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Molecular Neurobiology

, Volume 40, Issue 1, pp 55–72 | Cite as

Myelin Proteomics: Molecular Anatomy of an Insulating Sheath

  • Olaf Jahn
  • Stefan Tenzer
  • Hauke B. Werner
Open Access
Article

Abstract

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 myelin-associated 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 

Introduction

Neuronal signal propagation in vertebrates is sped up by the electrical insulation of axons with an ensheathing, specialized glial plasma membrane: myelin. Myelination of axons reduces their transverse capacitance and increases their transverse resistance [1]. Insulation is achieved by the multilayered arrangement of the myelin membrane (Fig. 1) and its special molecular composition, mainly its very high lipid content. In myelinated axons, action potentials are restricted to periodically spaced small segments spared from coverage with myelin, termed the nodes of Ranvier [2]. In the central nervous system (CNS), any individual oligodendrocyte myelinates up to 50 axon segments, termed internodes [3]. Oligodendrocyte precursor cell division, migration, and regular alignment along the axons have been recently visualized in vivo in zebrafish [4], which today complement rodents as an important model organism for myelin research [5, 6, 7, 8]. Myelin formation proceeds with outgrowth and retraction of glial cell processes, target axon recognition, stabilization of cellular contacts, rapid biosynthesis and trafficking of lipid and protein constituents of the myelin membrane, and its organization as a multilayered structure around the axon [9, 10]. Once myelinated, axons become dependent on glial support [11]. Some of the molecules involved in myelin development and function are known but a detailed molecular picture has not been gained yet.
Fig. 1

CNS myelin. a Purified mouse brain myelin was one-dimensionally separated in a 4–12% Bis–Tris gradient gel using a morpholineethanesulfonic acid buffer system. Proteins were visualized by colloidal Coomassie staining. Bands constituted by abundant myelin proteins are annotated. b Schematic depiction of an oligodendrocyte myelinating an axon, cross-sections in the internodal and paranodal segments, and subcellular localization of myelin proteins. Structural proteins of compact myelin (middle), cytoskeletal and vesicular proteins located in uncompacted regions (right), and adhesion proteins mediating association with the axon (bottom) are shown. CNP 2′,3′-cyclic nucleotide phosphodiesterase, Cntn contactin, Caspr contactin-associated protein, Cx29 connexin 29 kDa, DM20 small splice isoform of PLP, ERM ezrin, radixin, moesin, IPL intraperiod line, JAM3 junctional adhesion molecule 3, MAG myelin-associated glycoprotein, MBP myelin basic protein, MDL major dense line, Necl nectin-like protein, NF155 neurofascin 155 kDa, OSP oligodendrocyte-specific protein/claudin-11, PLP proteolipid protein, Rab3 Ras-related protein Rab3, SIRT2 sirtuin 2

That CNS myelin is important for normal sensation, cognition, and motor function is obvious considering that myelin-related disorders often affect humans lethally. Besides the inflammatory demyelinating disease multiple sclerosis [12], there are genetically inherited disorders that affect CNS myelin, collectively termed leukodystrophies [13]. This heterogeneous group of diseases is characterized by the loss of motoric, sensory, and mental capabilities and the susceptibility to seizures. A detailed knowledge of the molecular expression profiles of oligodendrocytes and myelin will be crucial to understand the pathomechanisms of white matter diseases. For example, the mRNAs [14, 15, 16] and proteins expressed in cultivated oligodendrocytes [17] and oligodendroglial exosomes [18] have been recently examined. This review focuses on systematic analyses of the molecular composition of mammalian CNS myelin, while no such compendium of peripheral nervous system (PNS) myelin proteins has been published yet. Proteomics approaches to myelin provide a valuable resource to understand its biogenesis, function, and pathology. Although only a few comparative studies have been reported to date, novel insights into the molecular basis of myelin-related diseases are beginning to emerge.

A Myelin-Enriched Fraction from the Central Nervous System

A comparatively simple method is available for the isolation of a myelin-enriched fraction from the CNS. Biochemically, myelin is defined as the lightweight membranous material accumulating at the interface between 0.32 and 0.85 M sucrose after sequential ultracentrifugation combined with osmotic shocks [19, 20]. The most commonly used protocol starts from brain homogenate contained in 0.32 M sucrose as the top layer, “spinning-down” myelin to accumulate at the interface with the bottom 0.85 M sucrose layer. One valuable modification is “floating-up” of myelin starting from brain homogenate contained in a more concentrated sucrose solution as the bottom layer (0.85, 1.2, 1.44, or 2 M). During ultracentrifugation, myelin also accumulates at the interface between the upper 0.85 and 0.32 M sucrose layers, while other fractions of interest assemble at higher sucrose concentrations. This method allows the simultaneous isolation of other brain fractions such as rough microsomes [21] or axogliosomes [22, 23]. The lightweight fraction from the interphase between 0.32 and 0.85 M sucrose is the most frequently used one for biochemical and proteomic experiments. This fraction is enriched in the most abundant proteins of compact myelin, proteolipid protein (PLP), and myelin basic protein (MBP), and as revealed by electron microscopy, mainly contains multilamellar membranes with a periodicity comparable to that of myelin in native or perfused brains [24, 25]. However, we suggest to term this fraction “myelin-enriched” rather than “compact myelin”, as it also contains proteins from the noncompacted cytosolic channels in myelin (i.e., adaxonal and paranodal myelin) and proteins associated with the axonal membrane. Myelin purification is very reproducible across different laboratories, even when applied to different species (e.g., mouse–rat) or to mutant mice with altered myelin protein or lipid composition, such as Cnp Cre/+ *Fdft flox/flox [26], Ugt3a1 null [27], Arsa null [28], and Plp null [29] (see below). Thus, the method has proven to be very robust, explaining why the original protocol from the early 1970s is still in common use. It is generally assumed that myelin purification relies on its special lipid content and composition.

Myelin Lipids

The molecular composition of myelin differs from other plasma membranes in that it contains 70–75% of its dry weight as lipid, unusually high compared to other eukaryotic plasma membranes. Also, its molar ratio of lipids with approximately 2:2:1:1 for cholesterol/phospholipid/galactolipid/plasmalogen [30, 31] distinguishes myelin from other cellular membranes. The abundance of cholesterol within a membrane affects its biophysical properties, including fluidity and curving [32]. Cholesterol has earlier been identified as unusually enriched in myelin and constitutes 24–28% of the total myelin lipids [19]. That the cellular cholesterol supply is rate-limiting for myelin membrane biogenesis has been shown in mice lacking squalene synthase (also termed farnesyl diphosphate farnesyl transferase [FDFT]) exclusively in myelinating glia [26]. FDFT mediates a crucial step of cholesterol biosynthesis. CNS myelination is severely delayed in Cnp Cre/+ *Fdft1 flox/flox mice, and that any myelin made in these mice is likely due to compensatory cholesterol uptake from other cells [26].

