Summary
Identification of proteins and characterization of posttranslational modifications are crucial steps for many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of choice for these analyses. Here we describe two methods for the on-membrane digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.
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Acknowledgements
We acknowledge support from NIH grants P30 NS050276 and P30 CA016087 to T.A.N., and the Ramón y Cajal Program (Ministry of Science and Technology, Spain) for support to J.L.L.-G.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Luque-Garcia, J.L., Neubert, T.A. (2009). On-Membrane Tryptic Digestion of Proteins for Mass Spectrometry Analysis. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_35
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DOI: https://doi.org/10.1007/978-1-59745-542-8_35
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Publisher Name: Humana Press, Totowa, NJ
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