Maintaining the protein high-order structures and interactions during the transition from aqueous solution to gas phase is essential to the structural analysis of native mass spectrometry (nMS).
Recently, a research group led by Prof. WANG Fangjun from the Dalian Institute of Chemical Physics (DICP) of the Chinese Academy of Sciences (CAS), in collaboration with Prof. SHUN Feng from Southwest Jiaotong University, systematically interrogated the effects of charge state and crown ether complexation on the gas-phase native-like protein structure by integrating nMS with 193 nm ultraviolet photodissociation (UVPD). They have revealed the molecular mechanism of crown ether microsolvation effect on the gas-phase native-like protein structure.
This study was published in Journal of the American Chemical Society on Dec. 30, 2022.
nMS and UVPD exploration of the molecular details of the CE microsolvation effect on the native-like protein structure in the gas phase (Image by LIU Zheyi and ZHOU Lingqiang)
The nMS is a cutting-edge technique for studying the composition and structure of proteins and their complexes. Non-denaturing electrospray ionization use biocompatible solutions to transfer proteins from liquid to gas phase while maintaining higher order structure and interactions.
However, positive charged residues in protein will form hydrogen bonds with protein backbone carbonyls to stabilize the positive charges of the side chains through intramolecular solvation.
Although the change of protein collision cross-sectional area caused by intramolecular solvation has been detected by ion mobility-mass spectrometry, there is still a lack of effective analytical means to accurately characterize the specific site where it occurs and the region that causes structural changes.
In this work, the researchers applied the self-built nMS-UVPD platform and home-developed MS data process software to monitor the conformational changes induced by intramolecular solvation. Based on the UVPD fragmentation pattern of myoglobin-heme complex, they determined the detailed microsolvation sites and the regions with conformational changes.
Then, they systematically characterized the protein structure alternations between protein ions with different charge states. They found that the gas phase structure of proteins in the high charge state was easily affected by the intramolecular solvation effect and deviated from the solution state structure, and the gas phase structure of low charge protein ions was closer to solution state.
Furthermore, they proved that the complexation of the crown ether 18C6 with protein mainly occurred in solution, and then played an important role in stabilizing the protein structure during the electrospray ionization process.
UVPD-nMS analysis revealed that the crown ether was mainly bound to protein region with high charge density, and the gas phase structure of the protein was closer to the solution state by blocking the intramolecular solvation of the charged side chain.
"This work demonstrates the significant advantages of UVPD-nMS in simultaneously obtaining protein sequence and dynamic structure information, and provides theoretical and technical insights for the maintenance and efficient characterization of protein solution structures in nMS," said Prof. WANG.