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Chr.9
| " We are looking for mass spectrometry evidence of missing proteins (MPs) in chromosome 9. Our group focuses on identifing the molecular and cellular roles of MPs by using halo tag purification system and imaging technology"PI : Je-Yoel CHO |
 | Chromosome-CentricHuman Proteome Project |
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We have been trying to reveal biological functions of MPs using human cell line models and immunoprecipitation-mass spectrometry (IP-MS) approach. This strategy is not only useful for neXt-CP50 uPE1 functional characterization project, but also next-MP50 MPs identification and validation project. Chromosome 9 contains a total of 103 missing protein candidates, and we focused on five proteins (FOXD4, ARID3C, OR1J1, ANKRD18A, and ZNF510). We constructed HaloTag tagged fusion protein expression vectors which expressed in mammalian cells to identify MPs’ subcellular localization and their binding partners.
- Five MPs have been turned out its subcellular localization. Two MPs (FOXD4, ARID3C) binding partner proteins were identified via our IP-MS strategy. Furthermore, we also found two MPs that are in the same family. Identified peptide length of each MP was 30 and 35 AA long, respectively. Currently, we have been generating increased measurement of additional technical replicates using three different fractionation methods.
- On the other hands, we have been proceed with the functional analysis of uPE1 proteins. Two strategies were used to reveal the intracellular functions of the uPE1 proteins. First, IP-MS, which is the same strategy as the MP project, was used to define general role of the uPE1 proteins in the mammalian cells at basic culture condition. Second, we investigated the function of the uPE1 under disease or specific-physiological conditions. Using overexpression or deletion of the uPE1 proteins, protein function was evaluated by screening post-translational modifications and stoichiometric analysis of protein-protein interaction.