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

doi:10.3724/SP.J.1123.2024.01021

摘要/Abstract

摘要：

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。

关键词: 非变性质谱, 紫外激光解离, 蛋白质结构, 蛋白质相互作用, 综述

Abstract:

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.

Key words: native mass spectrometry (nMS), ultraviolet photodissociation (UVPD), protein structure, protein-protein interaction, review

中图分类号:

O658

薛洁滢, 刘哲益, 王方军. 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[J]. 色谱, 2024, 42(7): 681-692.

XUE Jieying, LIU Zheyi, WANG Fangjun. Applications of native mass spectrometry and ultraviolet photodissociation in protein structure and interaction analysis[J]. Chinese Journal of Chromatography, 2024, 42(7): 681-692.

图/表 9

图 1 基于nMS的蛋白质结构解析技术时间发展图

Fig. 1 Time development of protein structure analysis technology based on native mass spectrometry (nMS) CID: collision-induced dissociation; ESI: electrospray ionization; SID: surface-induced dissociation; UVPD: ultraviolet photodissociation.

图 2 AtIspF与4-二磷酸核糖醇2-C-甲基-D-赤藓糖醇2-磷酸(CDP-MEP)、Zn2+结合的非变性ESI-MS图[41]

Fig. 2 Native ESI-MS spectra of AtIspF combined with CDP-MEP and Zn2+[41] a. native ESI-MS spectrum of the bare AtIspF; c. AtIspF incubated with CDP-MEP (T∶L=1∶2) in the absence of Zn2+, and the black number pointing to the peak of MS spectrum representing the numbers of CDP-MEPs bound to AtIspF; e. native ESI-MS spectrum of Zn2+-saturated AtIspF; g. AtIspF incubated with CDP-MEP (T∶L=1∶2) in the presence of Zn2+, and the red number pointing to the peak of MS spectrum representing the numbers of CMPs (one of the products in the reaction) bound to AtIspF; b, d, f, h. expanded spectra of the m/z 3800-4000 corresponding to a, c, e, g, respectively. CDP-MEP or L: 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; CMP: cytidine monophosphate; T: the bare AtIspF trimer.

图 3 (a)改进的Orbitrap Eclipse Tribrid质谱仪示意图和(b,c)AmtB在不同功率红外激光下的nMS图[52]

Fig. 3 (a) Schematic of the modified Orbitrap Eclipse Tribrid mass spectrometer and (b, c) nMS spectra of ammonia channel (AmtB) under different power infrared lasers[52] IR: infrared radiation.

图 4 CDK12/CDK13-CycK蛋白质复合物在有、无抑制剂SR-4835条件下的nMS图及SR-4835诱导的CDK12/CDK13-CycK蛋白质复合物解离示意图[20]

Fig. 4 nMS spectra of CDK12/CDK13-CycK protein complexes with and without inhibitor SR-4835, and schematic diagram of CDK12/CDK13-CycK protein complexes dissociation induced by SR-4835[20] a, b. nMS spectra of CDK12-CycK and CDK13-CycK protein complexes, respectively; c, d. nMS spectra of CDK12-CycK and CDK13-CycK protein complexes in the presence of SR-4835, respectively; e. SR-4835-induced allosteric regulation of CDK12/CDK13-CycK protein complexes dissociation. The concentration ratio of complex to inhibitor was 1∶3.

图 5 非变性血红素/肌红蛋白复合物的(a)CID、(b)HCD、(c)ETD、(d,e)UVPD二级质谱图及相应的解离序列匹配结果;(f)不同解离模式下所获得的序列覆盖度[29]

Fig. 5 MS2 spectra and corresponding dissociation sequence matching results of the native heme/myoglobin (Mb) complex activated by (a) CID, (b) HCD, (c) ETD, (d, e) UVPD; (f) the sequence coverages obtained under different dissociation modes[29] The filled blue circle represents the heme/myoglobin complex (9+) precursor ion and the unfilled blue circle represents apo-myoglobin (no heme) (8+). HCD: high energy collision dissociation; ETD: electron transfer dissociation.

图 6 肌红蛋白络合冠醚的UVPD分析[38]

Fig. 6 UVPD analysis of Mb complexed with crown ether (CE)[38] a. the sequence cleavages of Mb8+complexed with different numbers of CEs; b. the heatmap of residue fragmentation yields (FYs) of Mb8+ with different numbers of CE complexation; c, d. the residue-resolved FYs alterations (ΔFYs) between bare Mb and Mb complexed with 1 CE and 2 CEs, respectively; e, f. corresponding to the crystal structures of Mb in c and d, respectively, with red color indicating increased residue FYs and blue color indicating decreased residue FYs.

图 7 UVPD-nMS用于解析由DHFR突变体(P21L和W30R)引起的DHFR动态结构变化[75]

Fig. 7 UVPD-nMS analysis for the dihydrofolate reductase (DHFR) dynamic structure changes caused by DHFR variant (P21L and W30R)[75] a. the variations in UVPD FYs are shown across the backbone from the N-terminus to the C-terminus for each of the P21L and W30R constructs relative to wild type (WT); b, c. the DHFR crystal structure (1RX3) for P21L and W30R, respectively. The mutated residue is colored by purple.

图 8 蛋白激酶Cθ的C2结构域与pY218之间相互作用的UVPD-nMS分析[63]

Fig. 8 UVPD-nMS analysis for the interaction of the C2 domain of protein kinase Cθ (PKCθ) with pY218[63] a, b. nMS and UVPD-nMS spectra of C2 with 14AA-YY and 14AA-YpY, respectively; c. the sequence coverages of apo C2 and holo C2 obtained by UVPD; d. the different fragmentation efficiency between the holo C2 and apo C2; e. predicted possible pocket. 14AA-YY: a peptide consisting of 14 amino acids, which including a tyrosine residue; 14AA-YpY: a peptide containing 14 amino acids, which including a phosphorylated tyrosine residue; apo C2: the C2 of unbound 14AA-YpY; holo C2: the C2 of bound 14AA-YpY.

图 9 nMS和UVPD用于T4 gp32-ssDNA复合物结构的表征[94]

Fig. 9 nMS and UVPD for the structural characterization of the T4 gp32-ssDNA complex[94] a. the sequence map of gp32 with the backbone cleavage sites leading to the resulting dT12-containing fragment ions. Red, blue and green lines corresponding to the fragment ions containing the C-terminal, N-terminal, and bidirectional dT12-containing holo ions, respectively. b. the gp32 crystal structure with the backbone cleavage sites from which the C-terminal, N-terminal and both C-terminal and N-terminal holo fragment ions originate. The close ssDNA binding cleft is highlighted in a shade of purple.

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