谷胱甘肽与D型氨基酸非共价复合物的质谱

Probing Non-Covalent Complexes of Glutathione with D-Amino Acids by Mass Spectrometry

为了研究谷胱甘肽和D型氨基酸的非共价相互作用,将一定化学剂量比的还原型谷胱甘肽与D-苯丙氨酸、D-组氨酸或D-谷氨酰胺在室温下混合后,温育1h,使反应达到平衡.电喷雾质谱测量结果表明,在生理pH条件下,谷胱甘肽可以和D型氨基酸经反应生成非共价复合物.串级质谱中的碰撞诱导解离(CID)以及紫外光谱进一步确认了非共价复合物的生成.为了避免严重的离子化效率和质谱信号相互抑制作用,对谷胱甘肽和D-谷氨酰胺的相互作用作了定量的评估.配制一系列不同初始浓度的谷胱甘肽和D型氨基酸的混合溶液,并用电喷雾质谱测定混合溶液中不同质点的质谱峰强度,计算了谷胱甘肽与三个D型氨基酸结合形成的复合物的解离常数.计算结果表明,谷胱甘肽可以和D型氨基酸结合形成不同键合强度的非共价复合物,其稳定性按照D-谷氨酰胺、D-苯丙氨酸、D-组氨酸的次序逐渐增大.

To investigate the non-covalent interaction between glutathione and D-amino acids, stoichiometric reduced glutathione （GSH） and three D-amino acids, including D-phenylalanine （D-Phe）, D-histidine （D-His）, and D-glutamine （D-Gln）, were mixed respectively, and then incubated at room temperature for 1 h to reach equilibrium. The electrospray ionization mass spectrometry （ESI-MS） results indicated that glutathione and three D-amino acids could form non-covalent complexes in physiological pH conditions, respectively. The binding of glutathione to D-amino acid was further confirmed by collision-induced dissociation （CID） in a tandem mass spectrometer. Additionally, the complexes exhibited different features and properties from the reactants in UV spectroscopy, which also confirmed the results of ESI-MS. To avoid distinct ionization efficiency discrepancy and signal suppression in ESI-MS measurements, the interaction between glutathione （GSH） and D-glutamine （Gln） was quantitatively evaluated. A series of samples with different initial concentrations of glutathione and D-amino acids were mixed, and then series of peak intensities for different species in the mixture were achieved by ESI-MS. The dissociation constants of three complexes formed by glutathione and D-amino acids were calculated. The calculation results revealed that the reduced tripeptide γ-glutathione could interact with D-amino acids to form non-covalent complexes with different affinities, the stabilities of the three complexes increased gradually according to the order of D-glutamine, D-phenylalanine, and D-histidine. 【Keyword】：