Identification of phase-specific protein biomarkers during cell cycle progression

• Main Authors: Yu-Chiao Tseng, 曾郁喬
• Other Authors: Yeou-Guang Tsay
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/94d3kg

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 102 === Cell division is a basic and important event in vivo, and is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed. To understand how cell cycle is affected by various stimuli, we decided to identify what molecular properties may mark the progression of cell cycle. However, it has been difficult to identify cell cycle-regulated proteins due to several limitations, including poor specificity of protein purification, lack of comprehensive protein quantitative assays and deficiency of effective computational methods. In Chapter II, cells synchronized with various approaches were prepared for characterization for their cell cycle properties. In order to verify whether differential subcellular redistributions of specific proteins are associated with particular phases of cell cycle, we established a subcellular proteomics platform based on in situ protein extraction. Proteins in subcellular fractions were compared to find those with differential locations during cell cycle progression. In contrast to traditional purification methods based on centrifugation, our platform may provide another purification approach to discover the proteins with differential affinity toward cell architecture. To identify cell phase-specific biomarkers, we employed this platform to analyze five samples prepared using various cell synchronization techniques, which were enriched of G0, G1, S, G2 and M cells, respectively. Since the binding of a protein to cell architecture frequently correlates with its subcellular localization, we hypothesize that protein translocation biomarkers for cell cycle can be screened from those proteins that distribute among these subcellular fractions in a cell cycle-dependent manner. By subjecting HeLa cells to various synchronization procedures, we collected subcellular protein fractions from five phase cells. Then, gel-based protein analyses identified an average of 2500~3000 proteins in total from five subproteomics samples, showcasing the depth of this proteomics technology. Furthermore, we also employed peptide counting techniques to screen proteins whose distribution in subcellular fractions showed a significant change during G0-G1, G1-S, S-G2 or G2-M transition. With statistical evaluation, we identified 165, 199, 195 and 182 candidate proteins, respectively, and also found that components in the same protein complexes frequently showed similar distribution profiles. Finally, non-pharmacological studies have been used to verify that two proteins, DDX42 and annexin A11, indeed showed phase-specific changes during cell cycle progression, evidencing that our platform has the prowess to identify protein properties indicating the cellular functional status, including cell cycle progression.