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[Full Paper]Proteomic identification of oxidative-stress-reporting biomarkers differentiallysecreted from human neuroblastoma SH-SY5Y cells
Tosifusa Toda1, Megumi Nakamura1, Hiraku Morisawa1 and Mikako Hirota1, 2
SUMMARY

The free-radical theory predicts that the oxidative stress accelerates the rate of aging
and increases the onset of degenerative disorders in the elderly. Dopaminergic neurons areespecially vulnerable to age-related neuronal disorders due to reactive oxygen species generated in the pathway of dopamine metabolism. Biochemical changes occurring in substantia nigra of Parkinson’s disease patients suggest that the oxidative-stress-induced cell
damages may be involved in the neurodegeneration. In our previous researches, we foundthat the dephosphorylation of elongation factor-2 and phosphorylation of nuclear lamin A/
C might be neuronal cell specific response to oxidative stress. (Nakamura et al. BBA,1763(9), 977–989, 2006)
The dephosphorylation and phosphorylation of those proteins are significant biomarkers
for analyzing the molecular mechanisms of the stress response, however, such a phosphoproteome analysis is thought to be inappropriate for clinical investigation of neurodegeneration if it was not detectable in cerebrospinal fluid or serum of patients. Thus, weproceeded to the 2D-DIGE analysis of secretome, proteome of secreted proteins, using theculture system in which oxidative stress was applied to human SH-SY5Y neuroblastoma
cells. As the result of our secretome analysis, we identified ubiquitin-activating enzyme E1,ubiquitin-conjugating enzyme E2 N, ubiquitin C-terminal hydrorase-L1, 14-3-3 protein
isoforms, Rab GDP dissociation inhibitor β, Rho GDP-dissociation inhibitor 1, peroxiredoxin-2, glutathione S-transferase P, α enolase, LDH B chain as oxidative-stress-reporting
biomarker candidates.
Key words: proteome, secretome, oxidative stress, neuroblastoma, SH-SY5Y.
INTRODUCTION
Reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) are inevitably generated in all living cells as
by-products of electron transport in mitochondria and redox
enzyme reactions in the cytoplasm1, 2). Oxidative stressinduced with ROS and RNS is suspected to be a major causeof chronic neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. Dopamine neurons
are especially vulnerable to age-related disorders due to1
2

exposure to high level ROS generated normally as part of
dopamine metabolism3). Oxidative stress may cause neuronal cell dysfunctions through oxidative modification ofmacromolecules including proteins. The neuronal cells
must have a protective system against oxidative stress to
avoid inordinate cell death. However, little has been known
about which proteins in dopamine neurons are involved in
the response to oxidative stress.
In order to analyze proteins involved in the stressresponse, we established an in-vitro culture system inResearch Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology.
Faculty of Pharmaceutical Sciences, Teikyo Heisei University.
Correspondence address: Tosifusa Toda; Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology,35-2 Sakaecho, Itabashi, Tokyo 173-0015, Japan.
Abbreviations: 2D-DIGE, two-dimensional difference gel electrophoresis; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gelelectrophoresis; 6-OHDA, 6-hydroxydopamine; DTT, dithiothreitol; MALDI-TOF MS, matrix-assisted laser-desorption time-of-flightmass spectrometry; CHCA, α-cyano-4-hydroxycinnamic acid; TFA, trifluoroacetic acid; PBS, phosphate-buffered saline; D-MEM, Dulbecco’s minumum essential medium; HEPES, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonicacid; DMSO, dimethyl sulfoxide.
(Recieved October 1, 2007, Accepted October 15, 2007, Published December 15, 2007)

