The transfer of biomolecules from solution to vacuum using electrospray ionization (ESI) can result in structural rearrangement to accommodate intramolecular hydrogen bonding to newly desolvated charged moieties. Complexation reagents that bind selectively to these charged groups can reduce intramolecular solvation through local “microsolvation” of charged sites, enabling analysis of rearrangement processes or promoting the retention of condensed-phase structure for further characterization, for example by ion-mobility spectrometry. This has been previously demonstrated by the complexation of 18-crown-6 to cationic sites in proteins and peptides. However, an anion recognition reagent suited for mass spectrometry (MS) experiments has not been investigated. We present a potential reagent, N¹,N³-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-1,3-benzenedicarboxamide, or diserinol isophthalamide (DI), comprising an isophthalic acid moiety symmetrically linked to two serinol groups via amide bonds. DI can complex carboxylates, e.g., the C-terminus of peptides, through four direct hydrogen bonds, with the two remaining hydroxyl groups participating in an extended hydrogen bonding network, as predicted by density functional theory (DFT) calculations. Complexation of DI and 1,1’-(1,2-Phenylene)bis(3-phenylurea), an existing anionic complexation reagent to model peptide systems including valine-tyrosine-valine (VYV), leucine enkephalin (YGGFL), and aspartame was studied by ESI-MS (80/20 MeOH/H₂O v/v%) on a quadrupole MS. For all systems studied, appreciable binding to DI was observed, with a ratio of relative intensities of free peptide to complexed species of 10:1 and 1:1 for YGGFL and VYV, respectively. Interestingly, of the peptides studied, appreciable complexation of the urea-based compound was only found for VYV, at a 4:1 ratio. The affinity for complexation to VYV may be explained by additional stabilization from π-stacking interactions between the reagent and proximal aromatic moieties. NMR studies are in progress to quantify solution phase binding as compared to gas phase behavior. Overall, DI is a promising potential probe for intramolecular solvation and structural analysis by MS.

Daniel A. Thomas

Assistant Professor, University of Rhode Island

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