Reactions of Hydroxyalkyl Radicals with Cysteinyl Peptides in a NanoESI Plume

Abstract

In biological systems, carbon-centered small molecule radicals are primarily formed via external radiation or internal radical reactions. These radical species can react with a variety of biomolecules, most notably nucleic acids, the consequence of which has possible links to gene mutation and cancer. Sulfur-containing peptides and proteins are reactive toward a variety of radical species and many of them behave as radical scavengers. In this study, the reactions between alkyl alcohol carbon-centered radicals (e.g., •CH2OH for methanol) and cysteinyl peptides within a nanoelectrospray ionization (nanoESI) plume were explored. The reaction system involved ultraviolet (UV) irradiation of a nanoESI plume using a low pressure mercury lamp consisting of 185 and 254 nm emission bands. The alkyl alcohol was added as solvent into the nanoESI solution and served as the precursor of hydroxyalkyl radicals upon UV irradiation. The hydroxyalkyl radicals subsequently reacted with cysteinyl peptides either containing a disulfide linkage or free thiol, which led to the formation of peptide-S-hydroxyalkyl product. This radical reaction coupled with subsequent MS/MS was shown to have analytical potential by cleaving intrachain disulfide linked peptides prior to CID to enhance sequence information. Tandem mass spectrometry via collision-induced dissociation (CID), stable isotope labeling, and accurate mass measurement were employed to verify the identities of the reaction products.

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

“…9 Disulfide cleavage was the main reaction channel; however, the reaction yield was limited due to relatively low concentrations of primary radicals ( • OH) formed from 185 nm UV photolysis of ambient air. 9,26 An obvious way to improve the reaction yield is to use more efficient radical initiators, such as 2,2-dimethoxy-2-phenylacetophenone (DMPA) and benzophenone, which are commonly used in organic synthesis and radical polymerization. 27 These compounds go through Norrish type I cleavage at the α -carbon of the carbonyl group and form primary radicals for radical initiation upon UV irradiation.…”

“…S- hydroxymethyl product was verified by detecting signature formaldehyde loss (30 Da) from low energy CID (Figure S2, Supporting Information). 9 The extracted ion chromatogram (XIC) showed that the intact glutathione had a sharp drop from 100% to 0% within 30 s (UV irradiation 1.1−1.5 min), accompanied by steep increase of the reduced peptide ( m/z 308), the ion signal of which maximized at 1.2 min UV irradiation and then dropped to reach a steady state within 20 s. During this time, the ion signal of the hydroxymethyl substitution product ( m/z 338) increased rapidly and stayed constant afterward. These kinetic data suggest that reduced thiol might be further converted to S -hydroxymethyl product after its initial formation.…”

“…For the nanoESI setup, the lamp was placed orthogonally to the tip and off-axis by approximately 2−4 cm of the borosilicate glass emitter, as reported previously. 9 The flow microreactor was made from UV-transparently coated fused silica capillary (100 μ m i.d., 375 μ m o.d. ; Polymicro Technologies; Phoenix, AZ).…”

Tautomerization and Dissociation of Molecular Peptide Radical Cations

“…For example, regiospecific radical generation has been demonstrated from nitrate esters, diazo derivatives, peroxycarbamate derivatives, and diazirine rings . A variety of peptide ions with different radical sites can be formed at atmospheric pressure (AP) through ion–radical reactions . Furthermore, the formation of molecular peptide radical ions (i. e., M •+ , [M + H] •2+ , [M – 2H] •– ) can also be achieved through dissociative electron transfers of ternary metal–ligand–peptide complexes, which have been studied extensively and applied by our and other research groups …”

“…[98][99][100][101] A variety of peptide ions with different radical sites can be formed at atmospheric pressure (AP) through ion-radical reactions. [102][103][104][105][106][107][108][109] Furthermore, the formation of molecular peptide radical ions (i. e., M * + , [M + H] *2 + , [M -2H] *-) can also be achieved through dissociative electron transfers of ternary metal-ligand-peptide complexes, which have been studied extensively and applied by our and other research groups. [21, 24, 26-28, 31, 110-128] For example, CID of [Cu(II)(terpy)(M)] 2 + complexes (terpy = 2,2':6',2''-terpyridine; M = peptide) can lead to charge disproportion to give [Cu(I)(terpy)] + and [M] * + species.…”