Characterization of mature rat oligodendrocytes: a proteomic approach

Characterization of mature rat oligodendrocytes: a proteomicapproach

Debora Dumont, Jean-Paul Noben, Marjan Moreels, Joris Vanderlocht, Niels Hellings,Frank Vandenabeele, Ivo Lambrichts, Piet Stinissen and Johan Robben

Hasselt University, Biomedical Research Institute (BIOMED) and transnationale Universiteit Limburg, School of Life Sciences,

Diepenbeek, Belgium

Abstract

Oligodendrocytes are glial cells responsible for the synthesis

and maintenance of myelin in the central nervous system

(CNS). Oligodendrocytes are vulnerable to damage occurring

in a variety of neurological diseases. Understanding oligo-

dendrocyte biology is crucial for the dissemination of de- and

remyelination mechanisms. The goal of the present study is

the construction of a protein database of mature rat oligo-

dendrocytes. Post-mitotic oligodendrocytes were isolated

from mature Wistar rats and subjected to immunocytochem-

istry. Proteins were extracted and analyzed by means of two-

dimensional gel electrophoresis and two-dimensional liquid

chromatography, both coupled to mass spectrometry. The

combination of the gel-based and gel-free approach resulted

in confident identification of a total of 200 proteins. A minority

of proteins were identified in both proteomic strategies. The

identified proteins represent a variety of functional groups,

including novel oligodendrocyte proteins. The results of this

study emphasize the power of the applied proteomic strategy

to study known or to reveal new proteins and to investigate

their regulation in oligodendrocytes in different disease

models.

Keywords: mass spectrometry, oligodendrocytes, proteo-

mics, rat brain, two-dimensional gel electrophoresis, two-

dimensional liquid chromatography.

J. Neurochem. (2007) 102, 562–576.

Oligodendrocytes synthesize and maintain myelin in thecentral nervous system. Oligodendrocytes are vulnerable toinjury mediated by oxidative stress (Rosenberg et al. 1999),cytotoxicity (Griot-Wenk et al. 1991; Scolding and Comp-ston 1991; Jurewicz et al. 1998; Russell and Ley 2002)excitotoxicity (Matute et al. 2001; Werner et al. 2001),trophic factor deprivation and activation of apoptotic path-ways (Vartanian et al. 1995; Vanderlocht et al. 2006).Oligodendrocyte damage is observed in a variety of diseasesincluding multiple sclerosis (Buntinx et al. 2002), stroke(Aboul-Enein et al. 2003), dementia (Kurz et al. 2003),spinal cord trauma (Gomes-Leal et al. 2004), encephalopa-thies (El Hachimi et al. 1998), leukodystrophies (Ip et al.2006) and Alzheimer’s disease (Ness et al. 2005).

Although the composition of the myelin sheath has beeninvestigated by several proteomic strategies (Persson andOverholm 1990; Yamaguchi and Pfeiffer 1999; Taylor andPfeiffer 2003;Taylor et al. 2004; Vanrobaeys et al. 2005), nocomprehensive proteomic study of oligodendrocytes hasbeen published. Characterization of the oligodendrocyteproteome is on one hand crucial for a better understandingof molecular mechanisms in oligodendrocytes under normal

and pathological conditions. On the other hand, because ofthe limited availability of human brain biopsy material,animal models for disease (mainly rodents) are of utmostimportance for CNS research. Therefore, a comprehensiveproteomic map of rodent oligodendrocytes is expected to bea useful new tool aiding research of oligodendrocytepathology.

Received September 18, 2006; revised manuscript received December17, 2006; accepted February 6, 2007.Address correspondence and reprint requests to Johan Robben, Ph.D.,

Hasselt University, Biomedical Research Institute, Agoralaan building,A 3590 Diepenbeek,Belgium. E-mail: [email protected] used: 2D-GE, two-dimensional gel electrophoresis;

2D-LC, two-dimensional liquid chromatography; ACN, acetonitrile;EAE, experimental autoimmune encephalomyelitis; FCS, fetal calfserum; GalC, galactosylceramide; HAc, acetic acid; HSP, heat-shockprotein; IPG, immobilized pH-gradient; LC-ESI-MS/MS, liquid chro-matography-electrospray ionization-tandem mass spectrometry; MAG,myelin-associated glycoprotein; MBP, myelin basic protein; MIF,macrophage migration inhibitory factor; MS, mass spectrometry; Mw,molecular weight; PBS, phosphate buffered saline; pI, isoelectric point;SCX, strong cation exchange.

Journal of Neurochemistry, 2007, 102, 562–576 doi:10.1111/j.1471-4159.2007.04575.x

562 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 562–576� 2007 The Authors

To identify disease-related proteins in neurological disor-ders, studies using proteomic approaches are rapidly emer-ging (Harrington et al. 1985; Lubec et al. 1999; Choe et al.2002a,b; Davidsson et al. 2002; Guillaume et al. 2003; Jianget al. 2003; Dumont et al. 2004). Two-dimensional gelelectrophoresis (2D-GE) has become the standard approachfor the separation of complex protein mixtures. Indeed, thistechnique allows high-resolution protein mapping of variousbodily fluids, as well as tissue and cell types, therebyproviding valuable tools for identifying candidate diseasemarkers. However, proteins that are high or low in isoelectricpoints (pI), molecular weights (Mw), polarity or abundanceremain inadequately detected by 2D-GE. Other separationtechnologies have been explored to address some of theselimitations. Peptide-mapping by two-dimensional liquid chro-matography (2D-LC) emerged as a plausible alternative to2D-GE and has already proven its worth in CNS research(Maccarrone et al. 2004; Noben et al. 2006). In this study, aproteomic initiative was undertaken in our laboratory,applying 2D-GE as well as 2D-LC in combination withmass spectrometry for the construction of a proteomic map ofmature rat oligodendrocytes.

