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Kosaka, T. et al.

Kosaka, Okuyama, Sun, Ogata, Harada, Araki, Izumi, Yoshida, Okuno, Fujiwara, Ohsumi, Ichikawa,

Identification of molecular target of amp-activated protein kinase activator by affinity purification and mass spectrometry.

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase ( AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.[1]

References

Identification of molecular target of amp-activated protein kinase activator by affinity purification and mass spectrometry. Kosaka, T., Okuyama, R., Sun, W., Ogata, T., Harada, J., Araki, K., Izumi, M., Yoshida, T., Okuno, A., Fujiwara, T., Ohsumi, J., Ichikawa, K. Anal. Chem. (2005) [Pubmed]

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