The emerging role of mass spectrometry in molecular biosciences: studies of protein phosphorylation in fly eyes as an example.
Modern mass spectrometry (MS) streamlined with two-dimensional gel electrophoresis, in-gel digestion and HPLC-interfaced electrospray ionization quadrupole MS or matrix-assisted laser desorption ionization time-of-flight MS enables us to analyse proteins at a minuscule scale. We present here two examples of MS applications in which (1) we identified the in vivo phosphorylation site of Drosophila arrestin, phosrestin I (PRI), and (2) we revealed the identity of an 80 kDa phosphoprotein (80K) in Drosophila eyes to be the InaD gene product, a member of the PDZ domain proteins. Available evidence suggests that PRI quenches the activation of rhodopsin and that the InaD protein adjusts photoreceptor responsiveness by assembling/disassembling components involved in photoreceptor transduction in flies. PRI undergoes a reversible phosphorylation at a single site, and 80K at multiple sites. The phosphorylation states of PRI and 80K depend on the intensity and/or duration of light stimuli. From these results we postulate that these proteins function as a molecular switch adjusting the signalling cascade through phosphorylation. The combination of two-dimensional gel electrophoresis with MS will be a powerful tool for detailed investigation of such complex switching processes. The techniques described here can be applied also to other complex signalling systems.
Matsumoto
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Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
Kahn
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ES
Komori
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D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005136:Eye Proteins / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*
D019390:Arrestins; D029721:Drosophila Proteins; D005136:Eye Proteins; D010750:Phosphoproteins; C091568:inaD protein, Drosophila