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which cellular response to oxidative stress could be
detected by proteomic analysis. In our previous study, we
examined changes in whole proteins and phosphoproteinsof human dopaminergic neuroblastoma cell line SH-SY5Y
under oxidative stress induced with dopaminergic neurotoxin 6-OHDA4, 5). Total proteins of SH-SY5Y cells at various stages of oxidative stress were separated by 2-D PAGE
and compared quantitatively each other. Increases in cellular levels of glutathione S-transferase P and heat shock cognate 71 kDa protein were detected by computer-aided
image analysis. Stress-induced alterations in protein
phosphorylation were also detected by Pro-Q Diamondstaining. Elongation factor 2, lamin A/C, T-complex protein1, and heterogeneous nuclear ribonucleoprotein H3 wereidentified by MALDI-TOF MS as stress-responsive
phosphoproteins6).
However, those intracellular phosphoproteins are notdirectly applicable to clinical investigations of the earlystages of neurodegeneration. Thus in this paper, we report
the result of proteomic identification of oxidative-stressreporting biomarker proteins differentially secreted fromhuman neuroblastoma SH-SY5Y cell under 6-OHDAinduced oxidative stress.

were transferred to centrifuge tubes to remove cell debris
by centrifugation.
Fluorescent labeling of proteins
Fluorescent labeling (FL) of proteins in the culture
media was carried out according to the optimized proceduredescribed in Fig. 1. In brief, each culture medium (5-ml persample) was concentrated with Amicon Ultra 10,000
MWCO centrifugal filter device (Millipore) to be about
0.1 ml. Proteins were then precipitated with 10-fold volumeof cold acetone (–20°C) and re-dissolved in 50 µl of FL
Buffer (8 M urea, 2% (v/v) Triton-X100, 0.2% (w/v) SDS,
10 mM HEPES-NaOH, pH 8.6). The protein concentration
was assayed by a modified Lowry’s method using the RC
DC Protein Assay Kit (Bio-Rad), and adjusted to 10 mg/ml
by dilution. A 15-µl aliquot of the sample solution containing 0.15 mg protein was mixed with 1.5 µl of 0.4 mM IC3OSu or IC5-OSu dissolved in DMSO. The FL was carriedout at room temperature for 15 min in the dark, and thenterminated by the addition of 10 mM ethanolamine-HCl,
pH 8.6. The IC3-labeled samples were combined with theirMATERIALS AND METHODS
Materials
Immobiline DryStrips (pH 4–7, 18 cm) and Pharmalyte
were purchased from GE Healthcare Bioscience (Tokyo,Japan). IC3-OSu and IC5-OSu were products of Dojindo
Laboratories (Kumamoto, Japan). SYPRO Ruby Protein Gel
Stain was from Invitrogen. CoolPhorStar 2-DE Apparatus
System was from Anatech (Tokyo, Japan). Pharos FX,
PDQuest system and RC DC protein assay kit were fromBio-Rad Laboratories Inc. (Tokyo, Japan). SDS, Tris,Tricine, DTT, TFA, CHCA, DMSO and protease inhibitor
cocktail were from Sigma-Aldrich (St. Louis, MO, USA).
Other chemicals of HPLC grade were from Wako Pure
Chemical Industries Ltd. (Osaka, Japan). Trypsin was purchased from Promega (Madison, WI, USA).
Cell culture and induction of oxidative-stress response
The dopaminergic neuroblastoma cell line SH-SY5Y wasobtained from the Human Science Research Resources
Bank (Osaka, Japan). The cells were cultured in D-MEM(Gibco) supplemented with 10% heat-inactivated fetal
bovine serum, streptomycin (0.1 mg/ml), and penicillin(100 units/ml) in a CO2 incubator (5% CO2 /95% air) at37°C.
Before inducing oxidative stress in the SH-SY5Y cellswith 6-OHDA, attached cells and the culture dishes wererinsed twice with PBS to remove bovine serum proteins.
The cells were then exposed to various concentrations of 6OHDA (0, 10, 25 and 50 µM) in a serum-free D-MEM withN2-supplement. After 24-h incubation, the culture media

Fig. 1. The procedure for 2-D secretome mapping and 2D-DIGE
analysis.
For secretome mapping, non-labeled proteins were separated by 2-DE and stained with SYPRO Ruby. For 2D-DIGE
analysis, proteins were labeled with IC3-OSu or IC5-OSu,
combined each other and separated by 2-DE.

