Abstract
Combining antibody coated magnetic bead affinity purification as a separation tool with mass spectrometry as a protein identification tool has been a powerful method to determine the composition of interacting proteins and protein complexes. Receptor proteins and insoluble complexes offer added challenges, as they may contain hydrophobic membrane proteins. When strong detergents such as SDS are required to release the complexes from the magnetic beads or prevent their aggregation and precipitation, the subsequent required removal of anionic detergents appears to cause significant sample losses prior to MS analysis. We describe a procedure in which protein denaturation and trypsin digestion are performed directly on the affinity bound complex on the magnetic beads, circumventing detergents and reducing sample loss prior to LC/MS/MS analysis.
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Acknowledgments
This work was supported by the Intramural Research Programs of the National 24 Institute of Mental Health (MH000274). Dr. Ayse Dosemeci (NINDS) kindly provided immunoaffinity purified post-synaptic densities; her advice and assistance was most helpful. M.B., D.V. and A.B. and part of the work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
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Berberich, M.J. et al. (2011). Development of an On-Bead Digestion Procedure for Immunoprecipitated Proteins. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_7
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DOI: https://doi.org/10.1007/978-94-007-0828-0_7
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