Materials and methods

Primary rat oligodendrocyte cultures

Primary rat oligodendrocytes were isolated from whole brains of

adult Wistar rats (Harlan, The Netherlands) as described by Yong

and Antel (1992). Briefly, brain tissue was dissociated mechanically

and enzymatically for 30 min at 37�C. Glial cells were separated

from myelin debris and red blood cells by means of Percoll gradient

centrifugation. The mixed glial cell fraction was suspended in

Dulbecco’s modified Eagle medium (DMEM, Life Technologies,

Rockville, MD, USA) supplemented with 5% fetal calf serum

(FCS), penicillin (50 U/mL) and streptomycin (50lg/mL) and

seeded in culture flasks. The cultures were selectively enriched for

oligodendrocytes by means of differential adhesion to plastic. The

non-adherent oligodendrocyte fraction was plated at a density of

2 · 105 onto poly-L-lysine-coated glass slides and at a density of

106 cells/mL on poly-L-lysine-coated 24-well plates for immuno-

cytochemistry and proteomic analysis, respectively. Cells were

cultured for 12–14 days in DMEM supplemented with 5% FCS.

Immunocytochemistry

Cells seeded on glass slides were stained using the peroxide-based

Envision System (DakoCytomation, Glostrup, Denmark). Cells were

fixed in 4% formaldehyde (Unifix, Duiven, The Netherlands) for

20 min. After washing in 0.01 mol/L phosphate buffered saline

(PBS), cells were permeabilized (for intracellular staining) with

0.05% Triton X-100 (Boehringer, Mannheim, Germany) in PBS for

30 min followed by an additional wash step. Non-specific binding

sites were blocked with 3% normal goat serum (DakoCytomation) in

PBS for 20 min. After washing, cells were incubated for 1 h with

antibodies specific for myelin basic protein (MBP; 1/200; Serotec,

Oxford, UK), galactosylceramide (GalC; 1:50; kind gift from

Dr V.W. Yong, University of Calgary, Canada) or myelin-associated

glycoprotein (MAG; 1:500; Chemicon, Temecula, CA,USA), washed

thrice and incubated for 30 min with anti-mouse peroxidase-conju-

gated secondary antibody (DakoCytomation). A high sensitivity

diaminobenzidine chromogenic substrate system (DakoCytomation)

was used to visualize the immunoreactivity. Cells were counterstained

with Mayer’s hematoxylin, mounted in Aquatex (Merck, Darmstadt,

Germany) and examined using a photomicroscope equipped with

an automated camera (Nikon Eclipse 80i, Japan).

Two-dimensional gel electrophoresis (2D-GE)

Oligodendrocytes were lysed at 4�C in a buffer containing 7 mol/L

urea, 2 mol/L thiourea, 2% w/v CHAPS and homogenized with a

rotor stator mixer. Proteins were extracted using acetone precipita-

tion. The protein content was determined with the 2-D Quant kit

(GE Healthcare, Uppsala, Sweden). One part of the extract

containing 100 lg of proteins was concentrated using 5 kDa cut-

off Ultrafree columns (Millipore, Bedford CA, USA). The retentate

was suspended in 450 lL rehydration buffer (7 mol/L urea, 2 mol/L

thiourea, 2% w/v CHAPS, 200 mmol/L dithiothreitol, 0.5% v/v

immobilized pH-gradient (IPG) buffer pH 3–10 and bromophenol

blue) and spread out over the bottom of a strip holder. An IPG strip

(24 cm, linear pH 3–10; GE Healthcare) was applied on the protein

mixture and rehydrated for 12 h at 22�C. Two additional parts of thesame extract were treated in the same way. Isoelectric focusing,

SDS-PAGE, staining and software analysis were conducted as

described previously (Dumont et al. 2004). Spots were picked from

at least one of three gels run in parallel and gel plugs were trypsin-

digested individually or in combination (collecting corresponding

weak spots) according to the method described by Shevchenko et al.(Shevchenko et al. 1996).

Two-dimensional liquid chromatography (2D-LC)

Forty micrograms of the same protein extract (described in Two-

dimensional gel electrophoresis) was subjected to strong cation-

exchange (SCX) chromatography as described previously (Noben

et al. 2006). Briefly, protein extracts were denaturated, alkylated,

and digested with trypsin. Digests were trapped on a Hypercarb

column (0.5 cm · 200 lm i.d.; Nanoseparations, Nieuwkoop, The

Netherlands) and transferred to a polysulfoethyl aspartamide column

(12 cm · 200 lm i.d.; Nanoseparations) in 1 lL solution A (0.5%

(v/v) acetic acid (HAc) in water), containing 70% acetonitrile

(ACN). The analytical column was eluted with a linear salt gradient

(slope 15 mmol/L KCl/min) starting from 100% solution A to 100%

solution A containing 250 mmol/L KCl and 35% (v/v) ACN, to

100% solution A containing 500 mmol/L KCl and 35% (v/v) ACN

and 1 min fractions (2.5 lL) were collected. SCX fraction volume

was adjusted to �25 lL with a solution containing 5% (v/v) ACN in

100 mmol/L HAc. The second dimension separation involved the

reversed-phase chromatography described in Mass spectrometry.