J Electrophoresis

IC5-labeled counterparts for differential analysis. Proteinsin the coupled samples were precipitated again with 10-foldvolume of cold acetone, and dissolved in 20 µl of 2-DE Sample Buffer (8.5 M urea, 2% (v/v) Triton X-100, 0.2% (w/v)
SDS, 10 mg/ml DTT, 2% (v/v) Pharmalyte 3–10).

2007 ; 51 : 23

MALDI-TOF MS and protein identification
MALDI-TOF MS was performed using AXIMA-CFR(Shimadzu), and protein identification was done with thehelp of the Mascot search engine (Matrix Science).
RESULTS

2-D PAGE and protein staining
2-DE for secretome mapping and 2D-DIGE was carriedout according to our standard method7), of which the updated version was made public on our web site (http://www.proteome.jp/2D/2D_method.html). In the first
dimensional separation, immobilized pH-gradient isoelectric focusing was carried out using Immobiline DryStrip(pH 4–7, 18-cm long) in the CoolPhoreStar horizontal electrophoresis system (Anatech). Tricine-SDS-PAGE was run
on a 7.5%T slab gel in the second-dimensional separation.
Fluorescence imaging of IC3/5-labeled proteins was performed using the 532-nm laser and the 605-nm band-passfilter for IC3-labeled proteins, the 635-nm laser and the695-nm band-pass filter for IC5-labeled proteins, respectively on the Pharos FX system (Bio-Rad). Differential display and quantitative spot analysis were done using thePDQuest software. Non-labeled proteins for secretome
mapping were visualized by staining with SYPRO Ruby
after 2-DE.
Spot picking and in-gel digestion
Spot picking was performed using EXQuest Spot Cutter(Bio-Rad). In-gel tryptic digestion was carried according toour optimized procedure shown in our web site.

Secretome mapping of the human SH-SY5Y neuroblastoma cells under oxidative stressProteins, secreted from human SH-SY5Y neuroblastoma
cells into the serum-free medium containing 25 µM 6OHDA, were separated by 2-DE and visualized by stainingwith SYPRO Ruby (Fig. 2). The protein spots numbered in
the figure were excised from the slab gel. Protein identification was performed by in-gel tryptic digestion and peptide
mass fingerprinting. The identified proteins were listed inTable 1. The major spots pointed with arrowheads wereserotransferrin supplemented to the serum-free culture
medium as N2-supplement. Fifty-five spots on the map
were identified as secreted proteins from the SH-SY5Ycells.
Differential display of oxidative-stress-reporting biomarker proteins by 2D-DIGE
The IC3/5-labeled proteins were simultaneously separated on a single gel, and the differential gel image wasacquired and displayed using the PDQuest 2-D Gel Analysis
Software. The result of the differential display by 2D-DIGE
is shown in Fig. 3, in which the red color indicatesincreased proteins in the medium under the oxidativestress induced with 25 µM 6-OHDA for 24 h. The green
color indicates decreased proteins. The yellow color ofFig. 2. 2-D gel secretome map of SH-SY5Y cells under oxidative stress.
The major spots pointed with arrowheads were serotransferrin supplemented tothe serum-free culture medium as N2-supplement.

