Abstract: Objective To study the influence of sampling, handling, shipping and storage on low-molecular-weight serum proteome profiling，using magnetic beads and mass spectrometry. Methods Serum samples instantly separated and aliquoted, some stored at -80℃ for up to 6 months, others stored at 4 ℃ or room temperature (25℃) from 2h to 72h. The variations of protein profiling among these conditions on WCX magnetic beads were profiled. Profiling influenced by hemolysis，multi freeze-thaw cycles，storage conditions. Results Different handling procedures and storage conditions can cause different effects on serum profiling. Serum stored at -80℃ up to 6 months, stored at 4℃ or 25℃ for 2h or one freeze-thaw cycle, had little effects on serum proteomic analysis. If serum diluted into 9 M urea buffer at room temperature, the result is stable within 24 hours. Hemolysis can cause great effect on serum profiling. Statistics analysis shows that 35.9% peaks have significant changes (p < 0.01) in hemolytic samples. Conclusion The sampling procedure and storage will affect the result of protein profiling. Serum should be prepared in 2 hours at 4℃. Serum could be shipped or experimentally processed at 25℃ after dilution with 9 M urea buffer for 24 hours. Serum may be stored at －80℃ for long-term purpose with one freeze-thaw cycle. Hemolytic sample should be avoided. Our data suggested that a standard procedure for proteomic research and biomarker discovery is necessary.

Key words: WCX magnetic beads, SELDI－TOF－MS, mass spectrometry