Abstract
Mass spectrometry-based proteomics is an increasingly valuable tool for determining relative or quantitative protein abundance in brain tissues. A plethora of technical and analytical methods are available, but straightforward and practical approaches are often needed to facilitate reproducibility. This aspect is particularly important as an increasing number of studies focus on models of traumatic brain injury or brain trauma, for which brain tissue proteomes have not yet been fully described. This text provides suggested techniques for robust identification and quantitation of brain proteins by using molecular weight fractionation prior to mass spectrometry-based proteomics. Detailed sample preparation and generalized protocols for chromatography, mass spectrometry, spectral counting, and normalization are described. The rat cerebral cortex isolated from a model of blast-overpressure was used as an exemplary source of brain tissue. However, these techniques may be adapted for lysates generated from several types of cells or tissues and adapted by the end user.
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Acknowledgements
The authors would like to thank David Tabb, Matt Chambers, and Jay Holman, for advice as well as development and public licensing of the Bumberdash Suite and IDPicker; Joy Cagmat-Guingab for her expertise in analytical chemistry and mass spectrometry; Mr. Eric Maudlin-Jeronimo, SGT Myint, SPC Vincent Donkor, for exemplary technical skills; and Raymond Genovese, Stephen Ahlers, David W. Johnson, and Kara E. Schmid for advice and support. This research is funded by Combat Casualty Care Research Program and Congressionally Directed Medical Research Program (Contract #: W81XWH-12-2-0134).
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There are no potential conflicting interests to declare in the preparation of this chapter.
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This material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The views of the authors do not purport or reflect the position of the Department of the Army or the Department of Defense (para 4-3, AR 360-5).
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Boutté, A.M., Grant, S.F., Dave, J.R. (2016). A Simplified Workflow for Protein Quantitation of Rat Brain Tissues Using Label-Free Proteomics and Spectral Counting. In: Kobeissy, F., Dixon, C., Hayes, R., Mondello, S. (eds) Injury Models of the Central Nervous System. Methods in Molecular Biology, vol 1462. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3816-2_36
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