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.

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. [ABSTRACT FROM AUTHOR]