The biophysical properties of myelin are also influenced by its unusually high concentration of the galactolipids galactosylceramide (GalC), its sulfated form 3-O-sulfogalactosylceramide (SGalC), and their hydroxylated forms GalC-OH and SGalC-OH. Together, they add up to 20–26% of total myelin lipids. Myelination is moderately delayed in mice lacking UDP-galactose:ceramide galactosyltransferase (Ugt3a1), an enzyme required for galactolipid synthesis. Additionally, impaired glia–axonal interactions at the paranodes were observed [27, 33, 34]. Paranodal disruption was at least partly due to the lack of SGalC and hydroxylated galactolipids, since the long-term integrity of the sodium channel domain of the nodes of Ranvier was also impaired in mice lacking galactosylceramide-3-O-sulfotransferase (Gal3st1), the enzyme converting GalC into SGalC [35, 36, 37], and late onset myelin degeneration was also reported for mice lacking fatty acid 2-hydroxylase (Fa2h), the enzyme hydroxylating GalC and SGalC [38]. Absence of functional arylsulfatase A (ARSA), the enzyme degrading SGalC, causes metachromatic leukodystrophy (MLD), illustrating that a regulated galactolipid metabolism is required for long-term integrity of the white matter. SGalC accumulation and many pathological features of MLD are modeled in Arsa null mice and in transgenic mice overexpressing Ugt3a1 or Gal3st1 in neurons or oligodendrocytes [28, 39, 40]. Sulfatide metabolism with respect to myelin and MLD pathology was recently reviewed [41].

Also, the plasmalogen class of phospholipids is associated with white matter disease. Plasmalogens are ether-linked (as opposed to ester-linked) phospholipids, the main species being ethanolamine–plasmalogen. They are ubiquitous structural components of mammalian cell membranes and amount to 12–15% of total myelin lipid [19] and, when processed by plasmalogen-selective phospholipase A2, give rise to the second messengers arachidonic acid and eicosanoids [42]. At low concentrations, these metabolites have trophic effects, but at high levels, they are cytotoxic and may induce inflammation [43]. The reactivity of the alkenyl ether bond makes plasmalogens more susceptible to oxidative reactions than their fatty acid ester analogs. Thus, myelin plasmalogens may act as endogenous antioxidants protecting cells from oxidative stress [44]. Disrupted activity of peroxisomal plasmalogen synthesizing enzymes results in peroxisomal biogenesis disorders such as rhizomelic chondrodysplasia punctata (RCDP) in which hypomyelination of the optic nerve has been observed. Decreased plasmalogen levels [45, 46] and increased levels of reactive oxygen species [47, 48] may also contribute to the demyelination in X-linked adrenoleukodystrophy caused by the mutated peroxisomal transporter ABCD1, suggesting that a normal plasmalogen metabolism may prevent peroxisomal- and myelin-related disease. Mice lacking dihydroxyacetonephosphate acyltransferase (DAPAT) model several aspects of the RCDP pathology, including optic nerve hypoplasia [49]. Interestingly, the association of flotillin-1 and contactin with plasmalogen-deficient brain membrane microdomains was diminished in DAPAT null mice [49], suggesting that the local concentration of membrane lipids dictates the association of particular proteins.

Association of Myelin Lipids and Proteins

Cholesterol assembles with galactolipids and plasmalogens within the plane of the membrane, but how they are enriched to the levels found in myelin is unknown. It has been suggested that lipids are targeted to future myelin membrane by their association with myelin-bound proteins [9]. SGalC appears to be an example to the contrary. SGalC is associated with myelin and lymphocyte protein (MAL) [50]. Lack of SGalC and lack of MAL lead to similar paranodal malformation [35, 51]. The subcellular trafficking of MAL, as well as its abundance in myelin, is determined by SgalC [28], whereas SGalC abundance is not altered in Mal null myelin [51]. It is likely that other myelin proteins are also incorporated into the sheath by attachment with future myelin membrane because of its special lipid composition. Thus, whether myelin proteins dictate the fate of lipids or vice versa may not be generalized. It appears likely that the association of both molecule classes results in each other’s control of abundance and trafficking.

That myelin lipids and proteins are closely associated was suggested earlier after the characterization of two types of protein fractions isolated from the white matter based on their resistance to aqueous or organic solvents or to enzymatic proteolysis. One fraction behaved as a lipid with regard to its solubility and was termed PLP [52, 53]. PLP was later identified to be the most abundant protein of mammalian CNS myelin. It has a high affinity to phospholipids and cholesterol [54, 55, 56], and impaired interactions of mutant PLP with membrane lipids are a likely key step in the molecular pathogenesis of the leukodystrophy Pelizaeus–Merzbacher disease [57]. The other fraction, termed trypsin-resistant protein residue, was insoluble in organic solvent and attached to the membrane lipid phosphatidylinositol phosphate [58, 59]. The application of extraction methods by Folch became commonly used to categorize myelin proteins according to their biophysical properties.

More recently, the myelin-enriched brain fraction has been chemically subfractionated by differential detergent extraction at low temperatures, resulting in distinct nonidentical but overlapping assemblies of myelin-associated proteins and lipids that were suggested to represent myelin subcompartments [60, 61]. Cholesterol- and galactolipid-rich membrane microdomains (also referred to as “lipid rafts”) have been suggested to deliver myelin proteins to the plasma membrane [62, 63, 64]. The relevance of applying the analysis of biochemical characteristics established for membrane microdomains to such a large structure as myelin has remained debated. However, it is widely accepted now that lipid-associated cell signaling molecules, such as the protein tyrosine kinase fyn, have central roles in myelination [65, 66].

In oligodendroglial processes, fyn is activated by axonal signals via integrin alpha6beta1 [67]. Among other fyn substrates [68, 69], the protein translation repressor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 upon phosphorylation is released from its binding site in the 3′UTR of mRNA encoding MBP [70], the second-most abundant myelin protein. hnRNP A2 binding represses translation during the translocation of MBP mRNA to distal sites of the cell [71] where newly translated MBP is directly incorporated into the extending oligodendroglial process [21, 72]. It is generally assumed that MBP mediates the adhesion of the cytoplasmic surfaces between the individual layers of compact myelin [73] via binding of its many basic residues with the negatively charged headgroups of membrane lipids. Indeed, membrane association of MBP is controlled by the membrane lipid phosphatidylinositol-(4,5)-bisphosphate [74, 75, 76]. For over 30 years, it has been known that MBP is highly heterogeneous due to alternative splicing and multiple post-translational modifications (PTMs) [77]. More recently, modern mass spectrometric techniques have been used to compare the PTMs of MBP from normal and multiple sclerosis brains with respect to methylation, phosphorylation, and arginine deimination [78]. PTM alterations affect charge, conformation, and hydrogen bonding of MBP, which may modulate its affinity to the myelin membrane and play a role in myelin compaction and in the pathogenesis of demyelinating diseases. MBP is the only myelin protein that has been shown to be essential for myelin formation, as became obvious with the analysis of the natural mouse mutant shiverer and the rat mutant long evans shaker [79, 80], which are severely hypomyelinated. Interestingly, mice lacking fyn are also hypomyelinated [81, 82], likely due to affected translational regulation of MBP expression [70, 83]. Together, a multitude of factors affects mRNA transcription and transport, translation at axonal contact sites, or membrane binding of MBP, and we speculate that several myelin proteins with yet unidentified roles affect MBP abundance and function.

Systematic Analysis of the CNS Myelin Protein Composition

The relative abundance of myelin proteins has previously been calculated based on their binding to Buffalo black [84], Fast green [85], or Coomassie blue [86] after separation in one-dimensional (1D) sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). In these measurements, a small number of proteins was determined to be extraordinarily abundant in CNS myelin. PLP and its smaller splice isoform DM20 accounted for 30–45% of total myelin protein, two of the four MBP splice isoforms for 22–35%, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) for 4–15%, and all remaining proteins for 5–25% [19, 85, 87, 88]. Similarly, PNS myelin is also dominated by two proteins, myelin protein zero (MPZ, P0) and MBP, which have been estimated to account for 50–70% and 15%, respectively [89]. In comparison, the most abundant proteins in a brain fraction enriched for synaptic vesicles are synaptobrevin 2 and synaptophysin, which constitute 8% and 10% of the total synaptic vesicle proteins, respectively, as revealed by quantitative immunoblotting [90]. How and why myelin proteins are enriched to their unusual relative abundance is unclear, considering that PLP and CNP are not essential for the formation of normal amounts of CNS myelin [29, 91, 92].

