Acetone/Isopropanol Photoinitiating System Enables Tunable Disulfide Reduction and Disulfide Mapping via Tandem Mass Spectrometry

Abstract

Herein, we report the development of a new photochemical system which enables rapid and tunable disulfide bond reduction and its application in disulfide mapping via online coupling with mass spectrometry (MS). Acetone, a clean and electrospray ionization (ESI) compatible solvent, is used as the photoinitiator (1% volume) in the solvent system consisting of 1:1 alkyl alcohol and water. Under ultraviolet (UV) irradiation (∼254 nm), the acetone/alcohol system produces hydroxyalkyl radicals, which are responsible for disulfide bond cleavage in peptides. Acetone/isopropanol is most suitable for optimizing the disulfide reduction products, leading to almost complete conversion in less than 5 s when the reaction is conducted in a flow microreactor. The flow microreactor device not only facilitates direct coupling with ESI-MS but also allows fine-tuning of the extent of disulfide reduction by varying the UV exposure time. Near full sequence coverage for peptides consisting of intra- or interchain disulfide bonds has been achieved from complete disulfide reduction and online tandem mass spectrometry (MS/MS) via low energy collision-induced dissociation. Coupling different degrees of partial disulfide reduction with ESI-MS/MS allows disulfide mapping as demonstrated for characterizing the three disulfide bonds in insulin.

Figure 1.

Positive ion mode MS spectra of oxidized glutathione (10 μM) prepared in solutions containing 1% acetone as a photoinitiator. (a) Before and (b) after UV exposure of the aqueous solution in nanoESI emitter. (c) UV irradiation of the peptide in H₂O/MeOH ((v/v) = 1:1) and (d) extracted ion chromatogram (XIC) for the reaction shown in part c. UV irradiation of the peptide in (e) H₂O/EtOH ((v/v) = 1:1) and (f) H₂O/IPA ((v/v) = 1:1).

Figure 2.

(a) Flow microreactor setup for coupling acetone/IPA imitated disulfide bond reduction with ESI-MS. ESI-MS spectra of 10 μM of trypsin-digested somatostatin-14 in H₂O/IPA (1:1) with 1% acetone (b) before UV and (c) after 3 s UV irradiation in positive ion mode. d) Plot of % conversion of the intact peptide to disulfide reduced species as a function of UV irradiation time.

Figure 3.

MS spectra of 10 μM of selectin binding peptide dissolved in H₂O/IPA (1:1) with 1% acetone: (a) before UV, (b) after 5 s UV irradiation in flow microreactor, and (c) MS² CID of reduced peptide ions at m/z 524.7 ([^redM + 2H]²⁺). The fragmentation map of the peptide is indicated in the inset.

Figure 4.

MS spectra of trypsin-digested ribonuclease-B subjected to the radical reaction: (a) before UV and (b) after 5 s UV irradiation in flow microreactor. (c) Sequence and fragmentation maps of intact peptide and reduced A, B, and C chains.

Figure 5.

Positive ion mode ESI MS spectra of porcine insulin in 1% acetone, IPA/H₂O ((v/v) = 1:1). (a) Before UV, (b) after 1.8 s, and (c) 3.1 s UV irradiation in flow microreactor. (d−f) The zoomed-in region of 5+ insulin ions from parts a−c. Deconvoluted isotopic distributions of the intact (black dots), one disulfide reduced (green dots), and two disulfide reduced (red dots) species are overlaid with the experimental data and their relative contributions are listed in the insets.

Figure 6.

MS² CID of 5+ insulin ion populations derived from (a) intact, (b) 31% one disulfide reduced and 16% two disulfide reduced species, and (c) 28% one disulfide reduced and 60% two disulfide reduced species. The corresponding fragmentation maps are shown in parts d−f.

Scheme 1.

Proposed Reaction Pathways for Disulfide Bond Cleavage from Using Acetone/Alkyl Alcohol Photoinitiating System