Abstract: A method of on-line two-dimensional weak anion exchange chromatography-reversed-phase liquid chromatography (2D WAX-RPLC) was established, and applied for the comparative proteome study of antlers with four different growing stages. A five-protein-mixture was used as the sample to evaluate the reproducibility of the system. In addition, the capacity of such a system for the relative quantitative analysis was demonstrated by analyzing protein mixtures with different concentration ratios. Furthermore, five different lysis buffers were used to extract proteins from antlers with different growing stages. After the protein separation with 2D WAX-RPLC, the fractions with maximum peak height ratio larger than 2 were collected, digested, and further identified by micro-reversed-phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS). In total, 22 differential proteins were identified from 9 fractions. Our experimental results demonstrated that 2D WAX-RPLC based protein separation has the advantages of good reproducibility and automation, and might be a complementary method of two-dimensional gel electrophoresis for comparative proteome study.

Key words: antler, comparative proteomics , different growing stages, proteome, on-line two-dimensional liquid chromatography (2D LC)