Mass spectrometry

2D-GE protein digests and SCX fractions were analysed by liquid

chromatography-electrospray ionization-tandem mass spectrometry

(LC-ESI -MS/MS). Flow regulation was as described by Meiring

et al.(2002). Each digest/fraction was diluted with a solution

containing 5% (v/v) ACN in 100 mmol/L HAc. This solution

contained 4 pg/lL of cortisone as an internal analytical standard to

Proteomic analysis of rat oligodendrocytes 563

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 562–576

monitor flow stability (by retention time) and overall performance

(by peak height as derived from the selected ion chromatogram for

[M + H]+ = m/z 361.2) of the ion trap mass spectrometer (LCQ

Classic, ThermoElectron Corporation, San Jose, CA, USA). Of each

digest/fraction, 10 lL was injected (autoinjector AS3000, Thermo-

Electron). The trapped samples were separated on the analytical

column (Biosphere C18, 200 mm L · 0.05 mm i.d., 5 lm, Nano-

separations) using a linear gradient from 5 to 60% v/v ACN in water

containing 100 mmol/L HAc either in 55 min (2-DE protein

digests) or 120 min (SCX-separated fractions). The eluate from

the analytical column was introduced in a nanoelectrospray device

(ThermoFinnigan) and sprayed from a gold-coated fused silica

emitter (5 lm i.d., NanoSeparations). The LCQ automated protocols

were used to optimize the ion optics and calibration parameters. The

mass spectrometer was operated in a data-dependent acquisition

mode to automatically switch between MS (m/z 300–1500 Thomp-

son in centroid mode at a maximum injection time of 150 ms) and

MS/MS acquisition on the three most intense precursor ions,

controlled by Xcalibur 1.3 software.

Database management, evaluation and reporting

Peak lists in DTA file format were generated from mass spectro-

metric raw data files using the CreateDTA tool available in Sequest

v27 within BioWorks v3.0 (ThermoElectron).

2D-GE-derived MS data

DTA files were examined with the search engines Sequest v27

(Thermofinnigan) and Mascot (Matrix Science) using the database

NCBInr (subset Rodentia, 105,999 entries). Sequest parameters

were set as follows: Xcorr ‡ 1.8, ‡ 2.5 or ‡ 3.5 for singly, doubly

or triply charged peptide ions; delta Cn > 0.1; precursor and product

ion mass tolerance ± 3 and ± 1 Da; enzyme: trypsin; one missed

cleavage allowed; static chemical modification: cysteine-carbami-

domethylation; dynamic chemical modification: oxidation of methi-

onine, histidine, and tryptophan. Mascot search parameters were:

taxonomy rodentia, parent, and peptide ion mass tolerance ± 3

and ± 1 Da, enzyme: trypsin/P, one missed cleavage allowed. The

Mascot significance threshold was set at p £ 0.05.

2D-LC-derived MS data

DTA files derived from each SCX fraction were first examined with

the search engine Sequest as described above (2D-GE-derived MS

data). Sequest identifications were assembled in a DTASelect v1.9

report (Tabb et al. 2002). DTASelect licenses were obtained from

the Scripps Research Institute (La Jolla, CA, USA). Second, all DTA

files were examined with Mascot as described in 2D-GE-derived

MS data.

Evaluation

The number of sibling peptides was derived according to the

guidelines for peptides and protein identification data (Carr et al.2004). Only peptides assigned by both search engines were retained

in this study. Proteins assigned on the basis of two or more peptides

meeting the criteria described in 2D-GE-derived MS data and

2D-LC-derived MS data were considered as confidently identified.

Single-peptide protein identifications returned by both Sequest and

Mascot were re-examined with the de novo sequencing algorithm

Lutefisk1900 v.1.3.2. (Taylor and Johnson 1997) and the

tag-generating algorithm Inspect (Frank et al. 2005; Tanner et al.2005) in this order. In case the outcome was negative, the peptide

identification was rejected. Finally, an additional data analysis was

performed to validate the 2D-LC identifications. To calculate the

false-positive error rate, all DTA files were analyzed with Mascot

using the ‘sequence-reversed’ databases RV_NCBI, generated with

an in-house developed Perl script.

Results

Oligodendrocyte cultures

Oligodendrocytes were isolated from whole brain of adultrats and their phenotype was studied by means of immun-ocytochemistry. After 12–14 days, 90% of the cells in thecultures consisted of oligodendrocytes with a characteristicmature morphology. They were characterized by a roundphase bright nucleus and a small number of thick, irregularcell processes. Clusters of 3–10 oligodendrocytes were oftenobserved in culture (Fig. 1a). Mature oligodendrocytesexpressed MAG, MBP and GalC proteins (Figs 1b–d).Longitudinal flowcytometric analysis was performed show-ing that the vast majority of MBP positive cells were derivedfrom MBP/GalC positive cells while only a very smallincrease in NG2 precursor cells was observed. The amount ofprecursor cells mounted to 5% while contaminating astro-cytes, fibroblasts en endothelial cells constituted less than 5%(data not shown).

2D-GE and 2D-LC experiments

2D-GE experiments were performed on three oligodendro-cyte cultures. Silver staining of three gels from each culturerevealed reproducible protein patterns (Fig. 2). Proteins spotswere picked, trypsin digested and analyzed by means of ESI-LC-MS/MS. Only peptides assigned by both Sequest andMascot search engines were retained in this study.

From one culture of rat oligodendrocytes, an ultrafiltered,reduced, alkylated and trypsinized sample was prepared andfractionated by SCX. The resulting fractions were analyzedby LC-ESI-MS/MS. A total of 30,467 MS/MS spectra (DTAfiles) were recorded. First, these DTA files were examinedwith the search engine Sequest. Because of the stringentselection criteria (2D-LC-derived MS data) and the removalof contaminants (e.g. keratins) only 1% (306 DTA files) ofthe initial data set (30 467 DTA files) was assigned bySequest. Second, all DTA files were searched with Mascot.Only peptides assigned by both Sequest and Mascot wereretained in this study.

Validation of the proteomic data

Single-peptide protein identifications respectively amountedto 30% and 58% in the 2D-GE and 2D-LC experiment(Fig. 3) and were examined additionally with the de novo

564 D. Dumont et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 562–576� 2007 The Authors

sequencing algorithm Lutefisk. The sequence returned bySequest and Mascot matched the de novo sequence proposedby Lutefisk in 80% of all examined peptides (102). In allother cases, no quality de novo sequence could be derived(noisy spectra) or the de novo sequence with primary rankingdiffered from the one found by both search engines. In thelatter case, peptides identifications were additionallysearched with the tag-generating algorithm Inspect. In this

way, all but six peptide identifications were confirmed(Table 1).