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Table 1. Identified proteines excreted by SH-SY5Y under oxidative stress
Spot
NumberSwiss-ProtProtein ID

Accession
Number

101
310
311
313
1216
1217
1218
1219
1220
1404
1512
1513
1705
1706
1809
2213
2214
2412
2610
2611
2816
2817
3207
3409
3410
3411
3412
3508
3613
3614
3615
3711
3712
3821
4108
4206
4413
46154705
5107
5108
5209
5312
5510
5611
6007
6209
6412
6513
6514
7309
7508
8508
8509
8510
8711

CALM_HUMAN
C1QBP_HUMAN
1433E_HUMAN
HS90B_HUMAN
1433G_HUMAN
GDIR_HUMAN
1433Z_HUMAN
1433E_HUMAN
1433B_HUMANRSSA_HUMAN
TBB5_HUMAN
TBB3_HUMAN
GRP78_HUMAN
MMP2_HUMAN
HS90A_HUMAN
TMM31_HUMAN
LMNB1_HUMAN
ACTB_HUMAN
TBA1A_HUMAN
TBA1A_HUMAN
TERA_HUMAN
DDB1_HUMAN
UCHL1_HUMAN
ACTB_HUMAN
ACTB_HUMAN
IF4A2_HUMAN
ILF2_HUMAN
TRFE_HUMAN
TCPE_HUMANFKBP4_HUMANFKBP4_HUMAN
HSP7C_HUMAN
HSP74_HUMAN
UBE1_HUMAN
PRDX2_HUMAN
GSTP1_HUMAN
CSN4_HUMAN
HSP71_HUMAN
GRP75_HUMAN
STMN1_HUMAN
NDKA_HUMAN
PARK7_HUMAN
LDHB_HUMAN
PRS7_HUMAN
TCPA_HUMANUBE2N_HUMAN
GRB2_HUMAN
DCUP_HUMAN
TRFE_HUMAN
GDIB_HUMAN
GSTO1_HUMAN
GDIB_HUMAN
TCPB_HUMANENOA_HUMANENOA_HUMAN
TRFE_HUMAN

P62158Q07021
P62258P08238P61981
P52565
P63104
P62258
P31946P08865
P07437
Q13509P11021
P08253
P07900Q5JXX7P20700
P60709
Q71U36
Q71U36
P55072
Q16531
P09936P60709
P60709Q14240Q12905
P02787
P48643Q02790Q02790P11142
P34932
P22314
P32119P09211
Q9BT78P08107
P38646
P16949P15531
Q99497
P07195
P35998P17987
P61088
P62993
P06132
P02787
P50395
P78417
P50395
P78371
P06733
P06733
P02787

