J Electrophoresis

2007 ; 51 : 1[Full Paper]

The protein expression profile of cynomolgus monkey embryonic stem cells in
two-dimensional gel electrophoresis: a successful identificationof multiple proteins using human databases
Masako Nakahara1, Kumiko Saeki1, Yoshiko Yogiashi1, Akiko Kimura1,
Akiko Horiuchi1, Naoko Nakamura1, Asako Yoneda1, Koichi Saeki1,
Satoko Matsuyama1, Megumi Nakamura2, Tosifusa Toda2,
Yasushi Kondo3, Yasushi Kaburagi4 and Akira Yuo1
SUMMARY

Global gene and protein expression analyses have had great impacts on scientificprogresses in this new era of bioinformatics. Although studies using murine and humanmaterials can fully exploit the large volume of their databases, there are quite a few inconveniences for an investigation on non-human primate materials due to still insufficient datacollections. Here we examined the availability of human databases for the protein identification process using the two-dimensional electrophoresis-based proteomic study in cynomolgus monkey embryonic stem (ES) cells. Querying public human protein databases, we
successfully identified multiple protein spots via mass spectrometric analysis using
MALDI-TOF apparatus. The results of the protein identification were confirmed by western blotting using polyclonal antibodies raised against human epitopes. Interestingly, theresults of western blotting further identified the existence of previously unreported multiple isoforms of common proteins including glycolytic pathway enzymes. Thus, combined
analyses of the mass spectrometry querying the Homo sapience databases and the western
blotting using polyclonal antibodies is highly effective in determining protein expressions
in monkey cells. Our success in obtaining a draft protein expression profile of cynomolgus
monkey ES cells will contribute to the promotion of non-human primate ES cellresearches.
Key words: cynomolgus monkey, embryonic stem cells, proteomics, two-dimensional electrophoresis, mass spectrometric analysis.
INTRODUCTION
Recent years, researches on non-human primates are of
growing importance in the fields of life science. It has beenemphasized that animal studies with clinical concerns suchas a toxicological study of environmental factors and safety1evaluation of newly invented drugs should be performed
using primate, but not rodent, models. Although studies
using rodents are feasible in technical and economical
points of view, they do not always provide sufficient informations applicable to human cases. The differences inmetabolism and tissue sensitivity of the drug between

Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan.
Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
3
Advanced Medical Research Laboratories, Tanabe Seiyaku Co. Ltd., Osaka, Japan.
4
Department of Metabolic Disorder, Research Institute, International Medical Center of Japan, Tokyo, Japan.
Correspondence address: Akira Yuo; Department of Hematology, Research Institute, International Medical Center of Japan, 1-21-1,
Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
Abbreviations: ES, embryonic stem; 2-DE, two-dimensional gel electrophoresis; MEF, murine embryonic fibroblast; MMC, mitomycin
C; SDS, sodium dodecyl sulphate; PMF, peptide mass fingerprinting; HSP60, 60-kDa heat shock protein; HSC70, heat shock cognate
71-kDa protein; TIM, triosephosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VDAC-1, voltage-dependentanion-selective channel protein 1; PKM2, pyruvate kinase isozyme M2; PGK1, phosphoglycerate kinase 1.
(Received October 16, 2006, Accepted December 22, 2006, Published March 15, 2007)
2