The false-positive error rate for the DTA files derivedfrom the 2D-LC experiment was 0.7%. Since for the2D-LC experiment 50 single-peptide identifications werevalidated, the number of false-positive protein identifica-tions on the basis of one peptide is estimated at 0.35(0.7% of 50).

Mass(kDa)

93

67

43

30

20

14

3 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10p/

Fig. 2 Representative 2D map of mature

rat oligodendrocyte proteins. Post-mitotic

oligodendrocytes were isolated from total

rat brain and cultured for 12–14 days. Pro-

teins were extracted and separated

according to their isoelectric point (pI) in the

first dimension (IEF strips 24 cm, pH linear

3–10) and according to their molecular

weight by means of SDS-PAGE in the

second dimension. Protein spots were cut,

trypsin-digested and analyzed by means

of LC-ESI-MS/MS. Marked spots can be

found in the supplemental material,

together with a detailed protein inventory, of

which a summary is presented in Table 1.

(a) (b)

(c) (d)Fig. 1 Morphological and immunocyto-

chemical characterization of oligodendro-

cyte cultures. The primary cultures (day

12–14) isolated form adult Wistar rats were

characterized by clusters of small, phase-

bright oligodendrocytes with dark cell pro-

cesses (a). Oligodendrocytes showed

moderate to strong immunoreactivity for

MAG (b), MBP (c) and GalC (d). (Scale bars:

a–d: 20 lm).



Study outcome

Combining 2D-GE and 2D-LC experiments, 200 proteinswere identified in this study: 41 proteins were found incommon for both approaches, 114 proteins were found uniquefor 2D-GE and 45 unique for 2D-LC (Fig. 4, Table 1). Spotsannotations as well as protein identification details arepresented in the supplemental material. While the majorityof the 2D-GE identified proteins displayed a centered profilewith respect to pI and Mw parameters, 2D-LC identificationsspread towards the outer regions of this spectrum (Fig. 5). Inaddition, 2D-GE clearly favored the detection of cytoplasmicproteins (55%) while 2D-LC displayed a more distributedsubcellular localization (Fig. 6) including a prominent nuc-lear fraction (17%). Regarding the functional diversity, themajor part of the proteins identified via 2D-GE play a role incell metabolism. In contrast, 2D-LC identified proteins aremore evenly distributed over the different functional groups.Notably are the proteins belonging to the DNA/RNA-bindingand protein degradation group, which are more abundantlyencountered in the 2D-LC and 2D-GE analysis respectively(Fig. 7). The newly identified oligodendrocyte proteinsinclude proteins involved in Ca2+ binding (calumenin,translationally controlled tumor protein), protein folding(protein disulfide isomerase precursor A6), stress response(heat shock-related protein 1A/B, peroxiredoxins), proteindegradation (ubiquitin/proteasome pathway proteins, cathep-sin B) and CNS development (cysteine and glycine-richprotein 1, acyl-CoA-binding protein).

Discussion

Oligodendrocyte cultures

Study of oligodendrocyte development and biology withinnormal and pathological CNS tissue is generally hampered by

the limited availability of viable human brain tissue. Humanoligodengroglial cell lines represent suitable model systems tostudy oligodendrocyte injury (Buntinx et al. 2004). However,they display tumorigenic properties and often representoligodendrocytes in an immature developmental state (Bunt-inx et al. 2003). Moreover, they exhibit little or no morpho-logical resemblance with their in vivo counterparts. Incontrast, the primary oligodendrocytes used in this studydisplayed immunophenotypic characteristics of mature postmitotic oligodendrocytes (Snyder et al. 1980; Norton et al.1983;Vick et al. 1990). Caution was taken in the selection ofcell culture wells during harvesting as to minimize the numberof astrocytes and fibroblasts. Nevertheless, the presence of alimited number of contaminating cell types is inevitable. Wetried to deal with this drawback by searching literature andindicating proteins that have been reported in oligodendro-cytes or myelin (Taylor et al. 2004; Vanrobaeys et al. 2005)with the corresponding references in Table 1. Nevertheless,some of the newly identified proteins, including fatty acid-binding protein and peroxiredoxin 6 were previously des-cribed in astrocytes and could reflect the contribution of acontaminating population to the protein expression profile.These proteins have to be handled with caution and confirmedby immunocytochemistry. Another phenomenon encounteredwhen using cell cultures are cellular stress responses evokedby culture conditions. Although after 2 weeks in culture, therewas no evidence of lethal (cell death) or sublethal (retractionof processes) stress, we did identify a number of stress-associated proteins, including members of the heat shock andproteasome family (Table 1). It remains to be determinedwhether these proteins are also constitutively expressed byoligodendrocytes in vivo or represent a stress response toculture conditions. Although the latter could represent adrawback for the use of in vitro cultures, Hochstrasser andcoworkers exploited stress features by studying postmortemCSF in order to identify molecules related to ischemic andneurodegenerative conditions (Lescuyer et al. 2004).

Validation of proteomic data

The validity of the outcome of a proteomic experiment isdependent on the stringency of the validation process. In thisstudy, the validation process included the search enginesSequest and Mascot. The validity of single-peptides identi-fications in proteomic research is open to debate (Carr et al.2004) and therefore the de novo sequencing algorithmLutefisk and the tag generating algorithm Inspect were usedto evaluate these identifications (on average 44% of theproteins encountered). In 96 of the 102 cases examined, thesequence returned by Sequest and Mascot matched the topranking sequence proposed by Lutefisk or Inspect. Finally,the presence of 20 of these so called ‘one hit wonders’ in theoligodendrocyte cultures was confirmed with both 2D-GEand 2D-LC approaches while for 21 others, their occurrencein oligodendrocytes or myelin was reported in literature.

Fig. 3 Percentage of proteins matched by a given number of peptides.

