Photochemical Disulfide–Ene Modification Enhances Protein Sequencing and Disulfide Mapping by Mass Spectrometry

Yang, Xiaoyue; Zhang, Longfei; Xia, Yu

doi:10.1021/acs.analchem.1c04214

Cited by 5 publications

(1 citation statement)

References 27 publications

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“…In contrast to amide bonds, disulfide bonds do not readily dissociate upon traditional collisional activation, prohibiting characterization of the cyclized portions of proteins. − Although chemical reduction of disulfide bonds followed by alkylation of the free thiols prior to analysis is commonly used to circumvent some of these challenges, chemical modification of thiols often precludes distinction of non-native disulfides, disrupts native-protein structure, and may complicate sample handling. − Furthermore, achieving only partial alkylation of thiols owing to inefficient reduction hinders release of sequence fragments and dilutes protein signal across multiple alkylation products in top-down workflows. Other strategies include electrochemical or photochemical derivatization to similarly disrupt disulfide bonds. − …”

Section: Introductionmentioning

confidence: 99%

Investigation of Product Ions Generated by 193 nm Ultraviolet Photodissociation of Peptides and Proteins Containing Disulfide Bonds

Macias

Brodbelt

2022

J. Am. Soc. Mass Spectrom.

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Disulfide bridges are unique post-translational modifications (PTM) that contribute to protein architecture and modulate function. This PTM, however, challenges top-down mass spectrometry by cyclizing stretches of the protein sequence. In order to produce and release detectable product ions that contribute to the assignment of proteoforms, regions of a protein encapsulated by disulfide bonds require two fragmentation events: cleavage of the protein backbone and cleavage of the disulfide bond. Traditional collisional activation methods do not cleave disulfide bonds efficiently, often leading to low sequence coverage of proteins that incorporate this feature. To address this challenge, we have evaluated the fragmentation pathways enabled by 193 nm ultraviolet photodissociation (UVPD) and UVPD coupled to electron transfer dissociation for the characterization of protein structures incorporating disulfide bonds. Cleavage of disulfide bonds by either approach results in S–S and C–S dissociation products that result from a combination of homolytic cleavage and hydrogen-transfer processes. Characterization of these product ions elevates interpretation of complex top-down spectra of proteins that incorporate disulfide bonds.

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