J Electrophoresis

2007 ; 51 : 2rodents and primates are not negligible. In addition, theshort lifetime of rodents cannot afford the longtime, chronic
effect of drugs. The primate study is also essential tounderstand the molecular mechanism of diseases. An integrated study on genome, transcriptome or proteome, which
has become a powerful tool to elucidate the complicatedmechanisms of human multi-factorial diseases, should
better be performed using primate, but not rodent, models.
Indeed, growing numbers of pedigrees of cynomolgus monkeys that provide excellent models for human diseases
have been prepared1, 2).
In addition to the living individuals, cell lines establishedfrom monkey tissues play beneficial roles. For example,
embryonic stem (ES) cells, which are a valuable resource inregenerative medicine because of their high capacity todifferentiate into a broad range of cell types, have particularly large impacts. It is known that primate ES cells show
different characteristics from murine ES cells: they havedifferent extracellular and intracellular signaling pathways
for the maintenance of pluripotency3) and distinctive differentiation capacities4). Thus, the promotion of researches on
primate ES cells is now becoming exceedingly important.
Although studies using human ES cells are essential forclinical application, basic researches using monkey ES cells
still have importance because they can provide good allotransplantation models required for pre-clinical studies5, 6).
Moreover, ethical regulation that is heavily imposed on theusage of human ES cells is remissive concerning the use ofmonkey ES cells. Thus biotechnological manipulation,
including gene transfer, can immediately be applied to theusage of monkey ES cells, which will contribute to thefurther advance in our understanding of human ES cells.
Despite an increasing requirement to promote the monkey ES cell study, construction of integrated bioinformatics
on non-human primates is still underway, and only a small
volume of individually collected data is available at present.
As a result, we often experience difficulties in constructingmonkey polymerase chain reaction primers and the primers
designed from human databases do not necessarily work inmonkey samples. As compared with the informations on
genes or messages, data on proteins are rather compressed
due to three-to-one correspondence, where a train of three
nucleotides corresponds to one amino acid and the last
nucleotide in each train has a large redundancy. In addition,
degrees of freedom in amino acid sequence are lower thanthose in nucleotide sequences due to functional requirement of proteins. Thus, in contrast to the genomic and transcriptomic studies, proteomic analysis of monkey samples
might be effectively achieved querying human databases.
In the present study, we successfully determined a protein expression profile using the two-dimensional gel electrophoresis (2-DE) and human proteome databases in
undifferentiated cynomolgus monkey ES cells. Our results
will encourage the promotion of the monkey proteome

study in the present situation, without waiting for the
future accomplishment of the data construction of monkey
bioinformatics.
MATERIALS AND METHODS
1. Cells culture
Murine embryonic fibroblasts (MEFs), which had beentreated with Dulbecco’s modified Eagle’s medium containing mitomycin C (MMC) for 3 hours, were seeded on thedishes coated with 0.1% gelatin. Cynomolgus monkey ES
cells7) were maintained on MMC-treated MEF-coateddishes in DMEM/F12 medium supplemented with 20%
heat inactivated fetal bovine serum, 8 ng/ml fibroblast
growth factor 2, 10 ng/ml recombinant human bone morphogenic protein 4, 1 mM β-mercaptoethanol, 1 mM Lglutamine, 10 U/ml penicillin and 10 µg/ml streptomycin.
Monkey ES cells were passaged every 2 days using 0.25%
trypsin treatment for one minutes and were seeded at split
ratios of 1:2 to 1:4 on new MEF-coated dishes. For thecollection of ES cells for 2-DE, ES cells were detached by
0.2% EDTA treatment to avoid the contamination of MEFs.
2. Two-dimensional gel electrophoresis (2-DE)
ES cells were collected by 0.2% EDTA treatment. Afterwashing the cells with washing buffer (10 mM Tris-HCl
buffer, pH 8.0, 5 mM magnesium acetate), 4×107 cells
were suspended with 7 volumes of lysis buffer containing
2 M thiourea, 7 M urea, 4% (w/v) CHAPS and 1 mM Pefablc SC PLUS (Roche Diagnostics GmbH, Mannheim, Germany). The cell suspensions were kept for 10 minutes on
ice, sonicated intermittently and centrifuged at 12,000 g for
10 minutes at 4°C, and then the supernatant fractions werecollected. The protein concentration was determined in thelysis solution with a dye reagent from Amersham Biosciences using bovine serum albumin as a standard. The
lysate was alkylated with Ready PrepTM Reduction-Alkylation Kit (Bio Rad Laboratories, Hercules, CA). The 120 µg
protein lysate per gel were subjected to 2-DE. The firstdimensional isoelectric focusing was carried out using
Immobiline dry strip (18-cm long, pH 3–10 non-linear or
pH 6–11 linear) in a horizontal electrophoresis system,
Ettan IPGphor (Amersham Biosciences) according to the
manufacturer’s instructions. After the first dimensional
elecrofocusing, IPG gels were equilibrated with buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v)
glycerol, 2% (w/v) sodium dodecyl sulphate (SDS), 0.01%
BPB and 0.5% dithiothreitol, followed by alkylation withequilibration buffer containing 4.5% idoacetamide instead of
0.5% dithiothreitol at room temperature for 15 minutes.
The gels were subjected to the second-dimensional SDS
polyacrylamide gel electrophoresis (10% SDS). Proteins
were visualized in the gels by staining with SYPRO Ruby
Protein Gel Stain (Bio Rad) for overnight. The florescenceintensity of each protein spot was digitally recorded by

