Chemical derivatization is a simple approach for stable-isotope covalent labeling of proteins in quantitative proteomics. Herein we describe the development of a novel maleyl-labeling-based approach for protein quantification. Under optimized conditions, maleic anhydride can serve as a highly efficient reagent to label the amino groups of tryptic peptides. Furthermore, "click chemistry" was successfully applied to obtain the second modification of maleylated peptides via thiol-Michael addition reaction. Accurate quantification was further achieved via the first or/and second step stable-isotope labeling in this study. Our data thus demonstrate that the maleyl-labeling-based method is simple, accurate, and reliable for quantitative proteomics. The developed method not only enables an enhanced sequence coverage of proteins by improving the identification of small and hydrophilic peptides, but also enables a controllable, successive, second derivatization of labeled peptides or proteins, and therefore holds a very promising potential for in-depth analysis of protein structures and dynamics.

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Histone post-translational modifications (PTMs) have been considered to be a major group of important epigenetic marks and to play a critical role in the regulation of chromatintemplated biological processes. To date, novel strategies for the quantification of histone PTMs are still highly desirable. Here, we present an efficient approach to quantitatively characterize histone PTMs using stable isotope dimethyl-labeling coupled with mass spectrometry. At first, all the ε-amino groups of free lysines are derivated by heavy formaldehyde to enable an easy distinction of free lysines from those of naturally occurring lysine-dimethylation upon MS analysis. After tryptic digestion, a second derivatization was applied with heavy and light stable isotope dimethyl-labeling to respectively label the Ntermini of tryptic peptides from different sample sources. The mixture were further identified and quantified by HPLC/MS/MS. This method enables to compare histone PTMs from multiple sample sources and to quantify different PTMs at certain amino acid residues of histone in one single experiment. Thus it is highly attractive for the identification of epigenetic histone marks.