Mol. Cells, Vol. 22, No. 1, pp. 36-43
Molecules
and
Cells
©KSMCB 2006
Proteomic Analysis of the Hydrophobic Fraction of Mesenchymal
Stem Cells Derived from Human Umbilical Cord Blood
Ju Ah Jeong, Yoon Lee, Woobok Lee, Sangwon Jung, Dong-Seong Lee, Namcheol Jeong, Hyun Soo Lee,
Yongsoo Bae, Choon-Ju Jeon, and Hoeon Kim*
Creagene Research Institute, Creagene Co., Seoul 135-960, Korea.
(Received February 25, 2006; Accepted June 2, 2006)
Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering, but their
application has been impeded by lack of knowledge of
their core biological properties. In order to identify
MSC-specific proteins, the hydrophobic protein fraction was individually prepared from two different umbilical cord blood (UCB)-derived MSC populations;
these were then subjected to two-dimensional (2D) gel
electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time
of flight (TOF)-mass spectrometry (MS). Although the
2D gel patterns differed somewhat between the two
samples, computer-assisted image analysis identified
shared protein spots. 35 spots were reliably identified
corresponding to 32 different proteins, many of which
were chaperones. Based on their primary sub-cellular
locations the proteins could be grouped into 6 categories: extracellular, cell surface, endoplasmic reticular,
mitochondrial, cytoplasmic and cytoskeletal proteins.
This map of the water-insoluble proteome may provide valuable insights into the biology of the cell surface and other compartments of human MSCs.
Keywords: Human Umbilical Cord Blood; Hydrophobic
Fraction; Mesenchymal Stem Cells; Proteomic Analysis.
Introduction
Stem cells hold promise for cell-based therapy and tissue
engineering. Mesenchymal stem cells (MSCs) are a human stem cell type that has been rapidly introduced into
the clinic, because it can be easily cultured ex vivo, and
differentiates into multiple lineages in appropriate condi* To whom correspondence should be addressed.
Tel: 82-2-3461-0983; Fax: 82-2-3461-0981
E-mail: [email protected]
tions (Barry and Murphy, 2004). These cells are fibroblastic and adherent cells that express a characteristic panel of
cell surface proteins (Minguell et al., 2001; Tocci and
Forte, 2003). They have been isolated from a number of
different human tissues, including bone marrow (BM),
umbilical cord blood (UCB), adipose tissues, dermis, and
muscles (Barry and Murphy, 2004).
The core biological properties of human MSCs remain
poorly understood. A number of global gene expression
studies have been undertaken with human BM and UCBderived MSCs using serial analysis of gene expression
(SAGE) (Jia et al., 2004; Panepucci et al., 2004; Silva et
al., 2003), restriction fragment differential display (RFDD)
(Monticone et al., 2004), and DNA microarray techniques
(Jeong et al., 2005). In addition the whole cell proteomes
of BM and UCB-derived MSCs have been investigated
(Feldmann et al., 2005; Wang et al., 2004) using twodimensional (2D) gel electrophoresis (GE) coupled with
mass spectrometry (MS), currently a mainstream approach for analyzing the global proteomic profile. However visual inspection did not reveal a close resemblance
between the two 2D protein maps of the two cell populations, raising the possibility of substantial cell type originor individual-based variation in protein expression.
In the present study, we investigated the proteome of
the hydrophobic fraction of human UCB-derived MSCs.
In principle, this fraction should contain many important
proteins present on the cell surface and elsewhere that,
due to their low abundance, tend to escape analysis in
conventional proteomic studies. Prior to the proteomic
analysis, therefore, we eliminated abundant cytosolic proteins that might otherwise interfere with visualizing
weakly expressed proteins.
Abbreviations: MSCs, mesenchymal stem cells; UCB, umbilical
cord blood.
Ju Ah Jeong et al.
Materials and Methods
Sample preparation Human MSCs were obtained from fullterm UCB samples, as previously described (Gang et al., 2004a).
