Bifunctional Cross-linker For Peptide And Protein Structure Identification Studies

Chemical cross-linking mass spectrometry（XL-MS）has made significant contributions to the resolution of protein structures and the elucidation of protein-protein interactions（PPI）.Cross-linkers are an essential part of XL-MS and the most widely used cross-linkers are those containing succinimidyl esters,which target cross-linked the-NH₂ in lysine（K）and proteins.Although cross-linking strategies for glutamate,aspartate and cysteine were developed by researchers,few cross-linking strategies targeting the remaining amino acids were reported.To this end,two novel cross-linkers and cross-linking routes were designed and applied to analyze the three-dimensional structures of proteins by XL-MS in this paper.The ability to characterize protein interactions was enhanced and the utilization of XL-MS methodology was expanded.The detailed work is as follows:1.A new strategy for specific cross-linking with tyrosine（Y）and histidine（H）,respectively,was developed and a bifunctional cross-linker[4,4’-（disulfanediylbis（ethane-2,1-diyl））bis（1-methyl-1,2,4-triazolidine-3,5-dione）],referred to as DBMT,was synthesized.DBMT is capable of specifically targeting tyrosines（Y）residue through the electrochemical click reaction,targeting histidines（H）residue under white light irradiation.After the cross-linking reaction,the disulfide bonds in the cross-linked products（in the DBMT structure and in the cross-linked dipeptides）can be reduced and alkylated in an electrochemical flow cell,which simplifies the identification of the cross-linked products.Agngiotensin II,ubiquitin protein,glutathione S-transferase（GST）and bovine serum albumin（BSA）were chosen for the attempt of cross-linking experiments with DBMT,respectively.These results demonstrate the high reactivity and selectivity of DBMT.The bifunctional cross-linker that can selectively cross-link tyrosine and histidine was designed and synthesized in the work of this chapter to complement the existing library of cross-linkers,which will inevitably enrich the application of XL-MS.2.A tyrosine-targeting and mass spectrometry cleavable cross-linker[4,4’-（sulfinylbis（ethane-2,1-diyl））bis（1,2,4-triazolidine-3,5-dione）]（SBT）was designed and synthesized.Tyrosine could be cross-linked by SBT targeting at an oxidation potential of 0.44 V.The C-S bond in this cross-linker that is linked to the sulfoxide can be cleaved in the mass spectrum,hence it contains two symmetric dissociation sites.When the target peptide was broken,the C-S bond in the cross-linker is also cut and broken,and thus the linked cross-linked products are dissociated.Then the dissociated cross-linked products could be analyzed based on available search software and databases,and the cross-linked sites could be identified efficiently and simply.The mass spectrometry cleavable C-S bond in SBT converts the identification of cross-linked double peptides into cross-linked single peptides,simplifying the analytical work and time spent.In addition,both S and A of cross-linker fragments are generated when SBT was broken.For the intra-linked products,the cross-linked sites could be identified more conveniently and with higher confidence based on the identified b,y ions containing S or A.Angiotensin II,peptide（WNTQSTYSEA）,recombinant human growth hormone,glutathione S-transferase andβ-casein cross-linking with SBT were studied and cross-linking products were also analyzed and identified in part of the work.The cross-linking reactions involved in this paper were carried out at room temperature and in a buffer solution with p H 7.4.The physiological conditions required for the cross-linking reaction allow for maximum retention of the three-dimensional structure of the protein.The work in this paper not only enriches the application of XL-MS,but also will provide a new concept for the targeted modification of proteins.