Various proteomic techniques have been applied towards the systematic protein composition analysis of the myelin-enriched fraction. Traditionally, first insights into proteomes of subcellular structures often come from two-dimensional (2D) protein maps generated by utilizing isoelectric focusing (IEF) with immobilized pH gradients in the first and SDS-PAGE in the second dimension (2D-IEF/SDS-PAGE) (Fig. 2a). Proteins of interest are then excised from the gel, proteolytically digested in situ, and finally, identified by mass spectrometry (MS) [93]. Due to its high resolving power, 2D-IEF/SDS-PAGE can be routinely applied for profiling of proteins from complex mixtures and, as protein integrity is retained, also leads to information on protein abundance and processing [94]. However, major shortcomings of 2D-IEF/SDS-PAGE concern a limited dynamic range, the display of basic and hydrophobic proteins, and—most importantly—the under-representation of membrane proteins. As myelin is dominated by MBP (a highly basic protein) and PLP (a hydrophobic tetraspan protein), incremental improvements in 2D-IEF/SDS-PAGE technology were required before the first 2D mapping of myelin was presented [95]. By using the zwitterionic detergent amidosulfobetaine-14 (ASB-14) instead of the most commonly used 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) [96], it was possible to solubilize myelin proteins much more effectively and to identify 98 proteins (91 by MS and seven by immunoblotting) in the myelin-enriched fraction from mouse CNS [95]. This crucial effect of the solubilization conditions is further underscored by two more recent 2D-IEF/SDS-PAGE mapping studies of similar input material. Thirty-eight myelin-associated proteins were identified in one study after CHAPS solubilization [97], but 131 proteins were identified in another study with ASB-14 [25]. Thus, at least in the presence of appropriate detergents, myelin can now be considered as well accessible by 2D-IEF/SDS-PAGE, which not only facilitates protein cataloging but also paves the way for differential myelin proteomics on the basis of the 2D differential fluorescence intensity gel electrophoresis technology (2D-DIGE, see below). It is important to note that all conventional 2D mapping approaches mentioned above failed to appropriately display relatively abundant transmembrane myelin marker proteins such as PLP, myelin-associated glycoprotein (MAG) [98], myelin oligodendrocyte glycoprotein (MOG) [99], tetraspanin 2 [100], M6B [101], or oligodendrocyte-specific protein (OSP/claudin-11) [102, 103, 104]. A potential remedy is to perform the first dimension separation as nonequilibrium pH gradient electrophoresis for the 2D mapping of myelin proteins [105]. However, although this method appeared promising particularly for displaying highly basic proteins, it did not get as popular as 2D-IEF/SDS-PAGE with immobilized pH gradients, mainly due to limitations in reproducibility and resolution.
Fig. 2

Gel-based myelin proteome maps. Purified mouse brain myelin was two-dimensionally separated in different gel systems. Proteins were visualized by colloidal Coomassie staining, and spots constituted by selected myelin proteins are indicated. a 2D-IEF/SDS-PAGE with IEF in a nonlinear pH gradient (pH 3–10) as the first and gradient SDS-PAGE (8–16% acrylamide) as the second dimension. To improve resolution, myelin was delipidated and precipitated by a methanol/chloroform treatment prior to IEF [25]. b 2D-16-BAC/SDS-PAGE with separation in a 16-BAC gel (10% acrylamide) as the first and gradient SDS-PAGE (8–16% acrylamide) as the second dimension. c 2D-CTAB/SDS-PAGE with separation in a CTAB gel (10% acrylamide) as the first and gradient SDS-PAGE (8–16% acrylamide) as the second dimension. To deplete soluble and membrane-associated proteins, myelin was subjected to a multistep wash procedure before separation [25]. 16-BAC and CTAB resulted in similar spot patterns. 2D-IEF/SDS-PAGE provides good resolution but basic, hydrophobic, and transmembrane proteins are under-represented. 2D-16-BAC/SDS-PAGE and 2D-CTAB/SDS-PAGE lead to efficient representation of basic, hydrophobic, and transmembrane proteins but have a lower resolution since separation occurs by protein size in both dimensions

More complete proteome coverage while retaining the benefits of displaying intact proteins can be reached by the additional use of alternative 2D gel systems. Here, the charge-dependent separation in the first dimension (i.e., the IEF) is replaced by a size-dependent separation in the presence of cationic detergents such as 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC; Fig. 2b) [106] or cetyltrimethylammonium bromide (CTAB; Fig. 2c) [107]. Due to the similar separation principle in both dimensions, proteins are typically dispersed along a diagonal rather than distributed over the entire gel area. Accordingly, these gel systems have a lower resolution compared to 2D-IEF/SDS-PAGE, but can resolve highly basic and even membrane-spanning proteins [108]. Application of 2D-16-BAC/SDS-PAGE to mouse CNS myelin resulted in the identification of 62 proteins and readily enabled displaying of the transmembrane myelin proteins PLP, MAG, MOG, and OSP/claudin-11 [25]. Thus, the combination of 2D-IEF/SDS-PAGE and 2D-16-BAC/SDS-PAGE has, so far, yielded the most comprehensive gel-based proteome compendium of mouse CNS myelin, consisting of 162 nonredundant proteins [25]. Further technical refinements of the method were established in a recent systematic evaluation of five different cationic detergents for the 2D gel electrophoresis of myelin proteins. Here, 16-BAC was the most effective agent for the separation of myelin proteins in the first dimension, while CTAB was most effective for their solubilization [109, 110]. As resolution improves, 2D gel electrophoresis with cationic detergents may be combined with the DIGE technology as a future tool for monitoring abundance changes of highly basic and membrane-spanning myelin proteins [111].

To overcome the limitations of gel-based proteomic methods, in particular those of 2D-IEF/SDS-PAGE, gel-free techniques, commonly referred to as shotgun approaches, have emerged in recent years [93, 112]. Here, separation at the level of intact proteins is omitted and the protein preparation is proteolytically digested at the expense of information related to protein integrity, such as protein size and charge. Separation takes place at the level of proteolytic peptides before interfacing with MS. The tremendous complexity of such peptide mixtures requires a high resolving power and is, therefore, often addressed by the application of 2D liquid chromatography (2D-LC), usually consisting of strong cation exchange in the first and reversed-phase chromatography in the second dimension. In the first application of shotgun proteomics to the myelin-enriched fraction from the mouse CNS [97], 93 proteins were identified resulting—by combination with 2D-IEF/SDS-PAGE (see above)—in a myelin proteome compendium consisting of 103 proteins. The application of a similar shotgun approach to a myelin-enriched fraction from rat CNS led to the identification of 97 myelin proteins [23]. Both shotgun approaches yielded quite a high overlap of approximately 50% with the so far most comprehensive gel-based library [25] and contained transmembrane myelin proteins such as PLP, MAG, and MOG.

