Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry

This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with “shotgun”-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ-FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-β-d-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases

Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry

This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with “shotgun”-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ-FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-β-d-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases

Longitudinal Characterization of the Brain Proteomes for the Tg2576 Amyloid Mouse Model Using Shotgun Based Mass Spectrometry

Neurodegenerative disorders are often defined pathologically by the presence of protein aggregates, such as amyloid plaques composed of β-amyloid (Aβ) peptide in Alzheimer’s disease. Such aggregates are the result of abnormal protein accumulation and may lead to neuronal dysfunction and cell death. In this study, APPSWE transgenic mice (Tg2576), which overexpress the Swedish mutated form of human amyloid precursor protein (APP), were used to study the brain proteome associated with amyloid plaque deposition. The major aim of the study was to map and compare the Tg2576 model brain proteome profiles during pathology progression using a shotgun approach based on label free quantification with mass spectrometry. Overall, 1085 proteins were identified and longitudinally quantified. Principal component analysis (PCA) showed the appearance of the pathology onset between twelve and fifteen months, correlating with sharp amyloid plaque accumulation within the same ages. Cluster analysis followed by protein–protein interaction analysis revealed an age-dependent decrease in mitochondrial protein expression. We identified 57 significantly affected mitochondrial proteins, several of which have been reported to alter expression in neurological diseases. We also found ten proteins that are upregulated early in the amyloid driven pathology progression with high confidence, some of which are directly involved in the onset of mitochondrial apoptosis and may represent potential markers for use in human neurological diseases prognosis. Our results further contribute to identifying common pathological pathways involved in both aging and progressive neurodegenerative disorders enhancing the understanding of disease pathogenesis

Differences in regeneration of neurons, astrocytes and oligodendrocytes after injury.

<p>(A) Phase-contrast of an injured culture. The scalpel cut create clear injury (dashed line) with surrounding cells that remain unharmed. (B–D) Displaying the individual cell types' appearance 24 h after injury. (B) The neurons have regenerated new axons towards and along the laceration, without breaching the boundary of the cut. (C) Astrocytes reach towards and along the injury, and like neurons, do not grow into the laceration. (D) Oligodendrocytes are more hesitant in their regeneration of the injury and unless in its direct vicinity, does not grow along the cut but rather avert it. (E–M) The actin patterns of the cells 24 h after injury implicates it as an important regeneratory protein in wound healing. (E–G) The outgrowing new growth cones along the injury are highly reactive for actin. (H–J) The astrocytes have extended a multitude of actin-positive lamellipodia towards and along the cut at 24 h after injury. (K–M) Twenty-four hours after injury, oligodendrocytes in direct proximity to the injury have some lamellipodia-like extensions towards the cut, although they appear more reluctant in covering the cut compared to both neurons and astrocytes. Scale bars equal 50 µm (A–D) or 10 µm (E–M) and dashed lines represent the injury.</p

Previously shown functions for the proteins found in cells exclusively after injury.

<p>Previously shown functions for the proteins found in cells exclusively after injury.</p