Identification of ALDH2 Interacting Proteins by Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry
Sunday, March 4, 2018
South Hall A2 (Convention Center)
Jutaro Fukumoto, M.D. and Ph.D., Helena Hernández-Cuervo, M.D., Venkata Ramireddy Narala, Ph.D., Sahebgowda S. Patil, B.S., Ramani Soundararajan, Ph.D., Matthew Alleyn, B.S., Mason T. Breitzig, B.S., Richard F. Lockey, M.D., Narasaiah Kolliputi, PhD
RATIONALE: Recent studies have revealed that mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays an important role in protecting against oxidative stress in multiple organs, including the lung. Protein-protein interaction has long been known to affect critical cellular functions. Therefore, identifying yet unknown binding partners of ALDH2 is important to facilitate the understanding of how ALDH2 counteracts oxidative stress.

METHODS: H441 cells, a human lung epithelial cell line, were cultured under normal conditions. At 80-90% confluence, the cells were harvested and treated with or without a chemical cross-linker dithiobis(succinimidyl)propionate (DSP). The whole cell lysates were then prepared by adding lysis buffer. Each sample was immunoprecipitated (IP) with either anti-ALDH2 antibody or control IgG. The samples were then sequentially run on SDS-PAGE. Lastly, the entire gel lane from each sample was subjected to in-gel digestion and mass spectrometric (MS) analysis. To verify the results of MS analysis, we conducted Western blotting.

RESULTS: Several proteins [transglutaminase 2 (TGM2), heat shock protein 60 (Hsp60), etc.] were exclusively detected in samples immunoprecipitated with ALDH2 antibodies, regardless of the use of a chemical cross-linker. Other proteins [solute carrier family 25 member 5 (SLC25A5) etc.] were only detected in samples immunoprecipitated with ALDH2 antibodies after chemical cross-linking. Western blotting analysis confirmed these results.

CONCLUSIONS: Transglutaminase 2 (TGM2) and heat shock protein 60 (Hsp60) are binding partners of ALDH2 proteins. The combination of chemical cross-linking, immunoprecipitation, and mass spectrometry is a powerful tool for identification of unknown protein binding partners.