Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome (original) (raw)
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Myelin: Methods for Purification and Proteome Analysis
Oligodendrocytes, 2019
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 modifications, RNA, or myelin from peripheral nerves. Notably, technical advancements in solubilizing myelin are beneficial for gel-based and gel-free myelin proteome analyses. We conclude this article by exemplifying the exceptional power of label-free proteomics in the mass-spectrometric quantification of myelin proteins.
Journal of Proteome Research, 2005
The myelin sheath is an electrically insulating layer that consists of lipids and proteins. It plays a key role in the functioning of the nervous system by allowing fast saltatory conduction of nerve pulses. Profiling of the proteins present in myelin is an indispensable prerequisite to better understand the molecular aspects of this dynamic, functionally active membrane. Two types of protein, the myelin basic protein and the proteolipid protein, account for nearly 85% of the protein content in myelin. Identification and characterization of the other "minor" proteins is, in this respect, a real challenge. In the present work, two proteomic strategies were applied in order to study the protein composition of myelin from the murine central nervous system. First, the protein mixture was separated by 2D-gel electrophoresis and, after spot excision and in-gel digestion, samples were analyzed by mass spectrometry. Via this approach, we identified 57 protein spots, corresponding to 38 unique proteins. Alternatively, the myelin sample was digested by trypsin and the resulting peptide mixture was further analyzed by off-line 2D-liquid chromatography. After the second-dimension separation (nanoLC), the peptides were spotted "on-line" onto a MALDI target and analyzed by MALDI TOF-TOF mass spectrometry. We identified 812 peptides by MALDI MS/MS, representing 93 proteins. Membrane proteins, low abundant proteins, and highly basic proteins were all represented in this shotgun proteomic approach. By combining the results of both approaches, we can present a comprehensive proteomic map of myelin, comprising a total of 103 protein identifications, which is of utmost importance for the molecular understanding of white matter and its disorders.
Myelin Proteomics: Molecular Anatomy of an Insulating Sheath
Molecular Neurobiology, 2009
Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelinassociated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies. Keyword Oligodendrocyte . Leukodystrophy . Myelin . Internode . Proteome . Proteomics . Cytoskeleton . Neurodegeneration . Proteolipid protein . Myelin basic protein the axons have been recently visualized in vivo in
Proteomic mapping provides powerful insights into functional myelin biology
Proceedings of the National Academy of Sciences, 2004
Myelin is a dynamic, functionally active membrane necessary for rapid action potential conduction, axon survival, and cytoarchitecture. The number of debilitating neurological disorders that occur when myelin is disrupted emphasizes its importance. Using highresolution 2D gel electrophoresis, mass spectrometry, and immunoblotting, we have developed an extensive proteomic map of proteins present in myelin, identifying 98 proteins corresponding to at least 130 of the Ϸ200 spots on the map. This proteomic map has been applied to analyses of the localization and function of selected proteins, providing a powerful tool to investigate the diverse functions of myelin. M yelin is a dynamic, functionally active membrane (1), the loss or damage of which results in serious neurological disorders including leukodystrophies, central and peripheral neuropathies, and inflammatory demyelinating diseases such as multiple sclerosis (2-4). Rapid and efficient action potential conduction in the nervous system depends on myelin, which traditionally has been viewed as a passive contributor to conduction by increasing internodal membrane resistance and decreasing membrane capacitance (5). However, recent work has revealed additional active roles for myelin in nervous system development and function. For example, myelin regulates axon diameter and the formation of axon microtubular networks, and it is a key player in ion channel clustering at nodes of Ranvier (6-11). In return, the axon regulates myelin gene expression (12) and oligodendrocyte survival (13). The functional coupling of myelin and axons is further illustrated by the transfer of phospholipids (14) and N-acetylaspartate (15) from the axon to the myelin sheath.
