The development of electrospray ionization (ESI), which enables transfer of biomolecular ions from solution to vacuum with minimal energy deposition, has provided a method to readily carry out precision structural investigations on isolated biomolecular ions using mass spectrometry (MS). Although the characterization of biomolecular ions in vacuum provides exceptional control over experimental parameters, this approach must contend with the unintentional consequences of study under these conditions. Notably, anionic and cationic moieties often exhibit extensive ionic hydrogen bonding with solvent molecules in the condensed phase, and the absence of these hydrogen bonding partners in vacuum favors structural rearrangement to accommodate intramolecular ionic hydrogen bonding. This intramolecular “solvation” is typically observed in the most stable conformers of isolated biomolecular ions, leading to differences between condensed-phase and gas-phase low-energy structures. For monoalkylammonium functional groups (e.g. lysine), it is possible to modify this intramolecular solvation process to some extent through complexation with 18-Crown-6 ether. This approach has been demonstrated to minimize structural rearrangement of proteins upon transfer to vacuum and has also been used to directly modulate the low-energy gas-phase structure. Despite extensive development of anionic recognition reagents in organic solvents, no reagent has been investigated for MS experiments. We have developed a new reagent, N1,N3-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-1,3-benzenedicarboxamide, or diserinol isophthalamide (DIP), which exhibits favorable complexation with peptide anionic sites in ESI-MS experiments. In addition, this reagent was found to exhibit significantly more abundant complexation than a commercially available anion recognition reagent, 1,1’-(1,2-phenylene)bis(3-phenylurea), or PBP. Structural studies reveal that the poor performance of PBP in biomolecule complexation may arise from the steric hindrance of the terminal phenyl moieties. In contrast, DIP is able to bind anionic sites with significantly reduced steric limitations. However, the tendency of DIP to undergo deprotonation may interfere with complexation to anionic sites, and we are therefore developing a second-generation reagent in which the serinol moieties are replaced by serine N-methylamide, thereby reducing the number of hydroxyl groups available for deprotonation and favoring anion complexation.

New anion microsolvation reagents for the study of biomolecular ions in vacuum

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