TAILIEUCHUNG - Trypsin-induced proteome alteration during cell subculture in mammalian cells

It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. Methods: In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by. | Huang et al. Journal of Biomedical Science 2010 17 36 http content 17 1 36 a NSC Tha cost of publication In Journal of Blomodlcal Science Is bome by tlM National Science Council Taiwan JOURNAL OF BIOMEDICAL SCIENCE RESEARCH Open Access Trypsin-induced proteome alteration during cell subculture in mammalian cells Hsiang-Ling Huang 1 Hsiang-Wei Hsing1 Tzu-Chia Lai1 Yi-Wen Chen1 Tian-Ren Lee1 Hsin-Tsu Chan1 Ping- Chiang Lyu1 Chieh-Lin Wu1 Ying-Chieh Lu1 Szu-Ting Lin1 Cheng-Wen Lin2 Chih-Ho Lai3 Hao-Teng Chang4 Hsiu- Chuan Chou 5 and Hong-Lin Chan 1 Abstract Background It is essential to subculture the cells once cultured cells reach confluence. For this trypsin is frequently applied to dissociate adhesive cells from the substratum. However due to the proteolytic activity of trypsin cell surface proteins are often cleaved which leads to dysregulation of the cell functions. Methods In this study a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. Results 36 proteins are found to be differentially expressed in cells treated with trypsin and proteins that are known to regulate cell metabolism growth regulation mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated p53 and p21 are both up-regulated after trypsinization. Conclusions In summary this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design. Background Plasma membrane proteins are responsible for a wide variety of functions essential to maintaining normal physiological activities. For example when EGF receptor families a group of proteins located in the plasma membrane that

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