非变性质谱和紫外激光解离在蛋白质结构和相互作用分析中的应用

Applications of native mass spectrometry and ultraviolet photodissociation in protein structure and interaction analysis

蛋白质结构及相互作用的动态变化与其生物学功能密切相关,对蛋白质结构及相互作用进行精准探测和分析面临着巨大挑战。非变性质谱(nMS)是一种能够在近生理条件下将蛋白质及其复合物通过电喷雾离子化引入气相离子并进行质谱分析的方法。通过直接测定溶液中蛋白质及其复合物的组成或整合多种质谱解离技术,nMS可获取蛋白质及其复合物的计量关系、组装形式、解离常数、构象变化、结合界面及作用位点等关键信息,以揭示蛋白质相互作用与生物学功能之间的关系。紫外激光解离(UVPD)技术,特别是采用了193 nm准分子激光的UVPD是近年来迅速发展起来的质谱解离技术,其可以高效解离非变性蛋白质骨架,并保留碎片离子中的氢键等非共价相互作用力,从而实现单氨基酸位点分辨的蛋白质动态结构和相互作用质谱解析。本综述主要介绍了nMS和UVPD技术在蛋白质动态结构和相互作用分析中的应用和最新进展,包括由位点突变及配体结合等引起的蛋白质动态结构和相互作用变化,最后对蛋白质nMS表征的未来发展方向做出了展望。

Dynamic changes in the structures and interactions of proteins are closely correlated with their biological functions.However,the precise detection and analysis of these molecules are challenging.Native mass spectrometry(nMS)introduces proteins or protein complexes into the gas phase by electrospray ionization,and then performs MS analysis under near-physiological conditions that preserve the folded state of proteins and their complexes in solution.nMS can provide information on stoichiometry,assembly,and dissociation constants by directly determining the relative molecular masses of protein complexes through high-resolution MS.It can also integrate various MS dissociation technologies,such as collision-induced dissociation(CID),surface-induced dissociation(SID),and ultraviolet photodissociation(UVPD),to analyze the conformational changes,binding interfaces,and active sites of protein complexes,thereby revealing the relationship between their interactions and biological functions.UVPD,especially 193 nm excimer laser UVPD,is a rapidly evolving MS dissociation method that can directly dissociate the covalent bonds of protein backbones with a single pulse.It can generate different types of fragment ions,while preserving noncovalent interactions such as hydrogen bonds within these ions,thereby enabling the MS analysis of protein structures with single-amino-acid-site resolution.This review outlines the applications and recent progress of nMS and UVPD in protein dynamic structure and interaction analyses.It covers the nMS techniques used to analyze protein-small-molecule ligand interactions,the structures of membrane proteins and their complexes,and protein-protein interactions.The discussion on UVPD includes the analysis of gas-phase protein structures and interactions,as well as alterations in protein dynamic structures,and interactions resulting from mutations and ligand binding.Finally,this review describes the future development prospects for protein analysis by nMS and new-generation advanced extreme UV light sources with higher brightness and shorter pulses.