Protein extracts of mature rat oligodendrocytes were subjected to 2D-

GE or trypsin-digested and subjected to 2D-LC. Mass spectrometric

data coming from 2D-GE spots or from SCX-separated fractions

were validated, resulting in 155 and 86 confident protein identifications

for the 2D-GE and 2D-LC approach, respectively. Single-peptide protein

identifications amounted to 30% and 58% in the 2D-GE and 2D-LC

approach, respectively.



Table 1 Proteins identified in mature rat oligodendrocytes by means of 2D-GE and 2D-LC

Descriptiona Accession 2D-GEb 2D-LCc

1 14-3-3 Protein epsilon (Kawamoto et al. 2004; Satoh et al. 2003; Masters et al. 2002;

Berg et al. 2003)

P62260 5 5

2 14-3-3 Protein gamma P61982 7 1

3 14-3-3 Protein sigma Q9JJ20 3

4 14-3-3 Protein tau P68255 6 1

5 14-3-3 Protein zeta/delta P63102 7 2

6 26S Proteasome subunit p40.5 Q9WVJ2 3

7 26S Proteasome-associated pad1 homolog Q9CSU2 3

8 3(2), 5-Bisphosphate nucleotidase Q9Z1 N4 2

9 40S Ribosomal protein SA P38983 5

10 40S Ribosomal protein S20 P60868 1

11 60S Acidic ribosomal protein P0 P19945 3



14 6-Phosphogluconolactonase Q9CQ60 4

15 Actin, cytoplasmic 1 (Kajikawa et al. 1991) P60711 10 9

16 Actin-like protein 2 P61161 2

17 Acyl-CoA-binding protein P11030 1

AHNAK-related protein Q8VHJ0 1d

18 Alcohol dehydrogenase (Baslow et al. 2000) P51635 2

19 Aldolase reductase P07943 5

20 Allograft inflammatory factor-1 P55009 1

21 Alpha crystallin B chain (Goldbaum and Richter-Landsberg 2004; Dabir et al. 2004; Yaguchi

et al. 2003; Taylor et al. 2004)

