Fragmentation mechanisms of the singly protonated glutathione (γ-ECG) and its synthetic analogue peptides (ECG and PPECG) have been investigated by liquid chromatography tandem-mass spectrometry and theoretical calculations. In the mass spectra, similar fragmentation patterns were observed for γ-ECG and ECG, but a completely different one was found in the case of PPECG. The E–C amide bond cleavage is the predominant pathway for the fragmentation of γ-ECG and ECG, whereas the additional N-terminal prolyl residues in PPECG significantly suppress the E–C amide bond cleavage. Theoretical calculations reveal that the fragmentation efficiencies of the E–C bonds in the protonated γ-ECG and ECG are much higher than that in the protonated PPECG, being attributed to their lower barriers of the potential energy; clearly the introduction of two prolyl residues can increase substantially the potential energy barrier. In the proposed mechanism, the protonated E–C amide bonds in the three peptides are first weakened followed by a nucleophilic addition by the glutamyl carboxyl oxygen atom in side chain, leading to the breaking of the E–C amide bonds. However, the processes of E–C bond fragmentation for three protonated analogs were not collaborative. Protonated amide bonds first fragment, then the nucleophilic addition by the side chain of glutamyl carboxyl oxygen atom takes places. On the other hand, the prolyl residues in PPECG can largely diminish the nucleophilic addition, resulting in a much lower efficiency of its E–C amide bond breaking. Distance analysis indicates that breaking the E–C amide bonds in the protonated γ-ECG, ECG, and PPECG ions could not occur without the assistance from the nucleophilic attack, highlighting an asynchronous collaborative process in the bond breakings.

中文翻译：

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深入了解单质子化谷胱甘肽和相关肽基离子中谷氨酸侧链的亲核裂解机制

已经通过液相色谱串联质谱法和理论计算研究了单质子化谷胱甘肽 (γ-ECG) 及其合成类似肽 (ECG 和 PPECG) 的断裂机制。在质谱中，γ-ECG 和 ECG 观察到类似的碎裂模式，但在 PPECG 的情况下发现了完全不同的碎裂模式。E-C酰胺键断裂是γ-ECG和ECG断裂的主要途径，而PPECG中额外的N-末端脯氨酰残基显着抑制E-C酰胺键断裂。理论计算表明，质子化 γ-ECG 和 ECG 中 E-C 键的断裂效率远高于质子化 PPECG，这是由于它们的势能势垒较低；显然，引入两个脯氨酰残基可以显着增加势垒。在所提出的机制中，三个肽中质子化的 E-C 酰胺键首先减弱，然后侧链中的谷氨酰羧基氧原子亲核加成，导致 E-C 酰胺键断裂。然而，三个质子化类似物的 E-C 键断裂过程不是协同的。质子化的酰胺键首先断裂，然后发生谷氨酰羧基氧原子侧链的亲核加成。另一方面，PPECG 中的脯氨酰残基可以在很大程度上减少亲核加成，导致其 E-C 酰胺键断裂效率低得多。距离分析表明，在质子化的 γ-ECG、ECG、