Membrane Protein Identification in Rodent Brain Tissue Samples and Acute Brain Slices

Abstract

Acute brain slices are a sample format for electrophysiology, disease modeling, and organotypic cultures. Proteome analyses based on mass spectrometric measurements are seldom used on acute slices, although they offer high-content protein analyses and explorative approaches. In neuroscience, membrane proteins are of special interest for proteome-based analysis as they are necessary for metabolic, electrical, and signaling functions, including myelin maintenance and regeneration. A previously published protocol for the enrichment of plasma membrane proteins based on aqueous two-phase polymer systems followed by mass spectrometric protein identification was adjusted to the small sample size of single acute murine slices from newborn animals and the reproducibility of the results was analyzed. For this, plasma membrane proteins of 12 acute slice samples from six animals were enriched and analyzed by liquid chromatography-mass spectrometry. A total of 1161 proteins were identified, of which 369 were assigned to membranes. Protein abundances showed high reproducibility between samples. The plasma membrane protein separation protocol can be applied to single acute slices despite the low sample size and offers a high yield of identifiable proteins. This is not only the prerequisite for proteome analysis of organotypic slice cultures but also allows for the analysis of small-sized isolated brain regions at the proteome level.

Mass spectrometry and the cellular surfaceome

“…Substantial improvements were made by incorporating a countercurrent distribution (CD) (Schindler & Nothwang, 2006) through re‐extraction using fresh phases and pooling purified fractions. In a study on mouse brain membranes, single slices were subjected to two‐phase partitioning and CD was applied, resulting in 1378 identified proteins of which 804 (58%) were assigned to membranes, compared to 522 proteins (38%) that were not and a remaining 52 (4%) for which no subcellular localization information was available (Joost et al, 2019). To further increase specificity, ligands can be conjugated to the polymers used, a method known as two‐phase affinity partitioning (2PAP) (Schindler et al, 2006).…”

Oligodendrocyte Physiology and Pathology Function

“…To investigate mechanisms operant during de- and regeneration of the axon-oligodendrocyte-myelin compartment, and to develop effective MS treatment options, the following are required: (i) novel, dynamic technical platforms to investigate complex cell–cell interactions in a CNS-like microenvironment such as the oligodendrocyte-nanofiber platform described by Enz and colleagues [ 11 ]; (ii) unbiased evaluation systems to monitor disease progression and successful therapeutic interventions in pre-clinical models such as those described by Zhan an colleagues [ 12 ], Joost and colleagues [ 13 ], and Hochstrasser and colleagues [ 14 ]; and (iii) novel imaging modalities which would allow longitudinal studies as described by Khodanovich and colleagues [ 15 ]. Of note, a better understanding of the axon-oligodendrocyte-myelin compartment might lead to restorative therapies not just in MS but also other neuronal disorders such as Down Syndrome (reviewed by Reiche and colleagues [ 16 ]) or schizophrenia (reviewed by Raabe and colleagues [ 17 ]).…”