1. Neuroscience

Proteome profile of peripheral myelin in healthy mice and in a neuropathy model

  1. Sophie B Siems
  2. Olaf Jahn
  3. Maria A Eichel
  4. Nirmal Kannaiyan
  5. Lai Man N Wu
  6. Diane L Sherman
  7. Kathrin Kusch
  8. Dörte Hesse
  9. Ramona B Jung
  10. Robert Fledrich
  11. Michael W Sereda
  12. Moritz J Rossner
  13. Peter J Brophy
  14. Hauke B Werner  Is a corresponding author
  1. Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Germany
  2. Proteomics Group, Max Planck Institute of Experimental Medicine, Germany
  3. Department of Psychiatry and Psychotherapy, University Hospital, LMU Munich, Germany
  4. Centre for Discovery Brain Sciences, University of Edinburgh, United Kingdom
  5. Institute of Anatomy, University of Leipzig, Germany
  6. Department of Clinical Neurophysiology, University Medical Center, Germany
https://doi.org/10.7554/eLife.51406
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Cite this article
  1. Sophie B Siems
  2. Olaf Jahn
  3. Maria A Eichel
  4. Nirmal Kannaiyan
  5. Lai Man N Wu
  6. Diane L Sherman
  7. Kathrin Kusch
  8. Dörte Hesse
  9. Ramona B Jung
  10. Robert Fledrich
  11. Michael W Sereda
  12. Moritz J Rossner
  13. Peter J Brophy
  14. Hauke B Werner
(2020)
Proteome profile of peripheral myelin in healthy mice and in a neuropathy model
eLife 9:e51406.
https://doi.org/10.7554/eLife.51406
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7 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Proteome analysis of peripheral myelin.

(A) Schematic illustration of a previous approach to the peripheral myelin proteome (Patzig et al., 2011) compared with the present workflow. Note that the current workflow allows largely automated …

Figure 1—source data 1

Label-free quantification of proteins in wild-type PNS myelin fractions by three different data acquisition modes Identification and quantification data of detected myelin-associated proteins.

Tryptic peptides derived from four technical replicates (replicate digestion and replicate injection) per three biological replicate (20 sciatic nerves pooled from 10 animals) were analyzed by LC-MS (12 runs in total). Proteins (FDR < 1%; 2 peptides/protein) and peptides (FDR < 1%;≥7 amino acids) were identified by database search against the UniprotKB/SwissProt mouse database using PLGS. Data were post-processed with the software package ISOQuant to calculate absolute in-sample amounts for each detected protein based on the TOP3 approach. Reported abundance values are defined as the relative amount of each protein in respect to the sum over all detected proteins (ppm: parts per million (w/w) of total protein). Typical contaminant proteins like keratins were filtered. → sheet 1: protein identification details → sheet 2: WT myelin proteome by MSE → sheet 3: WT myelin proteome by UD-MSE → sheet 4: WT myelin proteome by DRE UD-MSE → sheet 5: 45 proteins additionally identified in WT myelin by 1D-gel-LC-MS.

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Figure 1—figure supplement 1
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Clustered heatmap of Pearson’s correlation coefficients for protein abundance comparing data acquisition modes.

The heatmap compares the log2 transformed ppm protein abundance values to assess peripheral myelin purified from wild type mice using three data acquisition modes (MSE, UDMSE, DRE-UDMSE). The inset …

Figure 2
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Relative abundance of peripheral myelin proteins.

MSE was used to identify and quantify proteins in myelin purified from the sciatic nerves of wild-type mice at P21; their relative abundance is given as percent with relative standard deviation (% …

Figure 3
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Developmental mRNA abundance profiles of myelin-associated genes.

(A) K-means clustering was performed for the mRNA profiles of those 1046 proteins in our myelin proteome inventory for which significant mRNA expression was found by RNA-Seq in the sciatic nerve of …

Figure 3—source data 1

Normalized developmental mRNA abundance data → sheet 1: normalized values for all individual 4 biological replicates per age → sheet 2: normalized values for biological replicates averaged to give mean per age.

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Figure 4
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Categorization of annotated protein functions.

All proteins identified in peripheral myelin by UDMSE (turquoise) and the respective developmental expression clusters (Figure 3; shades of red) were analyzed for overrepresented functional …

Figure 5 with 1 supplement
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Molecular analysis of myelin in the Prx-/- mouse model of CMT4F.

