Investigation of TOMM6 in brains from Tg2576 mice

The protein TOMM6 (translocase of outer mitochondrial membrane 6) was identified as an interacting protein of phospho-S670-GRK2 (G protein-coupled receptor kinase 2 phosphorylated at serine 670) in brain specimens (frontal cortex and hippocampus) of 12-month-old Tg2576 Alzheimer (AD) model mice. Mitochondrial proteins from brain specimens were solubilized and subjected to immuno-affinity enrichment with phospho-S670-GRK2-specific antibodies, which were covalently coupled to Affigel-10. After washing steps to remove unbound proteins, bound proteins were eluted from the immuno-affinity matrix and separated by SDS-PAGE. The immuno-affinity matrix with covalently coupled phospho-S670-GRK2-specific antibodies co-enriched a protein in the range of 6-12 kDa. The protein band was cut out and subjected to identification by nano-LC-ESI-MS/MS.