Relative Abundance of Myelin Proteins

To understand myelin biogenesis and pathology, a comprehensive knowledge of the proteins associated with myelin is a prerequisite. We have confirmed and expanded the previous myelin protein compendia by applying nanoscale 1D ultra performance liquid chromatography (1D-UP-LC) separation coupled to detection with a quadrupole time-of-flight (QTOF) mass spectrometer (Tenzer et al., unpublished). Data were acquired by LC-MS using an alternating low (MS) and elevated (MSE) collision energy mode of acquisition (LC-MSE), which allows simultaneous identification and label-free relative quantification of the proteins in the sample [113, 114, 115]. The identified peptides were annotated to a total of 294 myelin-associated proteins (Table 1) based on a minimum of two peptides per protein with an effective false-positive rate of <0.2%. They showed a very good overlap of 141 proteins that were also detected in previous myelin proteome analyses and included several established myelin markers (Table 1 and Fig. 3). We have calculated the relative abundance of the myelin-associated proteins based on the average intensity of the three most abundant peptides per protein. In the few cases where only two peptides were identified, their average intensity was used. Strikingly, PLP, MBP, and CNP constituted only 17%, 8%, and 4% of the total myelin-associated proteins, respectively (Fig. 4). All previously known myelin proteins together constituted 35%, while newly identified myelin-associated proteins accounted for 65%. These quantifications take into question previous estimates based on conventional techniques (Fig. 4b and see above). We suggest that the complexity of myelin protein composition has been overlooked because low abundant proteins did not constitute significant bands on gels when compared to the highly abundant PLP and MBP due to limitations concerning gel separation and/or protein staining.
Table 1

The CNS myelin proteome

Protein name

ID

Gene

Reference

A: Known myelin proteins

CD81

P35762

Cd81

E

CD9

P40240

Cd9

ND

Claudin 11, OSP

Q60771

Cldn11

B,S,T,E

CNP

P16330

Cnp

W,B,S,R,T,E

Contactin 1

P12960

Cntn1

B,S,R,T,E

Ermin

Q5EBJ4

Ermn

E

Ezrin

P26040

Ezr

W,T,E

Glycoprotein M6B

P35803

Gpm6b

E

Myelin and lymphocyte protein

O09198

Mal

ND, T (blot)

Myelin-associated glycoprotein

P20917

Mag

B,S,R,E

Myelin basic protein

P04370

Mbp

W,B,S,V,R,E

Myelin oligodendrocyte glycoprotein

Q61885

Mog

B,S,R,E

Myelin protein zero, P0

P27573

Mpz

R

Myelin proteolipid protein

P60202

Plp1

B,S,R,T,E

Myelin/oligodendrocyte basic protein

Q9D2P8

Mobp

E

Necl1, Ig superfamily member 4b

Q99N28

Cadm3

S

Necl4, Ig superfamily member 4c

Q8R464

Cadm4

S,E

Neural cell adhesion molecule 1

P13595

Ncam1

W,S,R,T,E

Neurofascin

Q810U3

Nfasc

B,R,E

Oligodendrocyte myelin glycoprotein

Q63912

Omg

ND

Opalin, TMP10

Q7M750

Opalin

R,E

Plasmolipin

Q9DCU2

Pllp

E

Ras-related protein Rab 3A

P63011

Rab3a

E

Ras-related protein Rab 3C

P62823

Rab3c

E

Sirtuin 2

Q8VDQ8

Sirt2

W,S,V,R,T,E

Tetraspanin 2

Q922J6

Tspan2

E

B: Newly identified myelin-associated proteins

14-3-3 protein beta

Q9CQV8

Ywhab

E

14-3-3 protein epsilon

P62259

Ywhae

S,R,E

14-3-3 protein eta

P68510

Ywhah

E

14-3-3 protein gamma

P61982

Ywhag

W,V,R,T,E

14-3-3 protein sigma, stratifin

O70456

Sfn

E

14-3-3 protein theta

P68254

Ywhaq

E

14-3-3 protein zeta delta

P63101

Ywhaz

S,R,E

Actin α cardiac muscle 1

P68033

Actc1

E

Actin α1

P68134

Acta1

E

Actin α

P62737

Acta2

R,E

Actin β

P60710

Actb

W,S,V,R,T,E

Actin γ1

P63260

Actg1

B,E

Actin γ2

P63268

Actg2

E

Acyl-CoA thioesterase 7

Q91V12

Acot7

R,E

ADAM 23

Q9R1V7

Adam23

E

Adenylate cyclase associated 1

P40124

Cap1

T

ADP ribosylation factor 1

P84078

Arf1

S,T,E

ADP ribosylation factor 2

Q8BSL7

Arf2

E

ADP ribosylation factor 3

P61205

Arf3

E

ADP ribosylation factor 4

P61750

Arf4

E

ADP ribosylation factor 5

P84084

Arf5

E

ADP ribosylation factor 6

P62331

Arf6

W,E

Aldehyde dehydrogenase 1A1

P24549

Aldh1a1

E

Aldolase A, fructose-bisphosphate

P05064

Aldoa

W,S,V,R,T,E

Aldolase C, fructose bisphosphate

P05063

Aldoc

R,T,E

Amphiphysin 2, bridging integrator 1

O08539

Bin1

E

Anillin

Q8K298

Anln

R,E

Annexin A2

P07356

Anxa2

E

Annexin A6

P14824

Anxa6

R,T

Argininosuccinate synthase 1

P16460

Ass1

B,E

α-Synuclein

O55042

Snca

E

Band 4.1 like protein 3

Q9WV92

Epb4.1l3

E

Brain acid soluble protein 1, NAP22

Q91XV3

Basp1

S,E

Breast carcinoma amplified seq 1

Q80YN3

Bcas1

S,E

β-Synuclein

Q91ZZ3

Sncb

E

Ca++ ATPase 1

Q3TSK3

Atp2b1

E

Ca++ ATPase 2

Q9R0K7

Atp2b2

E

Ca++ ATPase 3

Q0VF55

Atp2b3

E

Ca++ ATPase 4

Q6Q476

Atp2b4

E

Calmodulin CaM

P62204

Calm3

S,V,E

Calnexin

P35564

Canx

B,R

Calpain 5

O08688

Capn5

T

CaM kinase IIα

P11798

Camk2a

E

CaM kinase IIβ

P28652

Camk2b

E

CaM kinase IIδ

Q6PHZ2

Camk2d

E

CaM kinase IIγ

Q923T9

Camk2g

E

Cannabinoid receptor interacting 1

Q5M8N0

Cnrip1

W,E

Carbonic anhydrase 2

P00920

Car2

W,S,T,E

CD47, integrin signal transducer

Q61735

Cd47

E

CD82

P40237

Cd82

E

CDGSH iron sulfur domain 1

Q91WS0

Cisd1

E

Cell cycle exit and neuronal diff.

