Dual Maleimide Tagging for Relative and Absolute Quantitation of Cysteine-Containing Peptides by MALDI-TOF MS

A dual maleimide (DuMal) tagging method has been developed for both relative and absolute quantitation of cysteine-containing peptides (CCPs) in combination with MALDI-TOF mass spectrometry. A pair of maleimides with minimal differences in their chemical structures, N-methylmaleimide and N-ethylmaleimide, have been chosen to allow for the rapid (approximate to minutes) tagging of CCPs in the Michael addition reaction with high efficiency. It has been validated that the DuMal tagging technique is sensitive and reliable in the quantitative analysis of CCPs. Absolute quantitation of CCPs can be achieved with a detection limit as low as 7.3 nm. Relative quantitation of CCPs can be performed in various sample mixtures with consistent results (coefficient of variation <5%). The DuMal tagging technique provides a sensitive and accurate approach for the quantitation of biomolecules containing thiol reactive sites; thus it is promising for protein detection, disease diagnosis, and biomarker discovery associated with post-translational modifications of cysteines.