超电荷试剂在蛋白质紫外激光解离质谱表征中的应用研究

本文采用193 nm紫外激光解离质谱（UVPD-MS）研究非变性电喷雾条件下，超电荷试剂环丁砜对蛋白质序列、结构表征的影响。UVPD可在5 ns内对保持高级结构和非共价相互作用的不同价态碳酸酐酶进行快速激发解离，解离序列覆盖率达79.8%，具有较高的解离结构选择性。超电荷试剂环丁砜的引入可显著增加非变性电喷雾产生的碳酸酐酶离子的电荷数目，UVPD解离序列覆盖率提升至87.2%，实现对难解离区域的位点覆盖和序列、结构表征。加入环丁砜后，碳酸酐酶仍能保持高级结构谱学特征及与锌离子的非共价结合，锌离子结合区域等大部分结构区域的UVPD位点解离效率未见显著变化。但是，电荷数目的增加存在引起蛋白质局部结构变化的风险。

Investigation of Super Charging Reagent in the Characterization of Protein by Ultraviolet Photodissociation Mass Spectrometry

In this work, 193 nm ultraviolet photodissociation-mass spectrometry (UVPD-MS) was applied to investigate supercharging reagent sulfolane in protein sequence and structure characterization. All datasets were collected by using an Orbitrap MS system equipped with an ArF 193 nm EX50 excimer Laser. The 193 nm UV laser has the advantages of high brightness, good stability, and propagate in air, which can directly excite and dissociate the protein backbone within 5 ns (single pulse). Previous reports demonstrated that the partial protein high-order structures and non-covalent interactions could be retained in the UVPD fragments and the site-specific fragmentation yields (FYs) revealed the dynamic protein structure alterations under different conditions. Therefore, the combination of UVPD with nMS provides a new research paradigm for the characterization of protein structure changes. In this work, we further demonstrated 193 nm UVPD was superior to high-energy collision induced dissociation (HCD) in fragmenting and sequencing the non-denatured carbonic anhydrase II (CA) generated in native electrospray ionization (ESI). UVPD could efficiently activate and fragmentate the non-denatured CA with different charge states in just 5 ns, achieving cleavage sequence coverage 79.8% for CA Z10. Then, 0.25% supercharging reagent sulfolane was added into the protein solution and the CA charge distribution was significantly moved to higher charge states under identical native ESI conditions. However, higher-order structures (HOS) and non-covalent interactions could be obtained as the CA charge distribution was still concentrated and significantly different from the spectra features of denatured CA. The UVPD cleavage sequence coverage of the CA was increased to 87.2%, including the regions that were difficult to be dissociated and characterized without sulfolane addition. In addition, the non-covalent of CA and Zn²⁺ remained stable, indicating that the addition of sulfoxide had little influence on the Zn2+ binding region. The UVPD sequence coverage of CA Z11 was increased by 7.4% after the addition of sulfoxide, but the site FYs were almost not changed, demonstrating the corresponding local structures were nearly not changed. However, the higher charge states introduced by sulfolane also exhibited the risk of inducing specific structure alteration of part of protein regions. In conclusion, it was infered that supercharging reagent is efficient in modulating the charge state distribution of proteins in native ESI-MS characterization and can expand the MS detection mass range. The introduction of supercharging reagent can further improve the UVPD sequence coverage, including the structure-compact regions hard to be fragmented. Although part of the protein HOS and non-covalent interactions seems retained, the influence of higher charge states on protein structures should be considered in dynamic protein structure investigation.