Q9JKC6

Cend1

E

Cell division control protein 42

P60766

Cdc42

W,E

Centractin α

P61164

Actr1a

W

Choline transporter CD92

Q6X893

Slc44a1

E

Clathrin heavy chain

Q68FD5

Cltc

B,R,E

Cofilin 1

P18760

Cfl1

S,V,T,E

Cofilin 2

P45591

Cfl2

E

Contactin associated protein 1

O54991

Cntnap1

B,E

Coronin 1C

Q9WUM4

Coro1c

E

Creatine kinase brain

Q04447

Ckb

W,S,V,R,T,E

Crystallin α2

P23927

Cryab

W,S,T,E

Cyclophilin A

P17742

Ppia

W,S,V,E

Cysteine and glycine rich protein 1

P97315

Csrp1

E

Cytokeratin 1

P04104

Krt1

E

Cytokeratin 1B

Q6IFZ6

Krt77

E

Cytokeratin 5

Q922U2

Krt5

E

Cytokeratin 6A

P50446

Krt6a

E

Cytokeratin 6G

Q9R0H5

Krt71

E

Cytokeratin 10

P02535

Krt10

R,E

Cytokeratin 16

Q9Z2K1

Krt16

E

Desmin

P31001

Des

E

Destrin

Q9R0P5

Dstn

E

Dihydropyrimidinase-like 1,CRMP1

P97427

Crmp1

E

Dihydropyrimidinase-like 2,CRMP2

O08553

Dpysl2

W,B,S,V,R,T,E

Dihydropyrimidinase-like 3,CRMP4

Q62188

Dpysl3

E

Dihydropyrimidinase-like 4,CRMP3

O35098

Dpysl4

E

Dipeptidylpeptidase 6

Q9Z218

Dpp6

T

Down syndrome cell adhesion like 1

Q8R4B4

Dscaml1

E

Dynactin 2

Q99KJ8

Dctn2

V

Dynamin 1

P39053

Dnm1

W,B,R,T,E

Dynamin 2

P39054

Dnm2

E

Dynamin 3

Q8BZ98

Dnm3

R

Dynein heavy chain

Q9JHU4

Dync1h1

R

Ectonucleotide pyrophosphatase 6

Q8BGN3

Enpp6

E

EH domain containing protein 1

Q9WVK4

Ehd1

B,S,T,E

EH domain containing protein 3

Q9QXY6

Ehd3

B,E

EH domain containing protein 4

Q9EQP2

Ehd4

E

Elongation factor 1α1

P10126

Eef1a1

W,B,S,R,E

Elongation factor 1α2

P62631

Eef1a2

W,B,E

Elongation factor 1β

O70251

Eef1b2

T

Elongation factor 2

P58252

Eef2

T

Endonuclease domain containing 1

Q8C522

Endod1

E

Enolase 1, non-neuronal

P17182

Eno1

W,B,S,V,T,E

Enolase 2, neuronal

P17183

Eno2

W,S,V,T,E

Enolase 3, muscle

P21550

Eno3

E

Fascin

Q61553

Fscn1

W,E

Fatty acid synthase

P19096

Fasn

R

FK506 binding protein 1a

P26883

Fkbp1a

S,E

Flotillin 1

O08917

Flot1

ND, T (blot)

