Gas-Phase Intramolecular Protein Crosslinking via Ion/Ion Reactions: Ubiquitin and a Homobifunctional sulfo-NHS Ester

Abstract

Gas-phase intra-molecular crosslinking of protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of a homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin and crosslinker monoanions produces a stable, charge reduced complex. Covalent crosslinking is indicated by the consecutive loss of two molecules of sulfo-NHS under ion trap collisional activation conditions. Covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The presence of two unprotonated reactive sites within the distance constraint of the crosslinker is required for successful crosslinking. The ability to covalently crosslink is therefore sensitive to protein charge state. As the charge state increases, fewer reactive sites are available and protein structure is more likely to become extended due to intramolecular electrostatic repulsion. At high charge states, the reagent shows little evidence for covalent crosslinking but does show evidence for ‘electrostatic crosslinking’ in that the binding of the sulfonate groups to the protein is sufficiently strong that backbone cleavages are favored over reagent detachment under ion trap collisional activation conditions.

Ion/Ion Reactions with “Onium” Reagents: An Approach for the Gas-phase Transfer of Organic Cations to Multiply-Charged Anions

“…Activation of these complexes yields a signature loss of sulfo-NHS, indicating that a primary amine or guanidine within the analyte initiated a nucleophilic attack on the carbonyl carbon of the NHS reagent, ultimately resulting in stable amide bond formation [18]. This NHS reactivity in the gas phase allows for the modification of primary amine- or guanidine-containing peptides, as well as protein cross-linking in the gas phase [21]. Amide bond formation in the gas phase has also been reported using carbodiimide reagents to react with carboxylic acid-containing analytes in the negative mode [22].…”

Design of a TW-SLIM Module for Dual Polarity Confinement, Transport, and Reactions

“…Recently, ion/ion complex formation has been used for gas covalent modifications[22]. The covalent reactions have included such examples as gas phase Schiff base formation [22, 23], modification of amines with NHS-ester chemistry [24, 25], reactions with carboxylic acids [26, 27], and gas phase oxidations of disulfide bonds [28, 29]. Ion/ion reactions are showing great promise in moving site-specific traditionally solution phase bio-conjugation reactions into the gas phase.…”

Transformation of [M+2H]2+ Peptide Cations to [M – H]+, [M+H+O]+, and M+• Cations via Ion/Ion Reactions: Reagent Anions Derived from Persulfate

“…Site-selective covalent modification of peptides and proteins has also been demonstrated via ion/ion reactions. For example, N-hydroxysuccinimide (NHS) esters have been used to cross-link 12,13 and covalently label 14,15,16 various nucleophiles in peptide ions, 4-formyl-1,3-benzenedisulfonic acid (FBDSA) has been used to tag peptide ions via Schiff base chemistry, 17,18,19 and N -cyclohexyl- N ′-(2-morpholinoethyl)carbodiimide (CMC) has been used to selectively react with carboxylic acids 20 in various analytes. Ion/ion reactions occur on the typical reaction time-scale of ~100 ms and can either proceed via the long-range transfer of small charged particles, e.g., protons or electrons, or through the formation of a long-lived complex.…”