Probing Electrospray Ionization Dynamics Using Differential Mobility Spectrometry: The Curious Case of 4-Aminobenzoic Acid

Abstract

Here, we present the separation of two ions that differ only by the site of protonation of the analyte molecule using differential mobility spectrometry (DMS). Protonated 4-aminobenzoic acid molecules (4-ABA) generated by positive-mode electrospray ionization [ESI(+)] can exist with the proton residing on either the amine nitrogen (N-protonated) or the carboxylic acid oxygen (O-protonated), and the protonation site can differ on the basis of the solvent system used. In this study, we demonstrate the identification and separation of N- and O-protonated 4-ABA using DMS, with structural assignments verified by: (1) the presence of distinct peaks in the DMS ionogram, (2) the observed effects resulting from altering the ESI(+) solvent system, (3) the observed (13)C NMR chemical shifts arising from altering the solvent system, (4) the observation of distinct MS/MS fragmentation patterns for the two DMS-separated ions, (5) the unique hydrogen-deuterium exchange behavior for these ions, and (6) the fundamental behavior of these two ions within the DMS cell, linked back to the structural differences between the two protonated forms.

Studying Gas-Phase Interconversion of Tautomers Using Differential Mobility Spectrometry

“…Briefly, we prepared a 10-ng/mL solution of 4-ABA in 50/50 or 75/25 acetonitrile/water for ESI; these solvent ratios ensured that ESI would generate abundant populations of both N-and O-protonated 4-ABA [25]. To separate the two 4-ABA tautomers, we set the separation voltage (SV) at 3500 V and scanned the compensation voltage (CV) from −15 V to +15 V in 0.1 V increments.…”

“…Among these examples, we described the use of differential mobility spectrometry (DMS) [20][21][22][23][24] to separate the tautomers of protonated 4-ABA (N-and O-protonated) [25]. The primary confirmation for this separation came from the observed tautomers' behavior in the DMS cell and differences in their respective MS/MS fragmentation patterns.…”

Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution

“…To perform gas-phase HDX after separation with DMS, D 2 O or ND 4 OD was introduced in the resolving (throttle) gas line by bubbling nitrogen through a volume of either deuterating reagent at the interface between the DMS and TOF-MS [28]. In experiments where HDX during transit through the DMS cell was desired, 1:1 ND 4 OD:CH 3 OD (generated via bubbler) was infused at 1.5% (v/v) in the curtain gas as a chemical modifier together with nitrogen [28].…”

“…In experiments where HDX during transit through the DMS cell was desired, 1:1 ND 4 OD:CH 3 OD (generated via bubbler) was infused at 1.5% (v/v) in the curtain gas as a chemical modifier together with nitrogen [28]. For HDX in the DMS cell, it was also important to qualify the degree of mobility changes caused by ion/molecule clustering between the protein ions and HDX molecules.…”

“…A second 'throttle' gas can be introduced at the interface between the DMS and the mass spectrometer for resolution enhancement [3,28]. In principle, both of these inlets could be used to provide HDX reagents to the ion beam.…”