Simultaneous Quantitative Detection of Cysteine and Homocysteine Labeled by 1-Pyrenecarboxaldehyde Using MALDI-TOF MS

Cysteine (Cys) and homocysteine (Hcy) are two important reducing agents in living organisms and play crucial roles in many physiological processes. The quantitative analysis of Cys and Hcy holds significance in exploring the functions of biothiols in biological. In this work, 1-pyrenecarboxaldehyde (1-py) with high derivatization efficiency and ionization efficiency was used for quantitative analysis of Cys and Hcy by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). After 1-py derivatization, the detection limit of Cys and Hcy can reach as low as 250 amol/L. Without internal standards, the simultaneous quantitative detection of Cys and Hcy was achieved by analyzing the proportion of peak intensities of derivative products to total compounds. The linear quantitative ranges for Cys and Hcy were over the concentrations from 5 to 2500 μM. Moreover, the specific hydrogen loss of the derivatized products was observed in MALDI-TOF detection, and the potential fragment pathway and nitrogen protonation mechanism were demonstrated through density functional theory (DFT) calculations. Finally, this method was successfully applied to the quantification of Cys and Hcy in HepG2 cell lysate, offering a rapid and highly sensitive approach for the quantitative analysis of Cys and Hcy using MALDI-TOF MS.