J Electrophoresis

FluorImager 595 using Image QuaNT software and the protein expression was analyzed by PDQuest software.
3. Mass spectrometric analysis
The mass spectrometric analysis was performed according to the method reported previously8) with minor modifications. Briefly, each protein spot in SYPRO Ruby-stainedgels was picked by FluoroPhoreStar 3000 (Anatech, Tokyo,
Japan). The pieces of gels were dehydrated in 50% acetonitrile and 50% ammonium bicarbonate, and then in 100%
acetonitrile, and dried up. The proteins were digested with
5 µg/ml trypsin at 30°C. After the overnight protein digestion, peptide fragments in the digest were subjected to
MALDI-TOF mass spectrometer (AXIMA-CFR, Shimazdu
Corp., Kyoto, Japan) for peptide mass fingerprinting (PMF).
Protein identification process was accomplished by a twotiered approach using Mascot server (Matrix Science Ltd.,
Franklin St., Boston, MA) for selection of protein candidates and then using Protein Prospector (UCSF Mass Spectrometry Facility, San Francisco, CA) for its verification. In
the former, molecular weights and pI values were taken
into account as well as % coverage values during candidate
protein selection. In the latter, verification was performed
using MS-Digest software under a criterion that more thaneight m/z values were detected in major peaks of PMF.
The activated parameters used in Mascot server query
were as follows: primate database of SWISS-PROT and
NCBInr, peptide tolerance ±0.4 Da or ±1.0 Da, one missedcleavage and carbamidomethyl modification of cysteine.
During MS-Digest software query, acetylation of N-terminal end or lysine and phosphorylation of serine, threonine
or tyrosine was considered. Protein identification was
repeated at least once with spots from different gels.

2007 ; 51 : 3

4. Two-dimensional Western blotting
The SYPRO Ruby-stained proteins on gels were resolubilized and transferred according to our previously reported
method9). Briefly, the stained gel was incubated in resolubilization buffer (0.2% w/v SDS, 0.3% w/v Tris, 0.7% w/v
glycine) for 10 minutes and mounted onto a PVDF membrane in a semi-dry blotting apparatus (Bio Rad). Electrotransfer was carried out at 4 V/cm2 for one hour at room
temperature using buffer containing 0.3% (w/v) Tris, 1.5%(w/v) glycine, 0.1% (w/v) SDS. The fluorescence images ofthe blotted PVDF membranes were scanned and recorded
by FluorImager 595. The PVDF membranes were further
subjected to the immunoblotting using polyclonal antibodies against 60-kDa heat shock protein (HSP60), annexin A5,
heat shock cognate 71-kDa protein (HSC70), triosephosphate isomerase (TIM), 14-3-3 proteins, α-enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa
Cruz Biotechnology Inc.), annexin A2 (Abnova Corp.,
Taipei, Taiwan), voltage-dependent anion-selective channel protein 1(VDAC-1) (Abcam plc., Cambridge, UK), pyruvate kinase isozyme M2 (PKM2), phosphoglycerate kinase
1 (PGK1), serine/threonine-protein kinase 13 (Aurora-C)(Abgent Inc. San Diego, CA), nucleolin, thioredoxin reductase and GTP-binding nuclear protein Ran (Santa Cruz
Biotechnology).
RESULTS AND DISCUSSION
While the routine cell passage procedure was performed
detaching ES cells by trypsinization, we collected the ES
cells by EDTA treatment to prepare 2-DE samples to
exclude the contamination by MEFs. The typical 2-DE protein expression patterns of “ES cells” using strips of pH 3–
10 and pH 6–11 were shown in Figs. 1A and 1B, respec-