These cell populations were proven to possess broad differentiation potential ranging from mesenchyme-related multipotency
(Gang et al., 2004b) to neuroectodermal (Jeong et al., 2004) and
endodermal competency (Hong et al., 2005). For this study, we
chose the same cell populations as used previously for the DNA
microarray-based global gene expression analysis (Jeong et al.,
2005), and we further expanded them up to the 8th passage. The
cells around this stage were shown to retain the potential to
differentiate along at least a mesengenic lineage (Gang et al.,
2004b). About 1 × 107 cells of each population were harvested
by scraping the flasks with a cell scraper, and homogenized in a
Dounce homogenizer. Cell debris and unbroken cells were discarded by centrifugation at 6,000 × g for 10 min. The supernatant was centrifuged at 80,000 × g for 50 min in a Beckman
50 Ti rotor (Beckman Coulter, USA). The supernatant and the
pellet from this step were designated the hydrophilic (watersoluble) and hydrophobic (water-insoluble) fractions, respectively. The pellet was resuspended in 0.5 ml of lysis buffer consisting of 50 mM Tris, 7 M urea, 2 M thiourea, 4% (w/v)
CHAPS, and 16 μl protease inhibitor cocktail (Roche Molecular
Biochemicals, USA) for 60 min at 18°C in an orbital shaker,
and then centrifuged again at 80,000 × g for 30 min. Fifty units
of benzonase (250 units/μl; Sigma-Aldrich) was added to the
mixture, which was then stored at −80°C until use after protein
quantification by the Bradford method (Bio-Rad Laboratories,
USA). Samples of approximately 10 and 450 μg of protein were
used for one-dimensional (1D) and two-dimensional (2D) SDS
gel electrophoresis (GE), respectively.
2D gel electrophoresis For 2D GE, the IPG strips (pH 3−10,
Amersham Biosciences, Sweden) were hydrated with the sample
solution in a strip holder for 24 h. Swelling buffer, consisting of
7 M urea, 2 M thiourea, 0.4% (w/v) DTT, and 4% (w/v) CHAPS,
was added to approximately 450 μg of protein lysate in a final
volume of 350 μl, and the mixture was cup-loaded into the IPG
strips using a Multiphor II apparatus (Amersham Biosciences)
for a total of 57 kVh. The first dimension of isoelectric focusing
was performed with an Ettan IPGphor unit (Amersham Biosciences) at a constant voltage of 500 V for 10 h. The IPG strips
were then equilibrated for 30 min in 10 mg/ml of DTT and dissolved in an SDS equilibration buffer containing 50 mM Tris
base, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and a trace
of bromophenol blue, and sealed on the top of the gel with 0.5%
agarose. The 2-D separation was performed at a constant voltage of 100 V on 8−16% (v/v) linear gradient SDSpolyacrylamide (PA) gels that were pre-cast with 30% Duracryl,
0.65 % Bis, 10% SDS, 10% ammonium persulfate, and 0.375 M
Tris buffer at pH 8.8, and stopped once the bromophenol blue
front had disappeared from the gel.
Coomassie blue staining and image analysis After the gels
37
were fixed for 1 h in a solution of 40% methanol containing 5%
phosphoric acid, they were stained with colloidal Coomassie
Blue G-250 (Sigma-Aldrich) for 5 h, de-stained in 1% acetic
acid for 13 h and imaged with a GS-710 imaging calibrated
densitometer (Bio-Rad Laboratories). Protein spot detection and
2D pattern matching were performed with ImageMaster 2D
Platinum software (Amersham Biosciences). Matching of the
protein maps was achieved automatically but fine-tuned later by
manual registration.
Tryptic digestion and peptide extraction To identify proteins,
the protein spots were manually excised from the stained gels,
diced into small pieces (1 mm2) and placed into 0.65 ml siliconized tubes. Approximately 100 μl of 25 mM ammonium
bicarbonate buffer, pH 7.8, containing 50% (v/v) acetonitrile
was added, and the tubes were vortexted for 1 h at room temperature. After a brief spin, the supernatants were discarded with
GELoader tips (Eppendorf, Germany). After repeated washing,
gel pieces were dried with a SpeedVac for 10 min, and about 10
μl of 5 ng/μl trypsin (Promega, USA) freshly prepared in 25
mM ammonium bicarbonate buffer, pH 7.8, was added and incubated at 37°C overnight. After the tryptic peptides were extracted with 5 μl of 0.5% trifluoroacetic acid containing 50%
(v/v) acetonitrile for 40 min with mild sonication, the extracted
solution was reduced to approximately 1 μl in a vacuum centrifuge. Prior to MS analysis, the resulting peptide solution was
subjected to a desalting process using a reversed-phase column.