(A) Myelin purified from sciatic nerves dissected from Prx-/- and control mice at P21 was separated by SDS-PAGE (0.5 µg protein load) and proteins were visualized by silver staining. Bands …

Figure 5—source data 1

Label-free quantification of proteins in PNS myelin fractions from Prx-/- mice by MSE Identification and quantification data of detected myelin-associated proteins.

Tryptic peptides derived from four technical replicates (replicate digestion and replicate injection) per three biological replicate (20 sciatic nerves pooled from 10 animals) were analyzed by LC-MS (12 runs in total). Proteins (FDR < 1%; 2 peptides/protein) and peptides (FDR < 1%;≥7 amino acids) were identified by database search against the UniprotKB/SwissProt mouse database using PLGS. Data were post-processed with the software package ISOQuant to calculate absolute in-sample amounts for each detected protein based on the TOP3 approach. Reported abundance values are defined as the relative amount of each protein in respect to the sum over all detected proteins (ppm: parts per million (w/w) of total protein). Typical contaminant proteins like keratins were filtered. → sheet 1: protein identification details → sheet 2: Prx-/- myelin proteome by MSE.

https://cdn.elifesciences.org/articles/51406/elife-51406-fig5-data1-v1.xlsx
Figure 5—source data 2

Label-free quantification of proteins in PNS myelin fractions from WT and Prx-/- mice by DRE-UDMSE Identification and quantification data of detected myelin-associated proteins by DRE-UDMSE.

For each genotype, tryptic peptides derived from four technical replicates (replicate digestion and replicate injection) per three biological replicate (20 sciatic nerves pooled from 10 animals) were analyzed by LC-MS (24 runs in total). Proteins (FDR < 1%; 2 peptides/protein) and peptides (FDR < 1%;≥7 amino acids) were identified by database search against the UniprotKB/SwissProt mouse database using PLGS. Data were post-processed with the software package ISOQuant to calculate absolute in-sample amounts for each detected protein based on the TOP3 approach. Reported abundance values are defined as the relative amount of each protein in respect to the sum over all detected proteins (ppm: parts per million (w/w) of total protein). Typical contaminant proteins like keratins were filtered. The -log10-transformed q-value was plotted against the log2-transformed fold change to obtain the volcano plot shown in Figure 5D. As no imputation of missing values was performed, proteins exclusive for only one of the conditions do not appear in the volcano plot, but are appended at the end of the list. Criteria for statistically significant regulation were as follows: fold change of at least 1.5 and q-value below 0.05. → sheet 1: protein identification details → sheet 2: comparison of WT vs. Prx-/- myelin proteome by DRE-UDMSE.

https://cdn.elifesciences.org/articles/51406/elife-51406-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
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Clustered heatmap of Pearson’s correlation coefficients for protein abundance comparing genotypes.

(A) The heatmap compares the log2 transformed ppm protein abundance values from the DRE-UDMSE runs to assess peripheral myelin purified from wild type and Prx-/- mice. The inset shows the color key …

Figure 6
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Progressive loss and reduced diameters of peripheral axons in Prx-/- mice.

(A–D) Genotype-dependent quantitative assessment of light micrographs of toluidine-stained semi-thin sectioned quadriceps nerves dissected at 2, 4 and 9 months of age reveals progressive loss of …

Author response image 1
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Quantification of immunoblots in Figure 1B and Figure 5E.

Tables

Table 1
Known myelin proteins in the myelin proteome.

Proteins mass-spectrometrically identified in peripheral myelin are compiled according to availability of prior references as myelin proteins. Given are the official gene name, one selected …