Fig. 1. Protein expression patterns of cynomolgus monkey ES cells in 2-DE.
The SYPRO Ruby staining patterns in the 2-DE using a strip of pH 3–10 non-linear (A) and a strip of pH 6–11 (B) in the first-dimensional
isoelectric focusing. A typical result from four independent experiments for each was shown.

MALDI, WB
MALDI, WB
MALDI, WB
MALDI, WB

nucleophosmin

tropomyosin-4

tropomyosin α-314-3-3 protein ε

14-3-3 protein ζ

4

5

6

7

8

9

MALDI
MALDI
MALDI
MALDI
MALDI, WB
MALDI

MALDI
MALDI
MALDI
MALDI, WB
MALDI, WB

MALDI, WB
MALDI

zinc-finger FYVE domain-containing protein 19

ATP synthase β chain

ATP synthase β chain

60-kDa heat shock protein (HSP60)

60-kDa heat shock protein (HSP60)

T-complex protein 1 subunit εubiquitin carbonyl-terminal hydrolase isozyme

prohibitin

L-lactate dehydrogenase B chain

protein disulfide-isomerase A3 precursor

peroxiredoxin 6

glutathione S-transferase π

phosphoglycerate mutase

elongation factor 1-γ

α-enolase

α-enolase

phosphoglycerate mutase

triosephosphate isomerase

GTP-binding nuclear protein Ran

nucleolin

thioredoxin reductase

heat shock cognate 71-kDa protein (HSC70)

heat shock cognate 71-kDa protein (HSC70)

tublin β-2 chain

annexin A5

annexin A5

annexin A5

60-kDa heat shock protein (HSP60)

zinc-finger protein 192

pigment epithelium-derived factor

actin cytoplasmic 1

actin cytoplasmic 1

actin cytoplasmic 1

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

0.4

0.4

0.4

1

1

0.4

1

1

1

0.4

0.4

0.4

1

0.4

1

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

1

1

0.4

0.4

0.4

0.4

0.4

1

0.4

1

0.4

0.4

0.4

0.4

1

0.4

0.4

0.4

1

mass tolerance

19

19

19

14

6

13

24

24

24

17

17

17

16

13

37

29

30

50

29

13

20

48

21

22

23

28

15

20

13

13

12

12

10

11

17

29

22

22

24

33

11

11

18

21

% Coverage

Acc NoP60709

P60709

P60709P36955

Q15776

P10809P08758

P08758

P08758

P07437

P11142

P11142

Q16881

P19338P62826

P60174

P18669P06733

P06733

P26641

P18669P09211

P30041

P30101

P07195

P35232

P09936

P48643

P10809P10809P06576

P06576

Q96K21

P07437

P08670

P68363

P63104

P63104

P62258

P06753

P67936

P06748P08865

P63104

P27797

Funtion (as listed in Swiss-Prot)