G protein α transducing 1

P20612

Gnat1

E

G protein α transducing 2

P50149

Gnat2

E

G protein α transducing 3

Q3V3I2

Gnat3

E

G protein α11

P21278

Gna11

E

G protein α14

P30677

Gna14

E

G protein αI1

B2RSH2

Gnai1

E

G protein αI2

P08752

Gnai2

E

G protein αI3

Q9DC51

Gnai3

E

G protein αO1

P18872

Gnao1

S,T,E

G protein αO2

P18873

Gna0

B,T,E

G protein αq

P21279

Gnaq

T,E

G protein αS

P63094

Gnas

S,E

G protein αS olfactory

Q8CGK7

Gnal

E

G protein β1

P62874

Gnb1

W,S,V,T,E

G protein β2

P62880

Gnb2

W,V,R,E

G protein β3

Q61011

Gnb3

E

G protein β4

P29387

Gnb4

W,E

G protein β5

P62881

Gnb5

W

G protein γ12

Q9DAS9

Gng12

E

GAPDH

P16858

Gapdh

W,S,V,T,E

GAPDH sperm

Q64467

Gapdhs

E

Gelsolin

P13020

Gsn

V,R,T

Glial fibrillary acidic protein

P03995

Gfap

W,B

Glucose-6-phosphate isomerase

P06745

Gpi1

B,R,E

Glutamate oxaloacetate transaminase

P05201

Got1

E

Glutamate transporter GLAST

P56564

Slc1a3

E

Glutamate transporter GLT1

P43006

Slc1a2

R,E

Glutamine synthetase

P15105

Glul

W,S,V,T,E

Glutathione S transferase micros. 3

Q9CPU4

Mgst3

E

Glutathione S transferase Mu1

P10649

Gstm1

W,E

Glutathione S transferase Mu2

P15626

Gstm2

E

Glutathione S transferase Mu6

O35660

Gstm6

E

Glutathione S transferase P1

P19157

Gstp1

S,V,E

Glutathione S transferase P2

P46425

Gstp2

T

Growth associated protein 43

P06837

Gap43

T

GTPase Ran

P62827

Ran

E

H+/K+ ATPase α1

Q64436

Atp4a

E

H+/K+ ATPase α2

Q9Z1W8

Atp12a

E

Heat shock 70 kDa protein 1A

Q61696

Hspa1a

E

Heat shock 70 kDa protein 1B

P17879

Hspa1b

R,E

Heat shock 70 kDa protein 1L

P16627

Hspa1l

E

Heat shock 70 kDa protein 2

P17156

Hspa2

W,B,E

Heat shock 70 kDa protein 4

Q61316

Hspa4

T

Heat shock 70 kDa protein 5

P20029

Hspa5

W,T,E

Heat shock 70 kDa protein 8

P63017

Hspa8

W,B,S,V,R,T,E

Heat shock 70 kDa protein 12A

Q8K0U4

Hspa12a

E

Heat shock protein 90 kDa αA1

P07901

Hsp90aa1

B,E

Heat shock protein 90 kDa αB1

P11499

Hsp90ab1

T,E

Hexokinase 1

P17710

Hk1

T,E

Hexokinase 2

O08528

Hk2

E

Ig superfamily member 8, EWI-2

Q8R366

Igsf8

B,S,R,E

Internexin α, Neurofilament 66 kDa

P46660

Ina

W,B,V,R,T,E

Junctional adhesion molecule C

Q9D8B7

Jam3

S,E

K+ channel A1

P16388

Kcna1

E

K+ channel A2

P63141

Kcna2

E

K+ channel A3

P16390

Kcna3

E

K+ channel B2

P62482

Kcnab2

E

Lactate dehydrogenase A

P06151

Ldha

T,E

Lactate dehydrogenase B

P16125

Ldhb

W,T,E

Lactate dehydrogenase C

P00342

Ldhc

E

Leucine rich repeat containing 57

Q9D1G5

Lrrc57

E

Leucine rich repeat LGI 3

Q8K406

Lgi3

E

Limbic system associated membrane

Q8BLK3

Lsamp

S,E

Lymphocyte antigen 6H

Q9WUC3

Ly6h

E

Macrophage migration inhibitory factor

P34884

Mif

W,S,E

Malate dehydrogenase

P14152

Mdh1

W,S,V,T,E

MARCKS related protein

P28667

Marcksl1

S

Microtubule associated protein 1B

P14873

Mtap1b

E

Microtubule associated protein 6

Q7TSJ2

Mtap6

E

Microtubule associated protein tau

P10637

Mapt

E

Mitogen activated protein kinase 1

P63085

Mapk1

E

Moesin

P26041

Msn

W,E

Munc 18, syntaxin binding protein 1

O08599

Stxbp1

B,R,T,E

Myosin Id

Q5SYD0

Myo1d

B,R,E

Na+/K+ ATPase α1

Q8VDN2

Atp1a1

B,S,R,E

Na+/K+ ATPase α2

Q6PIE5

Atp1a2

B,R,E

Na+/K+ ATPase α3

Q6PIC6

Atp1a3

B,R,E

Na+/K+ ATPase α4

Q9WV27

Atp1a4

E

Na+/K+ ATPase β1

P14094

Atp1b1

B,S,R,E

Na+/K+ ATPase β3

P97370

Atp1b3

E

Na+/K+/Cl cotransporter

P55012

Slc12a2

E

N-ethylmaleimide sensitive fusion

P46460

Nsf

W,B,R,T,E

Neurocalcin δ

Q91X97

Ncald

S

Neurofilament H

P19246

Nefh

W,B,E

Neurofilament L

P08551

Nefl

W,B,V,R,E

Neurofilament M

P08553

Nefm

B,R,E

Neuroligin 1

Q99K10

Nlgn1

T

Neurotrimin

Q99PJ0

Hnt

E

N-myc downstream regulated

Q62433

Ndrg1

W,S,V,T,E

Nucleoside diphosphate kinase A

P15532

Nme1

W,S,T,E

Nucleoside diphosphate kinase B

Q01768

Nme2

W,S,T,E

Parkinson disease protein 7

Q99LX0

Park7

E

Peroxiredoxin 1

P35700

Prdx1

W,V,R,T,E

Peroxiredoxin 2

Q61171

Prdx2

W,V,E

Peroxiredoxin 5

P99029

Prdx5

S,E

Phosphatidylethanolamine binding 1

P70296

Pebp1

W,V,E

Phosphatidylinositol transfer α

P53810

Pitpna

W

Phosphofructokinase 1

P47857

Pfkm

E

Phosphoglycerate dehydrogenase

Q61753

Phgdh

W

Phosphoglycerate kinase 1

P09411

Pgk1

S,V,T,E

Phosphoglycerate kinase 2

P09041

Pgk2

E

Phosphoglycerate mutase 1

Q9DBJ1

Pgam1

W,S,T,E

Phospholipase Cβ1

Q9Z1B3

Plcb1

W,T,E

Phosphoserine aminotransferase

Q99K85

Psat1

R

Prion protein

P04925

Prnp

E

Prion protein dublet

Q9QYT9

Prnd

E

Programmed cell death 6 interacting

Q9WU78

Pdcd6ip

W

Prohibitin

P67778

Phb

W,B,E

Prohibitin 2

O35129

Phb2

E

Protein arginine deiminase 2

Q08642

Padi2

E

Protein disulfide isomerase A3

P27773

Pdia3

W,T

Protein kinase Cγ

P63318

Prkcc

E

Pyruvate kinase isozyme M2

P52480

Pkm2

W,S,V,T,E

Quinoid dihydropteridine reductase

Q8BVI4

Qdpr

E

Rab 1A

P62821

Rab1

E

Rab 1B

Q9D1G1

Rab1b

E

Rab 2A

P53994

Rab2a

R,E

Rab 2B

P59279

Rab2b

E

Rab 3B

Q9CZT8

Rab3b

E

Rab 3D

P35276

Rab3d

E

Rab 4A

P56371

Rab4a

E

Rab 4B

Q91ZR1

Rab4b

E

Rab 5C

P35278

Rab5c

E

Rab 7A

P51150

Rab7

R

Rab 8A

P55258

Rab8a

E

Rab 8B

P61028

Rab8b

E

Rab 10

P61027

Rab10

S,E

Rab 12

P35283

Rab12

E

Rab 13

Q9DD03

Rab13

E

Rab 14

Q91V41

Rab14

E

Rab 15

Q8K386

Rab15

E

Rab 18

P35293

Rab18

E

Rab 26

Q504M8

Rab26

E

Rab 30

Q923S9

Rab30

E

Rab 35

Q6PHN9

Rab35

E

Rab 37

Q9JKM7

Rab37

E

Rab 39B

Q8BHC1

Rab39b

E

Rab 43

Q8CG50

Rab43

E

Rab GDP dissociation inhibitor α

P50396

Gdi1

W,S,R,T,E

Rab GDP dissociation inhibitor β

Q61598

Gdi2

W,T,E

Rac1

P63001

Rac1

S,R,E

Rac2

Q05144

Rac2

E

Rac3

P60764

Rac3

E

Radixin

P26043

Rdx

W,E

Ras-related protein Ral A

P63321

Rala

B,E

Ras-related protein Ral B

Q9JIW9

Ralb

E

Ras-related protein Rap 1A

P62835

Rap1a

W,S,R,T,E

Ras-related protein Rap 1B

Q99JI6

Rap1b

E

Ras-related protein Rap 2a

Q80ZJ1

Rap2a

R

Reticulon 3

Q9ES97

Rtn3

R

Rho GDP dissociation inhibitor 1

Q99PT1

Arhgdia

V,T

RhoA

Q9QUI0

Rhoa

E

RhoB

P62746

Rhob

T,E

RhoC

Q62159

Rhoc

E

RhoG

P84096

Rhog

E

S-100β

P50114

S100b

R

Septin 2

P42208

Sept2

W,B,S,T,E

Septin 4

P28661

Sept4

W,E

Septin 7

O55131

Sept7

W,B,S,R,T,E

Septin 8

Q8CHH9

Sept8

W,B,S,V,R,T,E

Septin 11

Q8C1B7

Sept11

E

Sideroflexin 3

Q91V61

Sfxn3

E

Soluble NSF attachment protein α

Q9DB05

Napa

W

Soluble NSF attachment protein β

P28663

Napb

W,E

Soluble NSF attachment protein γ

Q9CWZ7

Napg

W

Spectrin α2

P16546

Spna2

B,T,E

Spectrin β2

Q62261

Spnb2

R,E

Stress induced phosphoprotein 1

Q60864

Stip1

W,T

Superoxide dismutase

P08228

Sod1

W,S

Synapsin 1

O88935

Syn1

W,E

Synapsin 2

Q64332

Syn2

W,E

Synaptic vesicle membrane protein

Q62465

Vat1

R,T

Synaptobrevin 2

P63044

Vamp2

E

Synaptobrevin 3

P63024

Vamp3

E

Synaptophysin

Q62277

Syp

E

Synaptosomal associated protein 23

O09044

Snap23

E

Synaptosomal associated protein 25

P60879

Snap25

W,S,V,R,E

Synaptotagmin 1

P46096

Syt1

E

Synaptotagmin 5

Q9R0N5

Syt5

E

Syndapin 1

Q61644

Pacsin1

W,E

Syntaxin 1A

O35526

Stx1a

E

Syntaxin 1B

P61264

Stx1b

S,R,E

T-complex 1α

P11983

Tcp1

W

T-complex 1β

