinity nor the best MS/MS match score. Instead of proton affinity, the transfer of protons during the electrospray process from the initial protonation site (i.e., mobile proton model) better explains the differences between the thermodynamic rationale and experimental data. Protomers that undergo fragmentation with lower energy barriers have greater contributions to experimental MS/MS spectra than their thermodynamic Boltzmann populations would suggest. Hence, in silico predictions still need to calculate MS/MS spectra for multiple protomers, as the extent of distributions cannot be readily predicted.

Figure 1.

Overall procedure of the CIDMD framework and analysis package.

Figure 2.

Scatter plots of calculated proton affinity and entropy similarity scores. In each plot, the molecular structure is shown. The numbers in black next to heteroatoms represent the protonation sites. The color blue represents the proton affinity values calculated in the acetonitrile and water 1:1 mixture ($\epsilon =56.13$), whereas red represents the values calculated in the gas phase.

Figure 3.

(A) Relationship between proton affinity and CIDMD entropy similarity scores for all bufotenine heteroatom protomers. Numbers 1–3 refer to protomer positions given in structures 3C. (B) Comparison of the HCD bufotenine experimental mass spectrum (top) at a collision energy of 8 eV with the CIDMD-predicted mass spectra of the three bufotenine heteroatom protomers (lower panels). (C) Main fragmentation pathways for the bufotenine protomers #1, #2, and #3. Green check marks: product ions observed in the experimental spectra. Red cross-mark: product ion not observed experimentally.

Figure 4.

Sorbic acid protomers. (A) Comparison of the CIDMD-predicted mass spectra to the experimental spectra for two sorbic acid protomer #1 (left) and protomer #2 (right). (B) CIDMD-predicted fragmentation pathways demonstrating CO and H2O losses for both protomers.

Figure 5.

Energy-dependent fragmentation of uracil and its protomers. Abundance of experimental positive-mode MS/MS product ions in the NIST20 library for increasing MS/MS collision energies.

Figure 6.

CIDMD fragmentation of uracil and its protomers. (A) Entropy similarity scores, proton affinities, structures, CIDMD product ion abundances, and MS/MS similarity scores of six higher ground-state energy uracil protomers (UH1–6). (B) Entropy similarity scores, proton affinities, structures, CIDMD product ion abundances, and MS/MS similarity scores of seven lower ground-state energy uracil protomers (UL1–7). (C) Extracted CIDMD fragmentation reaction pathways from the CIDMD simulations of uracil lower energy protomer UL7. (D) Head-to-tail graph of CIDMD-predicted and experimental mass spectra at 20 eV collision energy. Correctly predicted fragment ions are annotated.

Figure 7.

Proton migration and MS/MS fragmentation for aniline. (A) Transition-state calculations for a mobile proton transfer pathway from Cpara-protonation to nitrogen protonation. (B) Detailed proton migration (yellow) from C_para to nitrogen-protonated aniline, followed by a fragmentation reaction pathway of the nitrogen-protonated aniline. (C) Experimental MS/MS breakdown curves by collision energies using nitrogen or argon as collision gases, for triple-quadrupole (QQQ) or high-collision dissociation (HCD) Orbitrap mass spectrometers.