involved in various types of cell motility

involved in various types of cell motility

involved in various types of cell motility

Neurotrophic protein

May be involved in transcriptional regulation

mitochondrial protein import and macromolecular assembly

an anticoagulant protein

an anticoagulant protein

an anticoagulant protein

the major constituent of microtubules

Chaperone

Chaperone

CATALYTIC ACTIVITY: thioredoxin+NADP+=thioredoxin disulfide+NADPH

It induces chromatin decondensation by binding to histone H1

GTP-binding protein involved in nucleocytoplasmic transport

CATALYTIC ACTIVITY: D-glyceraldehyde 3-phosphate=glycerone phosphate

Interconversion of 3- and 2-phosphoglycerate with 2,3-bisphosphoglycerate

Multifunctional enzyme that, as well as its role in glycolysis

Multifunctional enzyme that, as well as its role in glycolysis

Probably plays a role in anchoring the complex to other cellular components

Interconversion of 3- and 2-phosphoglycerate with 2,3-bisphosphoglycerate

Conjugation of reduced glutathione

redox regulation of the cell

Catalyzes the rearrangement of -S-S- bonds

CATALYTIC ACTIVITY: (S)-lactate+NAD+=pyruvate+NADH.

DNA synthesis inhibitor

Ubiquitin-protein hydrolase

Molecular chaperone

mitochondrial protein import and macromolecular assembly

mitochondrial protein import and macromolecular assembly

Produces ATP from ADP

Produces ATP from ADP

unknown

the major constituent of microtubules

class-III intermediate filaments

major constituent of microtubules

Adapter protein

Adapter protein

Adapter protein

stabilizing cytoskeleton actin filaments

stabilizing cytoskeleton actin filaments

assembly and/or transport of ribosome.

unknown

Adapter protein

calcium binding chaperone

The protein name listed in UniProt (SwissProt), identification method (ID method), mass tolerance used as activated parameter during MASCOT sever query, percent of
the coverage (% coverage), accession number of the protein (Acc No), and function of the protein listed in UniProt (SwissProt) were shown.

MALDI

MALDI

MALDI

MALDI

MALDI

MALDI, WB

MALDI, WB

MALDI, WB

MALDI, WB

MALDI, WB

MALDI, WB

MALDI, WB

MALDI, WB

MALDI, WB

MALDI

MALDI

MALDI

MALDI

MALDI

MALDI

MALDI, WB

MALDI

vimentin

tublin β-2 chain

11

MALDI, WB
MALDI

14-3-3 protein ζtublin α ubiquitous

10

MALDI

MALDI

14-3-3 protein ζ

40S ribosomal protein SA

2

3

WB

ID method
MALDI

protein namecalreticulin

1#

Table 1. Identification of the proteins detected in 2-DE gel using a pH 3-10 strip

J Electrophoresis
2007 ; 51 : 4

MALDIMALDI, WB

MALDI
MALDI

α-enolase

GTP-binding nuclear protein Ran

phosphoglycerate mutase

annexin A2

annexin A2

septin 9

α-enolase

elongation factor 2

elongation factor 2

elongation factor 2

guanine mucleotide-binding protein β-subunit 2-like 1

annexin A2

pyruvate kinase isozyme M2 (PKM2)

annexin A2

phosphoglycerate kinase 1 (PGK1)

ATP synthase alpha chain, mitochondrial precursor

pyruvate kinase isozyme M2 (PKM2)

peroxiredoxin-1

voltage-dependent anion-selective channel protein 1 (VDAC-1)transcription elongation factor A protein 2

glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

paired box protein pax-8

annexin A8

glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

phosphoglycerate kinase 1 (PGK1)

heterogeneous nuclear, ribonucleoprotein A1 (herix-destabilizing protein)elongation factor 1-α 1transketolase

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

serine/threonine-protein kinase 13 (Aurora-C)

serine/threonine-protein kinase 13 (Aurora-C)

voltage-dependent anion-selective channel protein 1 (VDAC-1)

voltage-dependent anion-selective channel protein 1 (VDAC-1)

voltage-dependent anion-selective channel protein 1 (VDAC-1)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

pyruvate kinase isozyme M2 (PKM2)

phosphoglycerate kinase 1 (PGK1)

