Radical fragment ions in collision-induced dissociation-based tandem mass spectrometry

Collision-induced dissociation (CID) is a common fragmentation strategy in tandem mass spectrometry (MS²) analysis. A conventional understanding is that fragment ions generated in low-energy CID should follow the even-electron rule. As such, (de)protonated ([M+H]⁺/[M-H]^-) or even-electron precursor ions should follow heterolytic cleavages and predominately generate even-electron fragment ions with very few radical fragment ions (RFIs). However, the extent to which RFIs present in MS² spectra has not been comprehensively investigated. This work uses the annotated high-resolution MS² spectra from the latest NIST 20 tandem mass spectral library to investigate the occurrence of RFIs in CID MS² experiments. In particular, RFIs were recognized using integer double bond equivalent (DBE) values calculated from their annotated molecular formulas. Our study shows that 65.4% and 68.8% of MS² spectra of even-electron precursors contain at least 10% RFIs by ion-count (total number of ions) in positive and negative electrospray ionization modes, respectively. Furthermore, we classified chemicals based on their compound classes and chemical substructures, and calculated the percentages of RFIs in each class. As expected, compounds that can stabilize the radical site via resonance, such as aromatic and conjugated double bond-containing chemicals, are more likely to form RFIs. We also found four possible patterns of change in RFI percentages as a function of CID collision energy. Finally, we demonstrate that the inadequate consideration of RFIs in most conventional bioinformatic tools might be problematic during in silico fragmentation and de novo annotation of MS² spectra. This work provides a further understanding of CID MS² mechanisms, and the unexpectedly large percentage of RFIs suggests that the even-electron rule seems to be challenged in numerous cases where it is disobeyed.