Characterization Of Protein Denaturation/agglutination System Based On Capillary Electrophoresis-mass Spectrometry

The phenomenon of protein denaturation/aggregation is frequently encountered in fields including chemistry,biology,pharmaceutics,etc.It attracts increasing interests of researchers because of its pivotal role in human diseases as well as its effects on the efficacy and safety of bio-pharmaceuticals.Due to the high degree of complexity,heterogeneity and transient nature displayed by the denatured and aggregating protein systems,the task of characterizing them at the molecular level using conventional techniques remains extremely challenging,since their resolution and general applicability are rarely sufficient.In this work,by combining the separation power of capillary electrophoresis(CE),and the advantages of electrospray ionization-mass spectrometry(MS)in macromolecular characterization in terms of spatial resolution,accuracy,sensitivity,we developed a CE-MS-based platform with the aid of a flow-through microvial CE-MS interface,to enable characterization of protein denaturation/aggregation at multiple levels in great detail.We focused on method development for structural analysis of cysteine-rich proteins whose disulfide bonds undergo rearrangement during denaturation.We first developed an approach to deducing the disulfide patterns of native and non-native conformers of proteins based on tandem MS techniques,to identify protein conformation using top-down strategy.Second,we developed a conformer-specific characterization approach with CE-MS that allows multiple conformers of the same protein in a mixture to be separated prior to the online top-down MS analysis.In addition,aiming at analysis of complex mixtures comprising multiple protein identities,we developed a bottom-up proteomic approaching utilizing the unique advantages of CE-MS to identify proteins.The denaturation/aggregation products of Lysozyme(Lyz)and ?-lactoglobulin(?-Lg)yielded in heat-treatment of these proteins were selected as models for development of top-down approaches.We revealed the correlation between the disulfide patterns of protein with the modification patterns of the signature fragments resulted from gas-phase fragmentation,which was in turn applied to disulfide mapping of unknown protein conformers.By combining the CE methods we developed that can separate different conformers of heat-induced denaturation/aggregation products with the optimized tandem MS methods,we accomplished online CE-top-down analysis of intact proteins.While different conformers can be distinguished by analyzing charge state distributions of intact protein ions,the separated conformers can be individually delivered to the tandem MS stage for disulfide mapping using the methods we developed,which reveal the structural difference between different conformers.The hydrogen/detuterium exchange(HDX)technique integrated in our CE-MS platform provided complementary structural information for conformer-specific characterization.With HeLa proteome used as the model system,the performance of the bottom-up proteomic approach we developed is demonstrated to be superior to the conventional LC-MS based approach in term of the ratio of number of identified proteins to the quantity of sample consumption,and sensitive to the low-abundant species,thereby competent to analyze complex protein mixtures.The results of this project demonstrates that CE-MS platform we developed is suited for structural characterization of complex biomolecular systems,and the series of methods we developed can provide unique structural information for denaturation/aggregation systems comprising multiple protein species and various conformers,thus benefiting the deciphering of the mechanisms underlying these processes.