Protein nameGeneReferenceTMDCluster
2-hydroxyacylsphingosine 1-beta-galactosyltransferaseUgt8Bosio et al., 19962P6-up
Syntrophin α1Snta1Fuhrmann-Stroissnigg et al., 2012-P18-up
Annexin A2Anxa2Hayashi et al., 2007-Descending
Band 4.1 protein B/4.1BEpb41l3Ivanovic et al., 2012-Descending
Band 4.1 protein G/4.1GEpb41l2Ohno et al., 2006-P6-up
Breast carcinoma-amplified sequence 1Bcas1Ishimoto et al., 2017-P6-up
Cadherin 1/E-CadherinCdh1Fannon et al., 19951P18-up
Carbonic anhydrase 2Ca2Cammer and Tansey, 1987-Descending
Catenin α1Ctnna1Murata et al., 2006-U-shaped
Catenin ß1Ctnnb1Fannon et al., 1995-Descending
Caveolin 1Cav1Mikol et al., 20021P18-up
CD9, tetraspanin 29Cd9Ishibashi et al., 20044P18-p
CD59ACd59aFunabashi et al., 19941P18-up
CD47, integrin-associated signal transducerCd47Gitik et al., 20115P6-up
CD81, tetraspanin 28Cd81Ishibashi et al., 20044P18-up
CD82, tetraspanin 27Cd82Chernousov et al., 20134P18-up
CD151, tetraspanin 24Cd151Patzig et al., 20114P18-up
Cell adhesion molecule 4/NECL4Cadm4Spiegel et al., 20071P6-up
Cell division control protein 42Cdc42Benninger et al., 2007-P6-up
Cell surface glycoprotein MUC18McamShih et al., 19981Descending
Ciliary neurotrophic factorCntfRende et al., 1992-Late-up
CKLF-like MARVEL TMD-containing 5Cmtm5Patzig et al., 20114P6-up
Claudin-19Cldn19Miyamoto et al., 20054P6-up
Cofilin 1Cfl1Sparrow et al., 2012-Descending
Crystallin α2CryabD'Antonio et al., 2006-P18-up
Cyclic nucleotide phosphodiesteraseCnpMatthieu et al., 1980-P6-up
Sarcoglycan δSgcdCai et al., 20071Late-up
Dihydropyrimidinase related protein 1Crmp1D'Antonio et al., 2006-Descending
Disks large homolog 1Dlg1Cotter et al., 2010-Descending
Dynein light chain 1Dynll1Myllykoski et al., 2018-P6-up
DystroglycanDag1Yamada et al., 19941P6-up
Dystrophin/DP116DmdCai et al., 2007-P6-up
Dystrophin-related protein 2Drp2Sherman et al., 2001-P18-up
E3 ubiquitin-protein ligase NEDD4Nedd4Liu et al., 2009-Descending
EzrinEzrScherer et al., 2001-P6-up
Fatty acid synthaseFasnSalles et al., 2002-P6-up
Flotillin 1Flot1Lee et al., 2014-P18-up
Gap junction ß1 protein/Cx32Gjb1Li et al., 20024P18-up
Gap junction γ3 protein/Cx29Gjc3Li et al., 20021P6-up
GelsolinGsnGonçalves et al., 2010-Late-up
Glycogen synthase kinase 3ßGsk3bOgata et al., 2004-P6-up
Integrin α6Itga6Nodari et al., 20081P6-up
Integrin αVItgavChernousov and Carey, 20031Descending
Integrin ß1Itgb1Feltri et al., 20021Descending
Integrin ß4Itgb4Quattrini et al., 19962P18-up
Junctional adhesion molecule CJam3Scheiermann et al., 20071P18-up
Laminin α2Lama2Yang et al., 2005-P6-up
Laminin α4Lama4Yang et al., 2005-Descending
Laminin ß1Lamb1LeBeau et al., 1994-Descending
Laminin ß2Lamb2LeBeau et al., 1994-P18-up
Laminin γ1Lamc1Chen and Strickland, 2003-Descending
Membrane Palmitoylated Protein 6Mpp6Saitoh et al., 2019-P6-up
Microtubule-associated protein 1AMap1aFuhrmann-Stroissnigg et al., 2012-P18-up
Microtubule-associated protein 1BMap1bFuhrmann-Stroissnigg et al., 2012-P6-up
Mitogen-activated protein kinase 1/ERK2Mapk1Mantuano et al., 2015-Descending
Mitogen-activated protein kinase 3/ERK1Mapk3Mantuano et al., 2015-P18-up
MoesinMsnScherer et al., 2001-Unchanged
Monocarboxylate transporter 1Slc16a1Domènech-Estévez et al., 201511P18-up
Myelin associated glycoproteinMagFiglewicz et al., 19811P6-up
Myelin basic proteinMbpBoggs, 2006-P6-up
Myelin protein 2Pmp2Trapp et al., 1984-P18-up
Myelin protein zero/P0MpzGiese et al., 19921P6-up
Myelin proteolipid proteinPlp1Garbern et al., 19974P6-up
Myotubularin-related protein 2Mtmr2Bolino et al., 2004-P6-up
Noncompact myelin-associated proteinNcmapRyu et al., 20081P18-up
NDRG1, N-myc downstream regulatedNdrg1Berger et al., 2004-P18-uP
NeurofascinNfascTait et al., 20002P18-up
Nidogen 1Nid1Lee et al., 2007-Descending
P2X purinoceptor 7P2r×7Faroni et al., 2014-P6-up
PaxillinPxnFernandez-Valle et al., 2002-P6-up
PeriaxinPrxGillespie et al., 1994-P6-up
PlasmolipinPllpBosse et al., 20034P18-up
Profilin 1Pfn1Montani et al., 2014-Descending
Lin-7 homolog CLin7cSaitoh et al., 2017-P6-up
Rac1Rac1Benninger et al., 2007-U-Shaped
RadixinRdxScherer et al., 2001-Descending
RhoARhoaBrancolini et al., 1999-U-Shaped
Septin 2Sept2Buser et al., 2009-Descending
Septin 7Sept7Buser et al., 2009-U-Shaped
Septin 8Sept8Patzig et al., 2011-P18-up
Septin 9Sept9Patzig et al., 2011-P6-up
Septin 11Sept11Buser et al., 2009-Descending
Sirtuin 2, NAD-dependent deacetylaseSirt2Werner et al., 2007-P18-up
Spectrin alpha chain, non-erythrocytic 1Sptan1Susuki et al., 2018-P18-up
Spectrin beta chain, non-erythrocytic 1Sptbn1Susuki et al., 2018-P18-up
Tight junction protein ZO-1Tjp1Poliak et al., 2002-P6-up
Tight junction protein ZO-2Tjp2Poliak et al., 2002-P6-up
TransferrinTfLin et al., 19902Late-up
VimentinVimTriolo et al., 2012-Unchanged
VinculinVclBeppu et al., 2015-Descending
Table 2
Peripheral myelin proteins identified in PNS myelin involved in neuropathological diseases.

