High-Throughput Bioconjugation for Enhanced 193 nm Photodissociation via Droplet Phase Initiated Ion/Ion Chemistry Using a Front-End Dual Spray Reactor

Abstract

Fast on-line chemical derivatization of peptides with an aromatic label for enhanced 193 nm ultraviolet photodissociation (UVPD) is demonstrated using a dual electrospray reactor implemented on the front-end of a linear ion trap (LIT) mass spectrometer. The reactor facilitates the intersection of protonated peptides with a second population of chromogenic 4-formyl-1,3-benzenedisulfonic acid (FBDSA) anions to promote real-time formation of ion/ion complexes at atmospheric pressure. Subsequent collisional activation of the ion/ion intermediate results in Schiff base formation generated via reaction between a primary amine in the peptide cation and the aldehyde moiety of the FBDSA anion. Utilizing 193 nm UVPD as the subsequent activation step in the MS3 workflow results in acquisition of greater primary sequence information relative to conventional collision induced dissociation (CID). Furthermore, Schiff base modified peptides exhibit on average a 20% increase in UVPD efficiency compared to their unmodified counterparts. Due to the efficiency of covalent labeling achieved with the dual spray reactor, we demonstrate that this strategy can be integrated into a high-throughput LC-MSn workflow for rapid derivatization of peptide mixtures.

Dual‐spray hydrogen/deuterium exchange (HDX) reactions: A new method of probing protein structure

“…Ion/ion reactions can be carried out using a dual electrospray apparatus with opposite polarities on each ESI emitter . Covalent labelling of protonated peptides using 4‐formyl‐1,3‐benzenedisulfonic acid (FBDSA) anions can be achieved using a dual‐spray apparatus for enhanced ultraviolet dissociation . The ambient ionization technique known as extractive electrospray ionization (EESI) also uses a dual‐emitter setup to analyze untreated milk, urine, and non‐volatile compounds in breath .…”

Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent

“…Moreover, the applicability of this method for tryptic-like peptides, as well as previous demonstrations of reactor compatibility with high-throughput chromatographic workflows 39 hint at the potential utility of dual source integration into phosphoproteomic analysis.…”

“…39–43 This chemistry, as demonstrated in Figure S1, proceeds via the formation of long-lived electrostatic complexes arising from noncovalent interactions between the sulfonate moieties of FBDSA reagent anions and protonated sites of peptide cations. 40 Subsequent collisional activation of these ion/ion intermediates promotes nucleophilic attack on the FBDSA aldehyde by an unprotonated primary amine in the substrate peptide, resulting in concerted dehydration and imine bond formation.…”

“…39 This previous work relied on well-characterized gas-phase ion/ion-mediated covalent chemistry using 4-formyl-1,3-benzenedisulfonic acid (FBDSA) anions 40–42 to both facilitate rapid derivatization on a timescale compatible with chromatographic separations and to increase the intrinsic photoabsorption cross-section of peptides for enhanced photodissociation at 193 nm. 39 Herein, we demonstrate that this same chemistry can also be leveraged to modulate the collisional dissociation behavior of basic phosphopeptides in real time for improved sequence coverage and phosphosite localization relative to their unmodified counterparts. In a manner similar to the removal or selective derivatization of basic sites, this method relies on the preferential formation of noncovalent interactions between the sulfonate moieties of FBDSA and the basic sites within the peptide to overcome or partially disrupt the mechanisms leading to preferential phosphate loss by collisional activation.…”

Gas-Phase Oxidation of Neutral Basic Residues in Polypeptide Cations by Periodate

“…Recent examples of these modifications include the esterification of carboxylate groups via cation transfer [ 14 ], gas-phase conjugation of alkynes and azides via ‘Click’ chemistry reactions [15], the N- and C-terminal extension of peptides via conjugation with N-hydroxysuccinimide esters [ 16 ] and Woodward’s Reagent K [ 17 ], respectively, and the conjugation of aldehydes and amines via gas-phase Schiff base chemistry [18,19,20,21,22]. Gas-phase ion/ion reactions have several benefits over traditional solution-phase derivatizations including much shorter reaction times (<1 sec), reduction of side reactions via mass isolation of reactants, and control over the extent of reaction (viz., amount of product formed and number of modifications that occur) [1].…”