PROTEOMICS, 2003
Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2-DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins has slowed the identification of proteins from membrane-rich biological samples. Myelin is an unusually lipid-rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2-DE for the separation of myelin proteins. We have identified two detergents, the nonionic n-dodecyl b-D-maltoside and the zwitterionic amidosulfobetaine ASB-14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI-anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2-DE. We conclude that these detergents are effective tools for the 2-DE analysis of myelin, and that they may be more generally useful for the analysis of membrane-rich biological samples.
Journal of Cell Biology, 1990
The myelin-associated glycoproteins (MAG) are members of the immunoglobulin gene superfamily that function in the cell interactions of myelinating glial cells with axons. In this paper, we have characterized the structural features of these proteins. The disposition of MAG in the bilayer as a type 1 integral membrane protein (with an extracellularly disposed amino terminus, single transmembrane segment, and cytoplasmic carboxy terminus) was demonstrated in protease protection studies of MAG cotranslationally inserted into microsomes in vitro and in immunofluorescent studies with site specific antibodies. A genetically engineered MAG cDNA, which lacks the putative membrane spanning segment, was constructed and shown to encode a secreted protein.
Post-translational Modifications of Chicken Myelin Basic Protein Charge Components
Neurochemical Research, 2008
Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP's. Mammalian MBP's, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the posttranslational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial. The C1 component lacks any phosphorylated sites, a finding in agreement with the analysis of other MBP species. It also had a single methylation at R105 as did the components C2 and C3. The C2 component contains ten phosphorylated sites (S7,
Human myelin proteome and comparative analysis with mouse myelin
Proceedings of the National Academy of Sciences, 2009
Myelin, formed by oligodendrocytes (OLs) in the CNS, is critical for axonal functions, and its damage leads to debilitating neurological disorders such as multiple sclerosis. Understanding the molecular mechanisms of myelination and the pathogenesis of human myelin disease has been limited partly by the relative lack of identification and functional characterization of the repertoire of human myelin proteins. Here, we present a large-scale analysis of the myelin proteome, using the shotgun approach of 1-dimensional PAGE and liquid chromatography/tandem MS. Three hundred eight proteins were commonly identified from human and mouse myelin fractions. Comparative microarray analysis of human white and gray matter showed that transcripts of several of these were elevated in OL-rich white matter compared with gray matter, providing confidence in their detection in myelin. Comparison with other databases showed that 111 of the identified proteins/transcripts also were expressed in OLs, rather than in astrocytes or neurons. Comparison with 4 previous myelin proteomes further confirmed more than 50% of the identified proteins and revealed the presence of 163 additional proteins. A select group of identified proteins also were verified by immunoblotting. We classified the identified proteins into biological subgroups and discussed their relevance in myelin biogenesis and maintenance. Taken together, the study provides insights into the complexity of this metabolically active membrane and creates a valuable resource for future in-depth study of specific proteins in myelin with relevance to human demyelinating diseases.
Systematic approaches to central nervous system myelin
Cellular and Molecular Life Sciences, 2012
Rapid signal propagation along vertebrate axons is facilitated by their insulation with myelin, a plasma membrane specialization of glial cells. The recent application of 'omics' approaches to the myelinating cells of the central nervous system, oligodendrocytes, revealed their mRNA signatures, enhanced our understanding of how myelination is regulated and established that the protein composition of myelin is much more complex than previously thought. This review provides a meta-analysis of the >1200 proteins thus far identified by mass spectrometry in biochemically purified central nervous system myelin. Contaminating proteins are surprisingly infrequent according to bioinformatic prediction of subcellular localization and comparison with the transcriptional profile of oligodendrocytes. The integration of datasets also allowed the subcategorization of the myelin proteome into functional groups comprising genes that are coregulated during oligodendroglial differentiation. An unexpectedly large number of myelin-related genes cause -when mutated in humans -hereditary diseases affecting the physiology of the white matter. Systematic approaches to oligodendrocytes and myelin thus provide valuable resources for the molecular dissection of developmental myelination, glia-axonal interactions, leukodystrophies and demyelinating diseases.