Proteins mass-spectrometically identified in peripheral myelin were analyzed regarding the involvement of the ortholog human gene in neuropathological diseases. PMP22 was added, though it was not …

Protein nameGene nameOMIM#Gene locusNeuropathy
Monoacylglycerol lipase ABHD12ABHD1261359920p11.21Pharc
Apoptosis-inducing factor 1AIFM1300169Xq26.1CMTX4, DFNX5
Na+/K+ -transporting ATPase α1ATP1A11823101p13.1CMT2DD
Cytochrome c oxidase subunit 6A1COX6A160207212q24.31CMTRID
Dystrophin-related protein 2DRP2300052Xq22.1CMTX
Dynactin subunit 1DCTN16011432p13.1DHMN7B
Dynamin 2DNM260237819p13.2CMT2M, CMTDIB
Cytoplasmic dynein 1 heavy chain 1DYNC1H160011214q32.31CMT20, SMALED1
E3 SUMO-protein ligaseEGR212901010q21.3CMT1D, CMT3, CMT4E
Glycine-tRNA ligaseGARS (Gart)6002877p14.3CMT2D, HMN5A
Gap junction ß1 protein/Cx32GJB1304040Xq13.1CMTX1
Guanine nucleotide-binding protein ß4GNB46108633q26.33CMTDIF
Histidine triad nucleotide-binding protein 1HINT16013145q23.3NMAN
Hexokinase 1HK114260010q22.1CMT4G
Heat shock protein ß1HSPB16021957q11.23CMT2F, DHMN2B
Kinesin heavy chain isoform 5AKIF5A60282112q13.3SPG10
Prelamin A/CLMNA1503301q22CMT2B1
NeprilysinMME1205203q25.2CMT2T, SCA43
Myelin protein zero/P0MPZ1594401q23.3CHN2,CMT1B, CMT2I, CMT2J,CMT3, CMTDID, Roussy-Levy syndrome
Myotubularin-related protein 2MTMR260355711q21CMT4B1
Alpha-N-acetylglucosaminidaseNAGLU (NAGA)60970117q21.2CMT2V
NDRG1, N-myc downstream regulatedNDRG16052628q24.22CMT4D
Neurofilament heavy polypeptideNEFH16223022q12.2CMT2CC
Neurofilament light polypeptideNEFL1622808p21.2CMT2E, CMT1F, CMTDIG
Peripheral myelin protein 2PMP21707158q21.13CMT1G
Peripheral myelin protein 22PMP2260190717p12CMT1A, CMT1E, CMT3, HNPP,
Roussy-Levy syndrome
Ribose-phosphate pyrophosphokinase 1PRPS1311850Xq22.3Arts syndrome, CMTX5, DFNX1
PeriaxinPRX60572519q13.2CMT4F, CMT3
Ras-related protein Rab 7aRAB7A6022983q21.3CMT2B
Septin 9SEPT960406117q25.3HNA
Transitional ER-ATPaseVCP6010239p13.3CMT2Y
Tryptophan-tRNA ligase, cytoplasmicWARS19105014q32.32HMN9
Tyrosine-tRNA ligase, cytoplasmicYARS6036231p35.1DI-CMTC

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