College of Pharmaceutical Sciences, Zhejiang University , Hangzhou 310058, Zhejiang, China.
Department of Chemistry and Chemical Engineering, Beijing University of Technology , 100124, Beijing, China.
Anal Chem. 2017 Nov 21;89(22):11902-11907. doi: 10.1021/acs.analchem.7b03804. Epub 2017 Nov 7.
This paper describes a simple method to determine the absolute configuration of amino acids residues in peptides by mass spectrometry using a newly developed pair of mass-tagged chiral probes without the requirement of reference standards. A pair of benzylicaldehyde probes, 1-(S)-H in S configuration and 2-(R)-D in deuterium-labeled R configuration with the ratio of 1:1, were synthesized for in situ condensation with amino acid residues and transformed into a pair of stereodynamic imine products. The characteristic intensity difference observed in mass spectrometry can be used to determine the absolute configuration and to quantify the enantiomeric composition of chiral amino acid residues. Significant chiral recognition ability was achieved for 18 natural chiral amino acids and for one β-amino acid by comparing the ion intensity ratio of imine products I to I. For 16 kinds of amino acids, the L form of the amino acids was more reactive with 1-(S)-H, while D configuration amino acids preferred to react with 2-(R)-D. However, for three kinds of amino acid, the opposite result was obtained. The configurations of the residues in the peptides, Phe-Tyr-Ala, D-Phe-Tyr-Ala, Val-Pro-Phe-D-Leu-Met, Val-Pro-Phe-Leu-D-Met, as well as in a natural peptide with unknown chirality were determined by acid hydrolysis followed by the present method. In addition, molecular modeling results illustrate that the recognition process is mainly controlled by kinetic factors. Using the new probes coupled with a mass spectrometry approach avoids time-consuming workup and separation steps. We expect that the probes could be applied as tools to determine the absolute configuration of amino acid residues in proteins in future research.
本文描述了一种使用新开发的一对质量标记手性探针通过质谱法确定肽中氨基酸残基绝对构型的简单方法，而无需参考标准。合成了一对苄基醛探针 1-(S)-H（S 构型）和 2-(R)-D（氘标记的 R 构型），比例为 1:1，用于与氨基酸残基原位缩合，并转化为一对立体动力学亚胺产物。在质谱中观察到的特征强度差异可用于确定绝对构型并定量手性氨基酸残基的对映体组成。通过比较亚胺产物 I 和 I 的离子强度比，对 18 种天然手性氨基酸和一种 β-氨基酸实现了显著的手性识别能力。对于 16 种氨基酸，L 型氨基酸与 1-(S)-H 的反应性更强，而 D 构型氨基酸更喜欢与 2-(R)-D 反应。然而，对于三种氨基酸，得到了相反的结果。通过酸水解和本方法测定了肽、D-Phe-Tyr-Ala、Val-Pro-Phe-D-Leu-Met、Val-Pro-Phe-Leu-D-Met 以及一个未知手性天然肽中残基的构型。此外，分子建模结果表明，识别过程主要受动力学因素控制。使用新探针结合质谱法避免了繁琐的工作和分离步骤。我们期望这些探针将来能够作为确定蛋白质中氨基酸残基绝对构型的工具。