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

1

0.4

0.4

0.4

0.4

1

0.4

1

1

0.4

1

1

1

0.4

1

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

24

8

18

30

16

22

17

18

33

25

23

14

33

38

16

38

11

13

13

13

23

18

30

28

23

mass tolerance % Coverage

Acc NoP00558P14618P14618P14618P14618P14618P14618P14618P14618P14618P21796

P21796

P21796Q9UQB9Q9UQB9P06733

P04406

P29401

P68104

P09651

P00558P04406

P13928

Q06710P04406

P04406

Q15560P21796

Q06830

P14618

P25705

P00558

P07355

P14618

P07355

P63244

P13639P13639P13639P06733

Q9UHD8

P07355

P07355

P18669P62826

P06733

P06733

P06733

Funtion (as listed in Swiss-Prot)

glycolytic enzyme, polymerase alpha cofactor protein

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

Forms a channel through the mitochondrial outer membrane and also the plasma membrane.

Forms a channel through the mitochondrial outer membrane and also the plasma membrane.

Forms a channel through the mitochondrial outer membrane and also the plasma membrane.

May play a part in organizing microtubules during mitosis

May play a part in organizing microtubules during mitosis

Multifunctional enzyme that, as well as its role in glycolysis

CATALYTIC ACTIVITY: D-glyceraldehyde 3-phosphate+phosphate+NAD+=3-phospho-D-glyceroyl phosphate+NADH.

CATALYTIC ACTIVITY: Sedoheptulose 7-phosphate+D-glyceraldehyde 3-phosphate=D-ribose 5-phosphate+D-xylulose 5-phosphate.

promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes

Involved in the packaging of pre-mRNA into hnRNP particles, transport of poly(A) mRNA

glycolytic enzyme, polymerase alpha cofactor protein

CATALYTIC ACTIVITY: D-glyceraldehyde 3-phosphate+phosphate+NAD+=3-phospho-D-glyceroyl phosphate+NADH.

an anticoagulant protein

Transcription factor for the thyroid-specific expression of the genes

CATALYTIC ACTIVITY: D-glyceraldehyde 3-phosphate+phosphate+NAD+=3-phospho-D-glyceroyl phosphate+NADH.

CATALYTIC ACTIVITY: D-glyceraldehyde 3-phosphate+phosphate+NAD+=3-phospho-D-glyceroyl phosphate+NADH.

Necessary for efficient RNA polymerase II transcription elongation

Forms a channel through the mitochondrial outer membrane and also the plasma membrane.

Involved in redox regulation of the cell

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

Produces ATP from ADP in the presence of a proton gradient across the membrane

glycolytic enzyme, polymerase alpha cofactor protein

Calcium-regulated membrane-binding protein

CATALYTIC ACTIVITY: ATP+pyruvate=ADP+phosphoenolpyruvate.

Calcium-regulated membrane-binding protein

an intracellular receptor to anchor the activated PKC to the cytoskeleton

promotes GTP-dependent translocation

promotes GTP-dependent translocation

promotes GTP-dependent translocation

Multifunctional enzyme that, as well as its role in glycolysis

Involved in cytokinesis

Calcium-regulated membrane-binding protein

Calcium-regulated membrane-binding protein

Interconversion of 3- and 2-phosphoglycerate with 2,3-bisphosphoglycerate

GTP-binding protein involved in nucleocytoplasmic transport

Multifunctional enzyme that, as well as its role in glycolysis

Multifunctional enzyme that, as well as its role in glycolysis

Multifunctional enzyme that, as well as its role in glycolysis

The protein name listed in UniProt (SwissProt), identification method (ID method), mass tolerance used as activated parameter during MASCOT sever query, percent of the coverage (% coverage), accession
number of the protein (Acc No), and function of the protein listed in UniProt (SwissProt) were shown.

