Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

MS evidence acquisition of missing proteins in chromosome 9 using halo tag purification system and identification of cellular roles. (#140)

HuiSu Kim 1 , Dong Wook Kim 1 , Hyoung-Min Kim 1 , Yong-In Kim 2 , J. Eugene Lee 2 , Je-Yoel Cho 1
  1. BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science College of Veterinary Medicine, Seoul National University, Seoul, South Korea
  2. Bioanalysis center, Korea Research Institute of Standards and Science, Daejeon, South Korea

The NeXtProt release 2018-09-17 reports 2,890 of proteins are remaining in missing proteins level, also known as PE 2,3,4 level (14.2%).  Our goal is finding MS evidence of missing proteins  and identification of biological roles in cells. First, we tried to find missing proteins with no MS evidences in Chromosome 9 using NeXtProt database. High obstacles acquiring MS evidences of missing proteins due to the very low expression level or specific expression pattern in certain cell types or tissues drive us to take advantage of overexpression system. In brief, we constructed 5 plasmids DNA (FOXD4, ARID3C, OR1J1, ANKRD18A, ZNF510) harboring cDNA of missing proteins fused to Halo Tag and artificially transfected into HEK293T cells. Overexpressed missing proteins were purified with Halo tag purification system and subjected to LC-MS/MS analysis. This strategy gave us great advance in detection of MS evidences for missing proteins. Using this method, we definitely get 2 MS evidences of missing protein (FOXD4, and ARID3C) under the rule of Human Proteome Project Data Interpretation Guidelines. In short, we used followed search parameter; peptide FDR < 0.01, protein FDR < 0.01, and 2 or more unique peptides with 7 amino acids. Additional cellular analysis identified that FOXD4 and ARID3C were localized in cytosol and nucleus respectively. In conclusion, overexpression and purification system we used in the study may be alternative method to identify MS evidences of missing proteins.