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Quantitative proteomics is the study of protein expression levels between different proteomes, in which mass spectrometry (MS) is one of the most powerful tools for high sensitive identification.To reduce errors resulting from sample preparation and MS analysis, the development of isotopic labeling methods has been paid much attention, because of its low cost and high labeling efficiency.Heck et al [1] standardized three types of dimethyl labeling as labeling in solution, on-line labeling and on column labeling.However, the pretreatment processes of protein samples were not taken into consideration in all of these protocols.