WB

WB

WB

WB

WB

WB

WB

WB

WB

WB

WB

WB

WB

WB

MALDI, WB

WB
WB

glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

α-enolase

32

MALDI

MALDI

MALDI

WB

MALDI

MALDI

MALDI

WB

WB

MALDI

MALDI, WB

MALDI

MALDI, WB

MALDI

MALDI, WB

MALDI, WB

MALDI, WB

MALDI, WB

MALDI

MALDI

WB

WB

MALDI, WB

MALDI, WB

WB
MALDI, WB

α-enolase2

ID method
WB

protein nameα-enolase1#

Table 2. Identification of the proteins detected in 2-DE gel using a pH 6–11 strip

J Electrophoresis
2007 ; 51 : 5

J Electrophoresis

2007 ; 51 : 6

tively. Protein spots clearly visualized by SYPRO Ruby
staining were picked from the gels and subjected to themass spectrometric analysis using a MALDI-TOF apparatus.
Table 1 and Table 2 show the summary of the results. We
could identify totally 45 and 48 protein spots in 2-DEs using
pH 3–10 and pH 6–11 strips, respectively. These proteins
were either components of cytoskeleton (tropomyosin-4, βtubulin, vimentin, β-actin), enzymes involved in energy
regulation (ATP synthase, L-lactate dehydrogenase, α-enolase, transketolase, phosphoglycerate mutase, TIM,GAPDH, PKM2, PGK1), proteins involved in redox regulation (peroxiredoxin, glutathione S-transferase, thioredoxin
reductase), chaperones (T-complex protein, heat shockproteins, calreticulin precursor), components of the translation machinery (elongation factors 1 and 2), regulators ofdynamisms in nuclear events (prohibitin, nucleolin, GTPbinding nuclear protein Ran, Aurora-C), apoptosis-related
proteins (VDAC-1, annexin A5) or adaptor proteins (14-3-3
ε and 14-3-3 ζ). These proteins are major actors requiredfor cell survival in general. An exception is pigment

epithelium-derived factor, a neurotrophic factor thatinduces extensive neuronal differentiation in retinoblastoma cells. Its expression in undifferentiated ES cells might
explain, at least in part, the well-known characteristics ofmammalian ES cells that they prone to undergo neuronaldifferentiation when signals required for the maintenance of
undifferentiated states are eliminated.
We next performed the two-dimensional western
blotting to confirm the results of mass spectrometry using
commercially available polyclonal antibodies having widecross-reactivity concerning HSP60, Annexin A5, HSC70,
TIM, 14-3-3 ε and 14-3-3 ζ, α-enolase, GAPDH, annexin
A2, VDAC-1, PKM2, PGK1 and Aurora-C. As shown in
Fig. 2 and Fig. 3, each protein spot was eventually recognized by the corresponding antibody, proving the validity of
the MALDI-TOF-based protein identification process using
human databases. On the other hand, usage of non-humanprimate databases, in which cynomolgus monkey’s data are
included, provided no better information. They seldom gave
us candidate proteins or only gave the same candidate as

Fig. 2. Two dimensional western blotting after a pH 3–10 strip-using 2-DE gel.
The 2-DE gel was trimmed (an upper left panel) and transferred to the PVDF membrane. Western blotting was performed using indicatedpolyclonal antibodies, which are shown to have broad cross-reactivity among human, mouse and rat by the manufacturer. The PVDF
membrane was re-used after stripping the previously used antibody. The multiple spots and slurs in an area surrounded by a dotted line
in TIM and HSC70 antibody reactions are the background spots created during the anti-TIM antibody reaction. The irregular marks in thearea shown by a dotted line with a gray arrowhead in anti-HSC70 antibody reaction are non-specific stains created during this antibody
reaction.