Protein name

Synonums

Calmodulin
CaM
Complement component 1 Q subcomponent-binding protein Mitochondrial matrix protein p32
14-3-3 protein epsilon
14-3-3E
Heat shock protein HSP 90-beta
HSP 84, HSP 90
14-3-3 protein gamma
Protein kinase C inhibitor protein 1
Rho GDP-dissociation inhibitor 1
Rho-GDI alpha14-3-3 protein zeta/delta
14-3-3Z
14-3-3 protein epsilon
14-3-3E
14-3-3 protein beta/alphaProtein kinase C inhibitor protein 140S ribosomal protein SA
34/67 kDa laminin receptor
Tubulin beta chain
Tubulin beta-3 chain
78 kDa glucose-regulated protein
Heat shock 70 kDa protein 572 kDa type IV collagenase
Matrix metalloproteinase-2
Heat shock protein HSP 90-alphaRenal carcinoma antigen NY-REN-38
Transmembrane protein 31
Lamin-B1Actin, cytoplasmic 1Beta-actin
Tubulin alpha-1A chain
Tubulin alpha-1A chain
Transitional endoplasmic reticulum ATPase
DNA damage-binding protein 1
UV-damaged DNA-binding factor
Ubiquitin carboxyl-terminal hydrolase isozyme L1
UCH-L1
Actin, cytoplasmic 1
Beta-actin
Actin, cytoplasmic 1
Beta-actin
Eukaryotic initiation factor 4A-II
ATP-dependent RNA helicase eIF4A-2
Interleukin enhancer-binding factor 2
Nuclear factor of activated T-cells 45 kDa
Serotransferrin
Transferrin
T-complex protein 1 subunit epsilon
TCP-1-epsilon
FK506-binding protein 4
Peptidyl-prolyl cis-trans isomerase
FK506-binding protein 4
Peptidyl-prolyl cis-trans isomerase
Heat shock cognate 71 kDa protein
Heat shock 70 kDa protein 8
Heat shock 70 kDa protein 4
Heat shock 70-related protein APG-2
Ubiquitin-activating enzyme E1
A1S9 protein
Peroxiredoxin-2
Thioredoxin peroxidase 1
Glutathione S-transferase P
GST class-pi
COP9 signalosome complex subunit 4Signalosome subunit 4
Heat shock 70 kDa protein 1
HSP70.1, HSP70-1/HSP70-2
Stress-70 protein, mitochondrial
Mortalin, Heat shock 70 kDa protein 9Stathmin
Phosphoprotein p19
Nucleoside diphosphate kinase ANDP kinase AProtein DJ-1
Parkinson disease protein 7L-lactate dehydrogenase B chain
26S protease regulatory subunit 7Proteasome 26S subunit ATPase 2T-complex protein 1 subunit alphaTCP-1-alphaUbiquitin-conjugating enzyme E2 NUbiquitin-protein ligase NGrowth factor receptor-bound protein 2SH2/SH3 adapter GRB2
Uroporphyrinogen decarboxylase
URO-D
Serotransferrin
Transferrin
Rab GDP dissociation inhibitor beta
Rab GDI beta
Glutathione transferase omega-1
GSTO 1-1
Rab GDP dissociation inhibitor beta
Rab GDI beta
T-complex protein 1 subunit beta
TCP-1-beta
Alpha-enolase
Plasminogen-binding protein
Alpha-enolase
Plasminogen-binding protein
Serotransferrin
Transferrin

J Electrophoresis

2007 ; 51 : 25Fig. 3. Differential display of oxidative-stress-reporting biomarker proteins by 2D-DIGE.
The pseudo-colored image was generated using the “Multi-Channel Viewer” function of PDQuest software. The red color indicates increased proteins in the culture
medium of SH-SY5Y cells under the oxidative stress induced with 25 µM 6-OHDA
for 24 h. The insert indicates the survival of SH-SY5Y cells under various concentrations of 6-OHDA in the serum-free medium.

serotransferrin spots means the abundance of the protein in
culture medium is conserved during the 24-h incubation.
DISCUSSIONAs the results of our in-vitro research, Parkinson’s disease protein 7 (DJ-1), ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2 N, 26S protease regulatory
subunit 7, 14-3-3 protein isoforms (beta/alpha, gamma,epsilon), Rab GDP dissociation inhibitor beta, Rho GDPdissociation inhibitor 1, COP9 signalosome complex subunit 4, peroxiredoxin-2, glutathione S-transferase P, heatshock cognate 71 kDa protein, heat shock protein HSP 90alpha, 78 kDa glucose-regulated protein were identified asoxidative-stress-reporting biomarker candidates.
We expect that these proteins will be a potent molecularbiomarkers for monitoring early stage of neurodegeneration occurring under oxidative stress in elderly people ofadvanced age.
ACKNOWLEDGEMENT
This work was supported by the budget for Long-Term
Research Project in TMIG. The authors thank Dr.
Sakakibara of Miyazaki University for his technical advice
and Dr. Iwamoto for her cooperation in optimization of IC3/5-labeling. We really appreciate the technical supports
given by Mr. Sato and Mr. Ohkata of Bio-Rad Laboratories,and Mr. Yamada and Mr. Nishine of Shimadzu Biotech.
We really appreciate the technical supports given by Mr.
Sato and Mr. Ohkata of Bio-Rad Laboratories, Mr. Yamada
and Mr. Nishine of Shimadzu Biotech for MALDI-TOF MS.
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