P23928 1

22 Alpha enolase (Taylor et al. 2004) P04764 12 1

23 Alpha-centractin P61164 4

24 Alpha-soluble NSF attachment protein Q9DB05 4

25 Annexin A1 P07150 5

26 Annexin A2 Q07936 4

27 Annexin A3 P14669 8 1

28 Annexin A4 (Eberhard et al. 1994) P55260 4

29 Annexin A5 (Spreca et al. 1992) P14668 7

30 APC binding protein EB1 O35879 2

31 Apolipoprotein E precursor (Calenda et al. 1995) P02650 1

32 ATP synthase coupling factor 6, mitochondrial precursor P21571 1

33 ATP synthase beta chain (Taylor et al. 2004) P10719 9

34 ATP synthase D chain (Taylor et al. 2004) P31399 1

35 Beta-galactoside-binding lectin P11762 1

36 Calmodulin (Kovacs and Gulya 2002) P62161 2

37 Calpain smal subunit 1 (Ray et al. 2002) Q64537 1

38 Calponin H2 Q08093 4

39 Calnexin precursor (Eckhardt et al. 2005), (Denzel et al. 2002) P35565 1

40 Calreticulin precursor (Simpson et al. 1997), (Gudz et al. 2002) P18418 4

41 Calumenin precursor O35783 1

42 Gelsolin-like capping protein Q6AYC4 3 2

43 Carbonic anhydrase 2 (Halmi et al. 2006), (Cerghet et al. 2006) P27139 3

44 Catechol O-methyltransferase (Karhunen et al. 1995) P22734 1

45 Cathepsin B (Matsui et al. 1990) P00787 2

46 Chloride intracellular channel protein 1 Q9Z1Q5 5

47 Chloride intracellular channel protein 4 Q9QYB1 6

48 Cofilin (Fox et al. 2006; Taylor et al. 2004) P45592 2 2

49 Cofilin 2 P45591 1

50 Creatine kinase (Manos and Bryan 1993), (Kuzhikandathil and Molloy 1994) P07335 7 4

51 Cystatin B (Riccio et al. 2005) P01048 1



Table 1 Continued


52 Cysteine and glycine-rich protein 1 P47875 1

53 Cytochrome b-5 P00173 1

54 Dermcidin Q71DI1 1

55 Destrin (Lena et al. 1991) Q9R0P5 1 1

56 Dihydropteridine reductase P11348 6

57 Dihydropyrimidinase related protein 2 (Vanrobaeys et al. 2005) P47942 7 1

58 Dimethylarginase 1 Q9CWS0 3

59 Disabled homolog 2 O88797 1

60 DJ-1protein O88767 5

61 DnaJ (HSP40) homolog subfamily B member 11 Q99KV1 1

62 EF-hand domain-containing protein 2 Q9D8Y0 3

63 Electron tranfer flavoprotein alpha sunubit Q99LC5 1

64 Elongation factor 1 - alpha 1 (Vanrobaeys et al. 2005; Barbarese et al. 1995) P62632 1

65 Elongation factor 1 - alpha 2 (Barbarese et al. 1995) P62630 2

66 Endoplasmic reticulum protein ERp29 precursor (MacLeod et al. 2004; MacLeod et al. 2004) P52555 5 1

67 Endoplasmin (GRP-94) (Duzhak et al. 2003) P08113 11

68 Enolase 2 (Sensenbrenner et al. 1997) P07323 7

Epididymal secretory protein 1 precursor Q8CHN5 1d

69 Eukaryotic translation elongation factor 1 beta 2 (Barbarese et al. 1995) XP_32044 1

70 Eukaryotic translation initiation factor 2, subunit 2 beta (Dietrich et al. 2005) Q6P685 1

71 Eukaryotic translation factor 1 delta Q68FR9 2

72 Eukaryotic translation initiation factor 3 subunit 5 Q9DCH4 1

73 F-actin capping protein alpha-1 subunit P47753 2

74 F-actin capping protein alpha-2 subunit P47754 2

Fatty acid binding protein, heart P07483 1d

75 Fatty acid-biding protein, brain P51880 1

76 Fatty acid-binding protein,epidermal (Scarlato et al. 2000) P55053 1

77 Ferritin light chain (Quintana et al. 2006) Q7TP54 1

78 Filamin A Q8BTM8 1

79 Fructose-bisphosphate aldolase A P05064 5 1

80 Fumarylacetoaetate hydrolase P25093 4

81 Galactokinase 1 Q9R0N0 1

82 Galectin-3 (Deininger et al. 2002) P08699 4 3

83 GDP dissociation inhibitor beta 2 Q61598 3

84 Gelsolin (Liu et al. 2003) Q68FP1 1 1

85 Glia maturation factor, beta (Scarlato et al. 2000) Q63228 2

86 Glucose-regulated protein 78 (GRP-78) (Aquino et al. 1997) P06761 12 7

87 Glutamine synthetase (Warringa et al. 1988), (Werner et al. 2001) P09606 7 2

88 Glutathione S-transferase P (Cammer and Zhang 1992) P04906 3 1

89 Glyceraldehyde-3-phosphate dehydrogenase (Grinspan et al. 1993) P04797 4 1

90 Glycerol-3-phosphate dehydrogenase (Cheng and De Vellis 2000) P13707 8

91 Glycolipid transfer protein (GLTP) Q9JL62 1

92 Glyoxalase 1 Q6P7Q4 1

93 Guanidinoacetata methyltransferase (Tachikawa et al. 2004) P10868 2

94 Guanine nucleotide-binding protein beta subunit 2-like 1(Vanrobaeys et al. 2005) P63245 6

95 Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1 (Vanrobaeys et al. 2005) P54311 4

96 Guanine nucleotide-binding protein G(o),alpha subunit 2 (Taylor et al. 2004) P30033 5

97 Heat shock-related 70 kDa protein 2 P14659 1

98 Heat-shock protein beta-1 (HSP27) (Aquino et al. 1997) P42930 1

99 Heat-shock 70 kDa protein 1A/1B (Cwiklinska et al. 2003),(Goldbaum and Richter-Landsberg 2001) Q07439 1

100 Heat-shock protein 86 ( heat-shock protein 1-alpha) Q91XW0 3

101 Hemiferrin Q64599 5 2

102 Heterogeneous nuclear ribonucleoprotein P61980 4 1

103 High mobility group protein 1 (Daston and Ratner 1994) P63159 1



Table 1 Continued


104 Histon H2B (Shen et al. 2005) Q00715 1

105 Hypoxanthine-guanine phosphoribosyltransferase P27605 1

Hypothetical protein LOC500088 Q5M7U5 1d

106 Inositol-1-monophosphatase P97697 3

107 Insulin-like growth factor binding protein 2 P12843 1

108 Isocitrate dehydrogenase 3 (Vanrobaeys et al. 2005; Minich et al. 2003) Q99NA5 5

109 LIM and SH3 domain protein Q61792 3

110 LIM protein P52944 2

111 L-lactate dehydrogenase A chain (Warringa et al. 1988) P04642 2

112 L-lactate dehydrogenase B chain (Warringa et al. 1988) P42123 6

113 Lymphocyte cytosolic protein 1 (L-plastin) Q5XI38 1

114 Macrophage migration inhibitor factor (Vanrobaeys et al. 2005) P30904 1

115 Malate dehydrogenase, cytoplamic (Taylor et al. 2004) P14152 5

116 Malate dehydrogenase,mitochondrial precursor (Oh et al. 1991) P04636 5

117 Microtubule-associated protein 4 (Vouyiouklis and Brophy 1995) Q5M7W5 1

118 Mitochondrial H+-ATP synthase alpha subunit P15999 1

119 Myelin basic protein (Allinquant et al. 1991) P02688 2 1

120 Myosin light chain Q64119 1 2

121 Myosin regulatory light chain 2-A (Thomas et al. 2002) P13832 1 1

122 Myristoylated alanine-rich C-kinase substrate (Bhat 1995) P30009 1 1

123 N-acetylneuraminic acid synthase Q3TFB5 1

124 NAD-dependent deacetylase sirtuin 2 (Vanrobaeys et al. 2005) Q8VDQ8 5

Nestin P21263 1d

125 Non-muscle caldesmon Q62736 3

126 NSFL1 cofactor p47 O35987 2

127 Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) Q9EPJ0 1

128 Nuclease sensitive element binding protein 1 P62961 3

129 Nucleic acid binding factor pRM10 Q9Z0U8 1

130 Nucleolin P13383 2

131 Nucleophosmin P13084 2

132 Nucleoside diphosphate kinase B (Vanrobaeys et al. 2005) Q01768 2

133 Peptidyl-prolyl cis-trans isomerase A (Vanrobaeys et al. 2005) P10111 3 1

134 Peroxiredoxin 1 (Taylor et al. 2004) Q63716 4

135 Peroxiredoxin 2 (Vanrobaeys et al. 2005) Q61171 2

136 Peroxiredoxin 5 Q9R063 1 2

137 Peroxiredoxin 6 O35244 2

138 Phosphatidyl inositol transfer protein (Vuletic et al. 2003) P16446 2

139 Phosphatidylethanolamine-binding protein (Moore et al. 1996) P31044 6 4

140 Phosphoglycerate kinase (Taylor et al. 2004);(Vanrobaeys et al. 2005) P16617 3

141 Phosphoglycerate mutase (Taylor et al. 2004);(Vanrobaeys et al. 2005) P25113 1 2

142 Plasminogen activator inhibitor 1 RNA-binding protein Q62989 1

143 Poly(rc)-binding protein 1 P60335 4

144 Proinhibitin P67779 7

145 Protasome activator complex subunit 1 or 2 (Q63798) P97371 1

146 Proteasome subunit alpha type 1 P18420 1




150 Proteasome subunit alpha type3-like O70435 3

151 Proteasome subunit beta type 1 P18421 3

152 Proteasome subunit beta type 3 Q9R1P1 1

153 Proteasome unit alpha type 5 Q9Z2U1 4

154 Protein disulfide isomerase-related protein A6 Q63081 2

155 Protein disulfide-isomerase precursor (Bauer et al. 2002) P04785 3



2D-GE versus 2D-LC

2D-GE displays an unprecedented fractionation power.However, as stated in the introduction, 2D-GE does notprovide sufficient insight into several important protein

populations. Therefore, a 2D-LC approach using a gel-freefractionation of oligodendrocyte tryptic peptides was applied.

