0000000000427146
AUTHOR
Olaf Jahn
Myelin: Methods for Purification and Proteome Analysis
Molecular characterization of myelin is a prerequisite for understanding the normal structure of the axon/myelin-unit in the healthy nervous system and abnormalities in myelin-related disorders. However, reliable molecular profiles necessitate very pure myelin membranes, in particular when considering the power of highly sensitive "omics"-data acquisition methods. Here, we recapitulate the history and recent applications of myelin purification. We then provide our laboratory protocols for the biochemical isolation of a highly pure myelin-enriched fraction from mouse brains and for its proteomic analysis. We also supply methodological modifications when investigating posttranslational modifi…
In-depth evaluation of software tools for data-independent acquisition based label-free quantification.
Label-free quantification (LFQ) based on data-independent acquisition workflows currently experiences increasing popularity. Several software tools have been recently published or are commercially available. The present study focuses on the evaluation of three different software packages (Progenesis, synapter, and ISOQuant) supporting ion mobility enhanced data-independent acquisition data. In order to benchmark the LFQ performance of the different tools, we generated two hybrid proteome samples of defined quantitative composition containing tryptically digested proteomes of three different species (mouse, yeast, Escherichia coli). This model dataset simulates complex biological samples con…
Quantitative and integrative proteome analysis of peripheral nerve myelin identifies novel myelin proteins and candidate neuropathy loci
Peripheral nerve myelin facilitates rapid impulse conduction and normal motor and sensory functions. Many aspects of myelin biogenesis, glia–axonal interactions, and nerve homeostasis are poorly understood at the molecular level. We therefore hypothesized that only a fraction of all relevant myelin proteins has been identified so far. Combining gel-based and gel-free proteomic approaches, we identified 545 proteins in purified mouse sciatic nerve myelin, including 36 previously known myelin constituents. By mass spectrometric quantification, the predominant P0, periaxin, and myelin basic protein constitute 21, 16, and 8% of the total myelin protein, respectively, suggesting that their relat…
Myelin Proteome Analysis: Methods and Implications for the Myelin Cytoskeleton
Myelin, the multilayered membrane that enwraps and insulates neuronal axons for fast signal propagation, is a plasma membrane specialization of oligodendrocytes and Schwann cells in the central and peripheral nervous system, respectively. Here we provide our lab protocols for the puri fi cation of myelin from mouse brains and for gel-based and gel-free proteomic applications, as well as a brief discussion with respect to our current knowledge of the myelin cytoskeleton.
Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5
AbstractOligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here we find that expression of the tetraspan-transmembrane protein CMTM5 (Chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and CNS myelin. Genetic disruption of the Cmtm5-gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5-deficiency causes an early-onset progressive axonopathy, which we also observe in global and in tamoxifen-induced olig…