Development Of Novel Techniques Coupling Electrochemistry With Mass Spectrometry For Rapid Identification Of Protein/Peptide Structure

Significant advances in exploring the structures of proteins and protein complexes along with protein-protein interactions(PPIs)have drastically propelled the development of human pathology,diagnostics,and therapeutics.High-throughput structural sequencing of peptides containing disulfide bonds and precise assignment of disulfide bonds are a major difficulty.What's more,accurate identification of cross-linked peptides and unambiguous assignment of cross-linked sites with cross-linking mass spectrometry(XL-MS)remain extremely challenging owing to the difficulty in explaining convoluted tandem mass spectra stemming from the fragmentation of covalently linked peptides.Hence,a self-assembled electrochemical-mass spectrometer was presents to achieve rapid and high-throughput structure identification and disulfide linkage assignment of complex proteins in the study.Simultaneously,new cross-linkers and cross-linking strategies were proposed to explore the spatial structure of proteins.Four researchs have been carried out.?.Electrospray ionization(ESI)mass spectrometry has become an indispensable tool for qualitative and quantitative biological protein molecules due to its simple operation,high sensitivity,intuitive spectra,and rapid coupling with chromatographic instruments.However,cleavage of disulfide bond represents a challenge in de novo sequencing of peptide by tandem mass spectrometry because bond energy of disulfide bond is larger than that of the amide bond.Therefore,chemical reagents such as tris(2-carboxyethyl)phosphine(TCEP)or dithiothreitol(DTT),as well as alkylated reagents are often used.These complex processing steps limit the high-throughput analysis of proteins.Here,a novel system of electrochemistry coupled with mass spectrometry(EC-MS)was developed.An electrospray ion source with a grounded needle design was used as the interface of the EC-MS,which avoided the damage of the electronic components of the potentiostat and significantly simplified instrument configuration.The de novo sequencing octreotide and three acylated octreotides were easily obtained with followed tandem mass spectrometry by direct injection.Without any special sample preparation and any chemical reduction agents,disulfide bond in peptides was cleaved rapidly.The results demonstrate that this method is suitable for fast evaluation and characterization of peptide containing disulfide bonds.?.A novel approach for disulfide linkage assignment of disulfide-rich peptides and proteins via electrochemical reduction on a lead electrode with mass spectrometry(EC(Pb)/LC-MS)was presented for the first time.The method features partial electrochemical reduction and alkylation of peptides followed by alkylated peptide sequencing based on tandem mass spectrometry.Lead was chosen for the first time as an electrode material for disulfide bond reduction,because it has a higher hydrogen evolution potential and a wider electrochemical window,which greatly improved the reduction efficiency of disulfide bonds.What's more,it has the advantages of maintenance free(only infrequent polishing needed),easy operation(in DC mode),and stability(EC-MS can still reach 78% reduction efficiency after 8 hours).?.Cross-linking mass spectrometry(XL-MS)has attracted broad attention because of the capability to probe three-dimensional structure of proteins in living cells.At present,several amine-reactive cross-linkers have been developed for characterization of proteins and protein complexes.However,spatial information retrieved by XL-MS is still limited,partly because the strategies using an acidic residue(aspartic acid,Asp,and glutamic acid,Glu)reactive cross-linker have been rarely reported.Hence,an acidic residue-specific,disulfide bond-containing,cleavable cross-linker with a length of 13.1 ?,named 3,3'-dithiobis(propanoic dihydrazide),was presented for the first time.After cross-linked,the peptides could be electrochemically reduced,then characterized by high performance liquid chromatography mass spectrometry.The results showed that the cross-links displayed predictable fragmentation pattern upon collision induced dissociation(CID),thus admitting simplifying data analysis.In addition,the results suggested that the proteins analyzed can be accurately characterized even with a low-resolution mass spectrometer.?.Current XL-MS applications are limited to targeting lysine,glutamic acid,aspartic acid,and cysteine residues.There remains a need for the development of novel cross-linkers enabling selective targeting of other amino acid residues in proteins.Here,a novel simple cross-linker,namely,[4,4-(disulfanediylbis(ethane-2,1-diyl))bis(1,2,4-triazolidine-3,5-dione)](DBB),has been designed,synthesized,and characterized.This cross-linker can react selectively with tyrosine residues in protein through the electrochemical click reaction.The DBB cross-links produced the characteristic peptides before and after electrochemical reduction,thus permitting the simplified data analysis and accurate identification for the cross-linked products.To the best of our knowledge,DBB represents the first tyrosine-specific cross-linker without using photoirradiation or a metal catalyst,which undoubtedly enhances the capability of XL-MS for probing the 3D structures of proteins and protein complexes,along with mapping PPIs in the future.