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Various methods for sample preparation,HPLC-MS/MS analysis and data mining have been proposed for the quantitative profiling of differentially regulated proteins in biological samples.In our recent work,to improve the quantitation precision and accuracy,some new methods and integrated platforms have been developed.A double-chamber C18-SPE device was designed,to perform parallel sample denaturation,reduction,alkylation,digestion,labeling,and desalting,by which variations introduced during sample preparation could be minimized.A pseudo-isobaric labeling method was developed,by which peptides were respectively labeled by CD2O and reduced by NaCNBD3 or labeled by 13CD2O and reduced by NaCNBH3.Through the analysis of fragmental ions high resolution mass spectrometer,the average relative deviation and coefficient of variation (CV) for proteome quantitation could be decreased to 5% and 15%,respectively.An integrated platform for proteome quantitation was constructed,composed of dimethylated protein separation by a WAX/WCX column,digestion by an immobilized enzymatic reactor,and peptide analysis by nano-RPLC-ESI-MS/MS,to avoid variations introduced by multi-step off-line operation.All above mentioned strategies have been successfully applied into the analysis of syngenetic mouse hepatocarcinoma ascites cell lines with different metastasis risks,by which both the precision and accuracy for proteome quantitation were improved obviously,beneficial to find the potential biomarkers.