P80314

Cct2

W

T-complex 1δ

P80315

Cct4

R

T-complex 1ε

P80316

Cct5

W

T-complex 1γ

P80318

Cct3

W

Thy 1 membrane glycoprotein

P01831

Thy1

W,S,R,E

Transgelin 3

Q9R1Q8

Tagln3

W,E

Transitional ER ATPase

Q01853

Vcp

W,T,E

Transketolase

P40142

Tkt

W,B,S,T,E

Triosephosphate isomerase

P17751

Tpi1

S,E

Tubulin α1A

P68369

Tuba1a

W,B,R,E

Tubulin α1B

P05213

Tuba1b

W,S,V,T,E

Tubulin α1C

P68373

Tuba1c

E

Tubulin α3A

P05214

Tuba3a

E

Tubulin α4A

P68368

Tuba4a

E

Tubulin α8

Q9JJZ2

Tuba8

E

Tubulin β2A

Q7TMM9

Tubb2a

T,E

Tubulin β2B

Q9CWF2

Tubb2b

E

Tubulin β2C

P68372

Tubb2c

W,B,S,R,E

Tubulin β3

Q9ERD7

Tubb3

E

Tubulin β4

Q9D6F9

Tubb4

W,B,S,V,R,E

Tubulin β5

P99024

Tubb5

E

Tubulin β6

Q922F4

Tubb6

R,E

Tubulin polymerization promoting

Q7TQD2

Tppp

W,E

Tubulin polymerization promoting 3

Q9CRB6

Tppp3

S,E

Ubiquitin

P62991

Ub

W,S,E

Ubiquitin activating enzyme E1

Q02053

Uba1

T

Ubiquitin C-terminal hydrolase L1

Q9R0P9

Uchl1

W,T,E

Vacuolar ATP synthase A

P50516

Atp6v1a

W,E

Vacuolar ATP synthase B, brain

P62814

Atp6v1b2

W,E

Vacuolar ATP synthase C

Q9Z1G3

Atp6v1c1

T,E

Vacuolar ATP synthase E1

P50518

Atp6v1e1

T,E

Vimentin

P20152

Vim

E

Visinin like protein 1

P62761

Vsnl1

S,R,E

Visinin like protein 3

P62748

Hpcal1

S

WD repeat protein 1

O88342

Wdr1

W

Proteins identified in purified CNS myelin by MS

ID Swissprot or Trembl accession, Gene official NCBI Entrez gene name, Reference and method of detection, T 2D-IEF/SDS-PAGE or immunoblotting [95], V 2D-IEF/SDS-PAGE [97], W 2D-IEF/SDS-PAGE [25], B 2D-16-BAC/SDS-PAGE [25], R shotgun [23], S shotgun [97], E LC-MSE (Tenzer et al., unpublished), ND not detected by MS

Fig. 3

Assembling a compendium of myelin proteins. a The number of proteins identified by MS in different approaches to the CNS myelin proteome is plotted. The total number of myelin-associated proteins is unknown. Transmembrane proteins (black) have been categorized based on prior experimental studies or have been predicted using TMHMM and Phobius software. Proteins associated with mitochondria, which copurify with myelin, were omitted. T 2D-IEF/SDS-PAGE [95], V 2D-IEF/SDS-PAGE [97], W 2D-IEF/SDS-PAGE [25], B 2D-16-BAC/SDS-PAGE [25], R shotgun [23], S shotgun [97], E LC-MSE (Tenzer et al., unpublished). b Venn diagram comparing the number of myelin-associated proteins identified by MS after gel separation [25, 95, 97], previous gel-free shotgun approaches by LC/LC-MS/MS [23, 97], with those identified by LC-MSE (Tenzer et al., unpublished). Note the high overlap of proteins identified independent of the technique used. c Venn diagram showing our own experience with the identification of myelin-associated proteins by MS after combined 2D-IEF/SDS-PAGE and 2D-16-BAC/SDS-PAGE separation [25] or by LC-MSE with known myelin proteins according to the literature

Fig. 4

Relative abundance of myelin proteins. a The abundance of known myelin proteins was determined by LC-MSE. Note that known myelin proteins constitute less than 50% of the total myelin protein. Mitochondrial proteins were not considered. b Comparison of myelin protein abundance as quantified by LC-MSE with previous estimates based on band intensity after 1D-PAGE and various protein staining techniques [19, 85, 87, 88]. Note that the abundance of PLP and MBP was previously overestimated because low abundant proteins did not constitute significant bands due to limitations in the resolving power of the 1D gels and in the dynamic range of protein staining. c Simulated 2D map of myelin-associated proteins identified by LC-MSE. Proteins are indicated as dots at their molecular weight and isoelectric point as predicted from the amino acid sequence. The size of each dot reflects the relative abundance as determined by LC-MSE. Myelin-associated proteins without transmembrane domains are shown in blue and transmembrane proteins in green, the latter being usually under-represented or absent from conventional 2D gels. Mitochondrial proteins are shown in gray. The red frame indicates the portion of proteins that can be reproducibly displayed by 2D-IEF/SDS-PAGE (see Fig. 2a)

We conclude that modern LC-MS-based approaches—though technically more demanding than gel-based studies—appear to be appropriate for tackling the myelin proteome as they cover several orders of magnitude of protein abundance and detect highly basic, hydrophobic, and membrane-spanning proteins. This tackles the bias towards certain protein classes, which is the major shortcoming particularly of 2D-IEF/SDS-PAGE (Fig. 4c). Moreover, LC-MS-based approaches enable the gel- and label-free quantification of proteins from complex mixtures, which allowed for the systematic reassignment of protein abundance in CNS myelin (see above). Finally, they require only low amounts of sample, which is of special relevance for the proteome analysis of myelin purified from hypomyelinated model animals or human brain autopsy material.

Technical Limitations

How pure is the myelin-enriched fraction? Myelin-associated proteins are defined as proteins in the myelin-enriched fraction since all studies have operationally defined the term “myelin protein” without systematic experimental verification. Although the identification of new myelin proteins by more than one study and the detection of established myelin markers increase confidence, some of these proteins may only have copurified with myelin. The high dynamic range of LC-MSE leads to the new identification of proteins as myelin-associated, but also to the false-positive identification of contaminants. These mainly stem from copurifying mitochondria and synaptic vesicles. In reverse, proteomic compendia of mammalian brain mitochondria [116] or synaptic vesicles [90] include classical myelin proteins such as PLP, MBP, MOBP, and MAG. Notwithstanding that some of these proteins may have a dual localization, cross-contamination occurs likely due to similar floatation properties in sucrose or Percoll gradients and can only be excluded once improved separation protocols become available. Proteins of the axonal plasma membrane, such as potassium channels or Na+/K+-ATPases, have also been detected in the myelin fraction, which can be explained by the tight linkage of the membranes via adhesion proteins, sometimes referred to as the myelin–axolemma complex [24]. Indeed, some adhesion complexes are present in the myelin-enriched fraction, such as the glial neurofascin (NF155) and contactin and their axonal partner contactin associated protein 1 (Caspr) [117, 118, 119, 120] and the glial nectin-like protein Necl4 and its axonal counterpart Necl1 [121, 122, 123, 124]. Importantly, myelin proteome analysis also revealed novel candidate proteins to mediate intracellular or intercellular adhesion, such as the immunoglobulin domain superfamily protein Igsf8, also termed EWI-2 [23]. Igsf8 is associated with the myelin tetraspanins CD9 and CD81 and regulates integrin function, at least in vitro [125, 126], but its function in vivo remains to be shown. The experimental validation or falsification of newly identified myelin-associated proteins will be a matter of the systematic application of histological techniques, provided that reliable antibodies are available.