Trypsinization is not hampered by a gel matrix and noextraction of resulting tryptic peptides is needed. Therefore,

Table 1 Continued


156 Protein disulfide isomerase A3 GRP58 P11598 10

157 Protein phosphatase type 2A catalytic subunit alpha (Scarlato et al. 2000) P63331 4

158 Purine nucleoside phosphorylase P23492 3

Pyrroline-5-carboxylate reducase member 2 Q6AY23 1d

159 Pyruvate dehydrogenase E1 P49432 3

160 Pyruvate kinase, isozymes M1/M2 (Mostert et al. 1986) Q6P7S0 2

161 Reticulon 4 (Nogo) (Taketomi et al. 2002) Q9JK11 2

162 Rho GDP-dissociation inhibitor 1 (Taylor et al. 2004) Q5XI73 5 5

163 Ribonuclease inhibitor P29315 4

164 Ribosomal protein S27a (polyubiquitin) P62982 1 2

165 Ribosome-bindingprotein1 Q99PL5 1

166 S100a11 (calgizzarin) P50543 1 1

167 S100 protein, beta polypeptide (Hachem et al. 2005) P04631 1

168 Sec1311 protein Q5XFW8 2

169 Secernin Q9CZC8 3

170 Septin 2 (Taylor et al. 2004) P42208 1

171 Serine-threonine kinase receptor-associated protein Q9CZC8 1

172 SET protein (Scarlato et al. 2000) Q63945 2

173 Skeletal muscle LIM-3 protein 3 O70433 2

174 Sulfated glycoprotein 1 (prosaposin) P10960 3

175 Superoxide dismutase (Mn), mitochondrial precursor (Bernardo et al. 2003), (Baud et al. 2004) P07895 3 1

176 Superoxide dismutase Cu,Zn (Thaete et al. 1985), (Scarlato et al. 2000) P07632 2 2

177 Synaptic vesicle membrane protein VAT-1 homolog Q62465 1

178 Target of myb1 homolog Q5XI21 1

179 Thioredoxin P10639 1 1

180 Thioredoxin-related protein Q920J4 1

181 Transaldolase (Niland et al. 2005) Q9EQS0 2

182 Transforming protein RhoA (Liang et al. 2004) P61589 1

183 Transgelin P31232 7 1

184 Transgelin 2 Q9WVA 4 1

185 Transitional endoplasmic reticulum ATPase P63029 14

186 Translationally controlled tumor protein P14701 2 1

187 Triosephosphate isomerase P48500 5 2

188 Tropomyosin 1 alpha chain P04692 5

189 Tropomyosin alpha4 chain P09495 10

190 Tropomyosin isoform 6 (Had et al. 1993) Q63610 3

191 Trypsin I, anionic precursor P00762 1

192 Tubulin alpha 1 chain (Song et al. 1999) P68370 2 5

193 Tubulin beta (Song et al. 1999) Q6P9T8 3

194 Ubiquinol-cytochrome C reductase complex core protein I Q9CZ13 1

195 Ubiquinol-cytochrome C reductase complex 11 kDa protein P99028 1

196 Ubiquitin carboxyl-terminal hydrolase isoenzyme L1 (Taylor et al. 2004) Q00981 2

197 Ubiquitin thiolesterase protein Q7TQI3 1

198 Vimentin (Meyer et al. 1989) P31000 12

199 Vacuolar ATP synthase subunit d P51863 2

200 Voltage-dependent anion-selective channel protein 2 Q60930 4

aReferences of studies reporting the identified protein in oligodendrocytes or myelin are indicated. bNumber of sibling peptides identified

via 2D-GE. cNumber of sibling peptides identified via 2D-LC. dSingle-peptide protein identifications that could not be confirmed with Lutefisk

or Inspect.



higher peptide recoveries could be expected favouring thedetection of lower-abundant proteins in the oligodendrocyteproteome. Indeed, while only few DNA/RNA-binding pro-teins (representing proteins present in low-copy numbers)were detected with the 2D-GE approach, 2D-LC couldidentify multiple members of this functional group.

In addition, a gel-free approach holds promise to becapable to deal with membrane proteins. Indeed, 2D-LC diddetect a number of membrane proteins. The protein extractswere prepared in the presence of CHAPS, therefore it was

not surprising that a similar percentage of proteins wasdetected upon 2D-GE analysis. However, initial solubiliza-tion and extraction is not the only factor at play. Precipitationevents during IEF also strongly hamper 2D-GE analysis of

Fig. 4 Total counts of protein identifications in mature rat oligo-

dendrocytes for two proteomic strategies. Protein extracts of mature

rat oligodendrocytes were separated by 2D-GE or trypsin-digested

and subjected to 2D-LC, both followed by LC-ESI-MS/MS identifica-

tion and validation. Forty-one protein identifications were found in

common, while 114 and 45 proteins were only detected in the 2D-GE

and 2D-LC approach, respectively.

(b)

(a)

Fig. 5 Molecular mass (a) and isoelectric point (b) of oligodendro-

cyte proteins identified via 2D-GE and 2D-LC. Theoretical physico-

chemical parameters (pI: isoelectric point and Mw: molecular mass) of

the oligodendrocyte proteins identified performing the 2D-GE and

2D-LC approach are displayed.

