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Caveats of proteomics approaches in identifying novel spinophilin interacting proteins
Author(s) -
Baucum Anthony Jerome,
Ham AmyJoan Lorna,
Colbran Roger James
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.581.9
Subject(s) - immunoprecipitation , scaffold protein , proteomics , protein phosphatase 1 , phosphatase , microbiology and biotechnology , shotgun proteomics , biology , postsynaptic density , phosphorylation , postsynaptic potential , biochemistry , antibody , immunology , signal transduction , receptor , gene
Spinophilin is an F‐actin and protein phosphatase 1 (PP1) binding protein that targets PP1 to the postsynaptic density and to various dendritic substrates. PP1 binding to spinophilin is enhanced in an animal model of parkinsonism. Several other known spinophilin associated proteins (SpAPs) were not detected in spinophilin complexes isolated from rat or mouse striatum. Therefore, a de novo shotgun proteomics strategy was developed to identify SpAps. Complexes specifically isolated using goat, mouse or rabbit spinophilin antibodies contained spinophilin, PP1, and neurabin (a known SpAP), as well as novel candidate SpAPs. We selected one of the novel proteins for further validation: the thousand and one amino acid kinase (TAO1). While TAO1 and spinophilin were detected in the reciprocal precipitations, and the amount of co‐immunoprecipitation was reduced following dopamine depletion, TAO1 was also present in spinophilin immunoprecipitates isolated from extracts of spinophilin knockout (KO) mice. Therefore, to identify spinophilin‐selective interactions, spinophilin was immunoprecipitated from both WT and KO animals, using multiple antibodies, and these samples were analyzed by shotgun proteomics. Multiple proteins were present in complexes isolated from WT, but not KO, tissue and studies are ongoing to validate these interactions.