How many proteins can be considered true myelin proteins? Though proteomic compendia aim at completeness, the number can only be guessed at this time. As the dynamic range of current MS-based protein identification schemes is in the range of three to five orders of magnitude, detection of infrequent proteins remains a challenge. Additionally, some technical impediments remain. The myelin proteins CD9 [127, 128], oligodendrocyte myelin glycoprotein [22, 129], and MAL [51] have not yet been detected by proteomic approaches, and the appearance of MAL in one catalog [95] is due to the additional use of immunoblotting. Its nondetectability illustrates the limitations of proteome analysis. MAL is a very hydrophobic protein with four transmembrane domains and very small cytoplasmic and extracellular domains and is, therefore, hardly accessible by MS-based identification. Apart from the membrane-spanning peptides not visible in proteomic approaches, complete tryptic digest of MAL results in only four theoretically detectable peptides: one of 120 amino acids (which is too long for identification by MS), two of two amino acids each (too short to provide useful sequence information), and one of 29 amino acids, which is, in principle, appropriate for identification. However, to obtain a reasonable level of confidence for protein identification, the detection of two peptides per protein is usually set as a prerequisite in the algorithms. This suggests that all proteome approaches requiring protease cleavage have an inherent bias against very small polypeptides or proteins with an unusual cleavage site pattern. In future experiments, the lack of suitable trypsin cleavage sites may be circumvented by the use of endopeptidases with different specificities (e.g., GluC or AspN), although they create proteolytic peptides lacking a basic C-terminal amino acid and are difficult to sequence [130]. This suggests that the detection of more myelin-associated proteins is not just a matter of higher resolving power but also of other technical refinements.

Newly Identified Myelin-Associated Proteins

The compendium of proteins identified in the myelin-enriched brain fraction represents a valuable reference for myelin research. The proteins are candidates for performing important functions in myelin biogenesis and integrity, molecular interactions between myelinating glia and neighboring cells, and white matter homeostasis. By gene ontology terms (http://david.abcc.ncifcrf.gov), many myelin-associated proteins are implicated in catalytic activities (48%), the cytoskeleton (20%), protein transport (21%), vesicular trafficking (6.8%), cell adhesion (6.3%), phospholipid binding (4.2%), or glycolysis/gluconeogenesis (5.1%). Among the recently identified myelin proteins, some were first and others subsequently detected using proteomic approaches. They include proteins of quite various anticipated functions, such as the NAD+-dependent deacetylase sirtuin 2 (SIRT2, see below), cytoskeletal proteins of the septin family [23, 25, 131], and ermin [132], regulators of intracellular vesicle transport in the secretory pathway, such as cdc42 and Rac1 [133], Rab3A, and other Rab-GTPases [134, 135], the paranodal transmembrane glycoprotein Opalin/TMEM10 with a suggested signaling or adhesive function [136, 137, 138], the nucleoside diphosphate kinases NM23A and NM23B [95], and a protein particularly abundant in the CNS myelin of teleost fish, the 36K protein, also termed short-chain dehydrogenase/reductase (SDR family) member 12 (DHRS12) [139]. Some of these are quite abundant myelin proteins as judged both by the spots constituted on 2D gels and LC-based quantification, and the challenge to establish their functions in vivo promises a deepened understanding of myelin. Besides, novel myelin proteins are candidates to cause (when mutated), enhance, or ameliorate white matter disease, such as leukodystrophies.

Differential Myelin Proteome Analysis in Myelin-Related Disease

The proteomic comparison of myelin from human patients or animal models with that of respective controls is a powerful approach towards the identification of secondary molecular changes that may contribute to the pathogenesis of myelin-related disease. Such a differential approach has first been applied to myelin purified from PLP null mice [25], which provide a genuine model for spastic paraplegia (SPG-2) in humans, a comparatively mild variant of the leukodystrophy Pelizaeus–Merzbacher Disease with progressive axonal degeneration in the presence of normal amounts of CNS myelin [29, 140]. In that study, 2D-DIGE [141] was used to screen for candidate proteins that could be involved in the oligodendroglial failure to support the long-term integrity of myelinated axons. Three distinct proteins of the cytoskeletal septin family were found to be reduced, and the deacetylase SIRT2 was virtually absent from PLP null myelin. SIRT2 is an abundant myelin protein in the CNS and the PNS [23, 25, 142] and regulates microtubule dynamics during oligodendrocyte development [143]. Whether acetylated α-tubulin is a relevant substrate of SIRT2 in vivo remains to be shown. Similar to PLP null mice, CNP null mice are also normally myelinated but develop length-dependent axonal loss [92, 144]. It is intriguing that CNP also modulates microtubule dynamics [145, 146]. Taken together, spatiotemporal control of microtubule stability in oligodendrocytes (by SIRT2, CNP, and likely other factors) seems critical for normal axon–glia interaction.

Acetylation is a reversible post-translational modification of numerous mammalian proteins [147, 148], and all acetylated myelin proteins (α-tubulin, MBP, MOG, and several nonidentified proteins of lower abundance) are candidate substrates for SIRT2 [25]. In oligodendrocytes and myelin, SIRT2 activation upon increased axonal NAD+ levels may remove acetyl residues from myelin-associated proteins with consequences for their net charge and function. Interestingly, SIRT2 has been recently shown to interact with 14-3-3 beta and gamma [149], which are myelin-associated as revealed by proteome analysis (Table 1). Their interaction is strengthened by the serine/threonine kinase AKT [149], which is a central signaling molecule for CNS myelination [150]. 14-3-3 proteins have been implicated in membrane protein transport, exocytosis [151], and stress response [152], but their function in myelin has not yet been investigated. 14-3-3 proteins are homologs of the C. elegans partitioning-defective polarity protein Par5 and bind to the tight junction-associated Par3 [153, 154], which is required for establishing polarity prior to myelination, at least by Schwann cells in the PNS [155]. To determine whether SIRT2, 14-3-3 proteins, Par-proteins, protein kinases, and tight junctions indeed interact in myelinating glia will be an important topic of future investigation. We speculate that the competence of oligodendrocytes to dynamically react to NAD+ level changes in white matter tracts is required for their role in maintaining long-term axonal integrity.

With the objective to identify novel therapeutic targets for the treatment of multiple sclerosis, a systematic proteomic profiling of tissue samples from three brain lesions affected to various degrees (acute plaque, chronic active plaque, and chronic plaque) has recently been performed [156]. Material from the respective lesion type was collected by laser-capture microdissection and extracted proteins were separated by 1D gel electrophoresis followed by mass spectrometric protein identification. Unexpectedly, five coagulation proteins, including tissue factor and protein C inhibitor, were only present in chronic active plaque characterized by concomitant inflammation and degeneration, a finding that provided new insights in the relationship between the coagulation cascade and inflammation. Most importantly, administration of inhibitors to tissue factor (i.e., hirudin) and protein C inhibitor (i.e., activated protein C [aPC]) indeed ameliorated the disease phenotype in experimental autoimmune encephalomyelitis, a model of multiple sclerosis. The anti-inflammatory treatment with engineered aPC variants may develop into an alternative route to a therapy of multiple sclerosis. Together, differential proteome analysis has identified secondary molecular changes that contribute to understanding the pathogenesis of myelin-related disease and support the design of rational treatment strategies.

Notes

Acknowledgements

We thank S. Wichert, W. Möbius, J. Patzig, I. Ionescu, and K.-A. Nave for the discussions. ST is supported by the Deutsche Forschungsgemeinschaft (SFB 490 Z3) and the Forschungszentrum Immunologie (FZI) at the University of Mainz, and HW is supported by the BMBF (DLR-Leukonet).

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Copyright information

© Humana Press Inc. 2009

Authors and Affiliations

  1. 1.Proteomics GroupMax Planck Institute of Experimental MedicineGoettingenGermany
  2. 2.DFG Research Center for Molecular Physiology of the BrainGoettingenGermany
  3. 3.Institute of ImmunologyUniversity Medical Center of the Johannes Gutenberg University MainzMainzGermany
  4. 4.Department of NeurogeneticsMax Planck Institute of Experimental MedicineGoettingenGermany

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