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Mass Spectrometric Analysis of ALDH2 Interactome
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A2258 - Mass Spectrometric Analysis of ALDH2 Interactome
Author Block: J. Fukumoto, S. Patil, I. John, S. Krishnamurthy, M. Breitzig, M. Alleyn, H. Hernández-Cuervo, R. Soundararajan, R. F. Lockey, N. Kolliputi; Internal Medicine, University of South Florida, Tampa, FL, United States.
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, exploring uncharacterized ALDH2 interactome could improve our 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 whole cell lysates were prepared using a mild lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, pH 7.4) in order to maintain protein-protein interaction. The protein extracts were immunoprecipitated (IP) with either anti-ALDH2 antibody or control IgG. The protein elution from each group was obtained (4 duplicates of ALDH2 antibody-IP elutions and 3 duplicates of control IgG-IP elutions). The samples were sequentially run on SDS-PAGE. Lastly, the entire gel lane from each sample was subjected to in-gel digestion and mass spectrometric (MS) analysis. The profiles of the proteins detected in each group were compared and subjected to Student’s t-test. RESULTS: 23 distinct proteins were listed as significantly high in the ALDH2-IP group compared to controls. The group of distinct proteins includes, but is not limited to, DNAJB1 (DnaJ Heat Shock Protein Family (Hsp40) Member B1), Transglutaminase 2 (TGM2), and block of proliferation 1 (BOP1). CONCLUSION: ALDH2 may have an unidentified protein interaction network, which would help elucidate its importance in various cellular functions. The combination of immunoprecipitation and mass spectrometry will give us an insight that could not be easily attained otherwise.
Author Block: J. Fukumoto, S. Patil, I. John, S. Krishnamurthy, M. Breitzig, M. Alleyn, H. Hernández-Cuervo, R. Soundararajan, R. F. Lockey, N. Kolliputi; Internal Medicine, University of South Florida, Tampa, FL, United States.
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, exploring uncharacterized ALDH2 interactome could improve our 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 whole cell lysates were prepared using a mild lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, pH 7.4) in order to maintain protein-protein interaction. The protein extracts were immunoprecipitated (IP) with either anti-ALDH2 antibody or control IgG. The protein elution from each group was obtained (4 duplicates of ALDH2 antibody-IP elutions and 3 duplicates of control IgG-IP elutions). The samples were sequentially run on SDS-PAGE. Lastly, the entire gel lane from each sample was subjected to in-gel digestion and mass spectrometric (MS) analysis. The profiles of the proteins detected in each group were compared and subjected to Student’s t-test. RESULTS: 23 distinct proteins were listed as significantly high in the ALDH2-IP group compared to controls. The group of distinct proteins includes, but is not limited to, DNAJB1 (DnaJ Heat Shock Protein Family (Hsp40) Member B1), Transglutaminase 2 (TGM2), and block of proliferation 1 (BOP1). CONCLUSION: ALDH2 may have an unidentified protein interaction network, which would help elucidate its importance in various cellular functions. The combination of immunoprecipitation and mass spectrometry will give us an insight that could not be easily attained otherwise.
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