A Simplified Workflow for Protein Quantitation of Rat Brain Tissues Using Label-Free Proteomics and Spectral Counting.
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.
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD, 20910, USA. angela.m.boutte.civ@mail.mil.
Angela M
AM
Boutté
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD, 20910, USA.
Shonnette F
SF
Grant
Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD, 20910, USA.
Jitendra R
JR
Dave
D000818:Animals; D015415:Biomarkers; D001921:Brain / Q000378:metabolism*; D000070642:Brain Injuries, Traumatic / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D013058:Mass Spectrometry; D020543:Proteome; D040901:Proteomics / Q000379:methods; D051381:Rats; D015203:Reproducibility of Results; D013057:Spectrum Analysis; D057188:Workflow
D015415:Biomarkers; D020543:Proteome
Biomarkers; Brain tissue; Electrophoresis; Mass spectrometry; Spectral counting; Traumatic brain injury