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, various new methods have been developed.

An isobaric dimethyl labeling (IDL) method based on the subtle mass defect differences between ¹²C/¹³C and ¹H/²H. Lys-C protein digests were labeled with CD_²O/¹³CD_²O and reduced with NaCNBD₃/NaCNBH₃ as heavy and light isotopologues, respectively. The fragment ion pairs with mass differences of 5.84 mDa were resolved by high-resolution MS/MS and used for quantification. The IDL strategy described here resulted in highly accurate and precise quantification results with approximately four orders of magnitude.

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.

Furthermore, the relative quantitation for both phosphoproteome and glycoproteome was carried out, with the combination of the selective enrichment of posttranslational modification peptides and chemical/enzymatic labeling strategies.

All above mentioned novel strategies have been successfully applied into the analysis of syngenetic mouse hepatocarcinoma ascites cell lines with high and low metastasis risks, by which both the precision and accuracy for proteome quantitation were improved obviously, and the potential related proteins were identified.