J Electrophoresis

2007 ; 51 : 7

Fig. 3. Two dimensional western blotting after a pH 6–11 strip-using 2-DE gel.
The 2-DE gel was trimmed (an upper left panel) and transferred to the PVDF membrane. Western blotting was performed using indicatedpolyclonal antibodies, which are shown to have broad cross-reactivity among human, mouse and rat by the manufacture. The PVDF membrane was re-used after stripping the previously used antibody. The multiple spots and slurs in an area surrounded by a dotted line in
PKM2, Aurora-C, PGK1 and VDAC-1 antibody reactions are the background spots stubbornly remained after the α-enolase antibody
reaction.

human databases did with lower % coverage values (datanot shown).
The results of the 2D western blotting further revealed
that some of the proteins were expressed as multiple spots
with similar molecular weights but different pI values, indicating that those proteins have multiple isoforms via modification such as phosphorylation and acetylation. Our study,
in which a combined analysis of western blotting and mass
spectrometry was performed, could identify more protein
isoforms in monkey ES cells than a report did on mouse ES
cells, where a sole mass spectrometry analysis was performed (Table 3). Although most of the proteins identified
in monkey ES cells were also detected in mouse ES cells10),
we found that some proteins were unique to the monkey ES
cells. For example, annexin A family proteins were notdetected in mouse ES cells, while annexins A2, A5 and A8
were clearly detected in monkey ES cells. Because the 2DE proteomic study on mouse ES cells10) was extensively
performed by mass spectrometry successfully identifying
as many as 123 protein spots in pH 3–10 strip-using gel, it

seems that annexin A family proteins are not expressed, or
if any, in undifferentiated mouse ES cells. Other examples
are Aurora-C and pigment epithelium-derived factor,
whose murine homologues have not been reported so far.
We conclude that the human proteome databases, consisting of a large volume of information with high quality organization, are of a significantly great service in identifying
monkey proteins.
We showed here a draft protein expression profile ofundifferentiated cynomolgus monkey ES cells by 2-DE
proteomic studies. We have successfully identified multiple
protein spots via a combined analysis of mass spectrometry
using human databases and western blotting using polyclonal antibodies. We also found that non-human primate
databases are not so useful as human databases in identifying monkey proteins. This finding coincides with a previous
report, which is the only one report that has ever reported
on the 2-DE-based proteomic analysis using monkey
samples11). In this report, heart samples of individual cynomolgus monkeys were subjected to 2-DE, and the protein

J Electrophoresis

2007 ; 51 : 8

Table 3. The protein isoform detected in monkey and mouse ES cells
protein namecynomolgus monkey

mouse

elongation factor 2
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
pyruvate kinase isozymes M2 (PKM2)α-enolase
voltage-dependent anion-selective channel protein 1 (VDAC-1)
serine/threonine-protein kinase 13 (Aurora-C)
annexin A2

3
4
10
7
4
2
5

3
1
2
3
1
0
0

The number of the multiple spots detected by a combined study of mass spectrometry and western blotting incynomolgus monkey ES cells (present study) and those detected by mass spectrometry in mouse ES cells
reported elsewhere (reference No. 10) was listed.

identification was achieved by a MALDI-TOF-mediatedmass spectrometry querying human databases. Thus,
human databases are substantially available for identifying
monkey protein spots in 2-DE.
By virtue of 2-DE western blotting analysis, we could
identify multiple isoforms of common cytoplasmic proteins
including enzymes involved in glycogenesis/glycolysis
pathways such as GAPDH, α-enolase and PKM2. Amongthese, GAPDH is recognized as a multi-function protein,playing roles in endocytosis, microtubule bunding, phosphotrasnferase, nuclear RNA transport, DNA replication,
DNA repair, vial pathogenesis, oncogenesis andapoptosis12). The multiple isoforms we identified in monkey
ES cells might be related to its multiple functions. Other
enzymes shown here to have multiple isoforms may also
have multi-functions other than glycolytic regulation.
In conclusion, 2-DE–based proteomic studies using monkey samples can sufficiently be achieved via a combined
analysis of mass spectrometry querying human databases
and western blotting using polyclonal antibodies raised
against human epitopes.
ACKNOWLEDGEMENTS
The authors greatly thank Etsuko Yasugi and Emiko
Okuma for technical assistance.
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