Divinylpyrimidine Re-bridges Protein Disulfide Bonds And Enhances Ultraviolet Photodissociation Of Disulfide Bonds

Objective: Disulfide bonds are post-translational modifications formed between the sulfur atoms of two cysteines of proteins,which maintain protein structure and function.Disulfide bonds are also critical for the quality control of antibody drugs,and are important for the development of biological drugs.The disulfide bond cyclizes the protein sequence between the cysteines,and the release of diagnostic fragment ions from the sequence containing the disulfide bond is hindered,making it difficult to directly decipher the sequence and structural information of protein.In addition,the low cleavage efficiency of disulfide bond results in fragment ions from one enzymolysis peptide that are linked to another intact peptide chain by disulfide bond.This complex fragmentation provides little information about the localization of disulfide bonds and cannot be readily analyzed using conventional databases and procedures.Therefore,enhancing disulfide bond dissociation can facilitate the characterization of disulfide bonds and protein structures.Since chromophores can enhance ultraviolet photodissociation(UVPD),this study introduced divinylpyrimidine(DVP)as a chromophore to re-bridge disulfide bond,which enhanced the ultraviolet photodissociation of disulfide bond.Methods: 1.The disulfide bonds in octreotide and several proteins were re-bridged by DVP,and the chemical reactivity of DVP linker in octreotide,lysozyme,ribonuclease A,β-lactoglobulin and insulin was investigated using mass spectrometry.2.The effects of reagent dosages and reaction time on the conversion rates of DVP modified products were investigated using mass spectrometry.3.High-energy collisional dissociation(HCD)and193 nm UVPD MS/MS analysis of unmodified and DVP modified lysozyme and ribonuclease A were performed using top-down mass spectrometry to systematically study cleavage pathways of re-bridged disulfide bonds,and to calculate disulfide bond cleavage efficiency and protein sequence coverage.4.The enzymolysis peptides from unmodified and DVP modified lysozyme and ribonuclease A were analyzed by LC-MS/MS using bottom-up mass spectrometry to match the fragment ions produced by each peptide and calculate disulfide bond cleavage efficiency for each peptide.Results: 1.DVP could re-bridge the disulfide bond in octreotide and proteins.Disulfide bonds with high solvent accessibility and low steric hindrance when reacting with DVP were easily modified by DVP under native condition;almost all the intrachain disulfide bonds in the proteins could be modified under denatured condition.2.The conversion rates of products were stable after the reaction of octreotide and proteins with DVP for 2 h.3.The increase of reagent dosages did not significantly improve the conversion rates.4.The re-bridged disulfide bonds were cleaved by C-S bonds to produce homolytic cleavage and hydrogen transfer product ions.5.Compared to HCD,UVPD significantly improved disulfide bond cleavage efficiency and sequence coverage of intact protein.6.Disulfide bond re-bridging improved intact protein disulfide bond cleavage efficiency by HCD or UVPD MS/MS analysis.7.The re-bridging of disulfide bonds of enzymolysis peptides could improve disulfide bond cleavage efficiency by HCD or UVPD MS/MS analysis.Conclusion: 1.DVP could efficiently re-bridge the disulfide bonds of proteins and the modification exhibited high structure selectivity.2.193 nm UVPD enhanced dissociation of disulfide bonds of intact proteins compared to HCD.3.Re-bridging enhanced the ultraviolet photodissociation of disulfide bonds of intact proteins and enzymolysis peptides.4.Re-bridging improved the ability of HCD to cleave disulfide bonds of intact proteins and enzymolysis peptides.