(a)

(b)

ProteasomeIysosome

Membrane

Membrane

Extracellular

Extracellular

Cytoskeleton

Cytoskeleton

Ribosome

Ribosome

Mitochondria

Mitochondria

Endoplasmaticreticulum

Endoplasmaticreticulum

Nucleus

Nucleus

Cytosol

Cytosol

Fig. 6 Subcellular localization of proteins in cultured mature rat

oligodendrocytes as identified via 2D-GE (a) and 2D-LC (b). Oligo-

dendrocyte proteins confidently identified with the 2D-GE or 2D-LC

strategy were classified according to their subcellular localization

using the ‘GO biological compartment’ annotation at SwissProt or via

literature references.

(a)

(b)

Other

Other

Cell signaling/cell cycleregulation

Cell signaling/cell cycleregulation

Metabolism

Metabolism

Proteinsynthesis/assembly/

modification

Proteinsynthesis/assembly/

modification

Calcium-binding

Calcium-binding

Stress

Stress

Cytoskeleton/actin-binding/filament

Cytoskeleton/actin-binding/filament

CNS development/homeostasis

CNS development/homeostasis

DNA/RNA-binding

DNA/RNA-binding

Proteolysis,protease (inhibitor)

Proteolysis,protease (inhibitor)

Fig. 7 Functional annotation of cultured mature rat oligodendrocyte

proteins as identified via 2D-GE (a) and 2D-LC (b). Oligodendrocyte

proteins confidently identified with the 2D-GE or 2D-LC strategy were

classified according to their function using the ‘GO function’ or ‘GO

biological process’ annotation at SwissProt or via literature references.



membrane proteins, intuitively rendering gel-free approachessuch as 2D-LC more suitable for analysis for membraneproteins. Nevertheless, additional measures should be takenin the sample preparation and isoelectric focusing procedures(e.g. SDS as a solubilization agent and an adjusted focusingmedium) to truly assess the potential of 2D-GE versus2D-LC for membrane proteomics.

Finally, the 2D-LC technique tends to extend to a largeridentification platform with respect to pI and Mw parameters(Dumont et al. 2006) evidenced by the higher percentages ofprotein identifications in the upper and lower pI and Mwrange.

Study outcome

The assembly of 2D-GE and 2D-LC experiments performedon the oligodendrocyte cultures resulted in the identification ofin total 200 proteins, including known oligodendrocytes ormyelin proteins. The novel oligodendrocyte proteins embodya wide variety of cellular functions. Notably, macrophagemigration inhibitory factor (MIF) belongs to the cytokineprotein family and has been implicated in the pathogenesis ofinflammatory and autoimmune disease. Oligodendrocyteshave traditionally been considered passive bystanders ininflammatory reactions. However, oligodendrocytes wereshown to display a diverse cytokine receptor repertoire(D’Souza et al. 1996; Bonetti and Raine 1997; Cannella andRaine 2004). In addition, the identification of MIF inoligodendrocytes could ad a new member to the small list ofproinflammatory cytokines produced by oligodendrocytes andsupports the growing evidence that oligodendrocytes are notimmunologically inert. The identification of MIF is supportedby a proteomic study on myelin (Vanrobaeys et al. 2005).

Future perspectives

The presented 2D-GE and 2D-LC maps of these culturedrodent oligodendrocytes will serve as a reference point forfuture quantitative proteomic studies in which we willanalyze the effect of various culture conditions (cytokines,neurokines, ischemia) on the proteome of oligodendrocytes.In addition, proteome maps of mature versus immaturerodent oligodendrocytes aided by quantitative proteomicapproaches may provide a tool to simultaneously study theregulation of an array of proteins involved in differentiation(e.g., epidermal fatty acid-binding protein, glia maturationfactor, polyubiquitin, protein phosphatase 2A and Setprotein, all identified in this study). Finally, comparison ofprotein patters of oligodendrocytes derived from controlversus diseased animals (eg experimental autoimmuneencephalomyelitis (EAE), an animal model for multiplesclerosis) may point to important players in disease patho-genesis. Taken together, differentially expressed proteins canshed light on the destructive, protective or differentiationmechanisms in oligodendrocytes and may gain furtherinsights into demyelinating CNS pathologies.

Final remarks

This study is the first proteomic study of mature rat oligo-dendrocytes. The assembly of 2D-GE and 2D-LC experi-ments performed on the oligodendrocyte cultures followedby the validation strategy resulted in a confident identifica-tion of in total 200 proteins. 2D-LC proved to be a valuablefractionation method allowing for the detection of proteinsthat may escape analysis with 2D-GE. This study illustratesthat 2D-GE and 2D-LC should be used in parallel to allowfor a larger identification platform and emphasize the powerof the used proteomic approach to study known and to revealnew oligodendrocyte proteins. The constructed proteomemap in addition provides a powerful tool to assess differen-tiation, survival or death of oligodendrocytes in experimentalmodels of diseases.

Acknowledgements

The authors thank Veronique Haesen, Erik Royackers, Pierre

Dumont, Natalia Kwasnikowska, Marc Jans, Jeanine Santermans,

Marie-Josee Sleypen, Igna Rutten and Wilfried Leyssens for

excellent technical assistance. Leen De Ryck, Jerome Hendriks,

Kurt Baeten and Leen Slaets are highly acknowledged for helpful

discussions. This work was financially supported by the Belgian

‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO)’ and

grants from the transnationale Universiteit Limburg and the

‘Bijzonder Onderzoeksfonds’ of Hasselt University. D. Dumont

holds a fellowship from the Belgian ‘Wetenschappelijk Onderzoek

Multiple Sclerosis’ (WOMS) Foundation.

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Characterization of mature rat oligodendrocytes: a proteomic approach

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