Application of high-resolution liquid chromatography-mass spectrometry in chemical modification of proteins
[Significance]High-resolution mass spectroscopy(MS)combines the high-efficiency separation capabilities of liquid chromatography(LC)with the high-sensitivity detection capabilities of MS.This combination is widely used in proteomics,transcriptomics,and metabolomics,particularly for studying protein chemical modifications.Proteins,as the main executors of life activities,undergo complex and spatiotemporal post-translational modifications and protein oxidative damage modifications that regulate various biological processes.Designing responsive chemical modification strategies for protein side chains can elucidate the molecular mechanisms underlying protein regulation of life processes.These strategies also enable the development of molecular tools for disease diagnosis and treatment.Protein chemical modifications involve chemically labeling the products of post-translational modifications or protein oxidative damage modifications,affecting the structure,function,stability,and intracellular localization of proteins.There are two main types of protein chemical modification methods:natural chemical conjugation strategies and specific amino acid modifications.Specific modifications include click chemistry,the introduction of post-translational modifications,and"bioorthogonal"reactions for labeling intracellular functional proteins.These modifications are increasingly significant in biological research and clinical applications pertaining to protein structure-function relationships,disease occurrence,and disease progression.[Progress]LC-MS/MS,located in analytical testing centers,plays an important role in detecting protein chemical modifications.When combined with MS analysis software such as pFind,it can intuitively analyze the modification sites,products,and possible reaction mechanisms of small molecule labeling reagents and protein modifications.For example,using the open search module of pFind,we analyzed methionine oxidation modifications in proteins and other posttranslational modifications.Our results identified 40 974 peptide segments in Hela cell lysates,with 222 peptide segments exhibiting methionine oxidation(0.61％).These oxidized segments corresponded to various oxidation-reduction or signal regulation-related proteins such as LDHA and KPYM.Further analysis revealed that the oxidation products included methionine sulfoxide in 8 peptide segments and methionine sulfone in 2 peptide segments.In-depth mining of MS data revealed that the protein thiol labeling reagents(Br-PPG,PPG-TPP)and protein amino labeling reagents(NC2-PPG)designed and synthesized in the study could successfully modify cysteine or lysine residues in β-lactoglobulin and bovine serum albumin.By configuring potential modification and photolysis products of these chemical modification reagents and proteins in the pFind open search module,we effectively identified specific modification sites,potential reaction mechanisms,and the effects of small molecule labeling reagents on target protein structures.Our analysis of NO2-PPG modification products with β-lactoglobulin and bovine serum albumin amino groups found that pFind could simultaneously identify various forms,including unstable chemical modification products.This capability is advantageous for studying protein structures and functions or imparting specific functions to proteins via small molecule modifications.The results illustrate that the chemical modification of protein thiols and amino groups can effectively introduce functional proteins into mitochondria within cells.This process enables the release of fluorescence signals at specific locations around the mitochondria,achieving carrier-free release of proteins.Consequently,this method allows for in-depth studies of redox signal transduction functions between proteins and mitochondria,providing a theoretical basis and drug design targets for the site-specific release of protein-based drugs and disease treatments.[Conclusions and Prospects]In summary,The above results demonstrate that LC-MS/MS combined with pFind analysis enables accurate identification of protein chemical modification sites,elucidation of reaction mechanisms,characterization of modification product types,and quantification of modification levels.In the future,ongoing advancements in research and technology suggest that LC-MS/MS will have broader applications.It is expected to reveal protein structure,function,and regulatory mechanisms,construct complex protein structures for targeted drug delivery and precise therapeutic interventions,and investigate protein-protein and protein-nucleic acid interactions to establish interaction networks.Moreover,it will enrich the research methods in fields such as chemical biology,life science,and structural biology.
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