A GELoader tip was packed with Poros 20 R2 resin (PerSpective Biosystems, USA). After an equilibration step with 10 ml of
5% (v/v) formic acid, the peptide solution was loaded on the
column and washed with 10 μl of 5% formic acid. The bound
peptides were eluted with 1 μl of elution buffer consisting 5
mg/ml α-cyano-4-hydroxycinnamic acid, 50% (v/v) acetonitrile
and 5% (v/v) formic acid.
Protein identification by MS Protein identification was achieved with peptide mass fingerprinting using matrix-assisted
laser desorption/ionization − time of flight − mass spectrometry
(MALDI-TOF-MS), as previously described (Shim et al., 2004).
In brief, about 1 ml of the tryptic digested peptide samples were
mixed with equal volumes of elution buffer consisting of 50%
acetonitrile, 0.1% trifluoroacetic acid and 5 μg/ml α-cyano-4hydroxycinnamic acid, and loaded onto a MALDI plate (Applied Biosystems, USA). Mass spectra were obtained on a Voyager-DE STR mass spectrometer (Perspective Biosystems,
USA) in reflection positive ion mode, and processed by MassLynx 4.0 software. The proteins were identified by peptide mass
fingerprint searching against the Swiss-Prot and NCBI databases,
using the search programs ProFound (http://129.85.19.192/profound_bin/WebProFound.exe, Rockefeller University), MASCOT
(http://www.matrixscience.com), or MS-Fit (http://prospector.
ucsf.edu/ucsfhtml4.0/msfit.htm, University of California San
Francisco). The following mass search parameters were set:
peptide mass tolerance, 50 ppm; a mass window between 0 and
100 kDa, allowance of missed cleavage, 2; consideration for
38
Hydrophobic Fraction Proteomics of UCB-derived MSCs
Fig. 1. 1D SDS-PAGE of the hydrophobic and hydrophilic fractions of UCB-derived MSCs. About 10 μg of protein per lane of
each faction was loaded onto a small-format SDS-PAGE gel and
visualized by CBB R-250 staining.
variable modifications such as oxidation of methionine and
propionamides of cysteines. Only significant hits as defined by
each program were considered initially with at least 4 matching
peptide masses.
Results
The hydrophilic and hydrophobic proteomes of UCBderived MSCs are mutually exclusive In order to determine the purity of the hydrophobic fraction of UCBderived MSCs, 1D PAGE was performed simultaneously
on the hydrophilic and hydrophobic fractions. As shown
in Fig. 1, the hydrophilic fraction contained a large number of proteins bands dispersed over an extended area,
whereas the hydrophobic fraction consisted of a relatively
small number of protein bands that tended to form a cluster between 40 and 60 kDa. There were hardly any protein
bands occupying the positions in the two lanes, indicating
that the protein species constituting the two fractions were
different from each other. This differential gel pattern
implies that the proteome of UCB-derived MSCs has been
divided into two mutually exclusive fractions.
The hydrophobic proteomes of UCB-derived MSCs
show inter-individual divergence In order to address the
possibility of inter-individual proteomic variation in the
UCB-derived MSC populations, we prepared hydrophobic
proteomes with an identical protocol from two different
MSC populations. To our surprise, a side-by-side com-
Fig. 2. Side-by-side comparison of the 2D gels of the hydrophobic fractions of two different UCB-derived MSC populations.
For this experiment, the same cell populations as used at the 5th
passage for the previous gene expression study (Jeong et al.,
2005) were chosen and culture-expanded up to the 8th passage.
About 450 μg of protein was used for a 2D GE, consisting of
isoelectric focusing in the pH range 3−10 and 8−16% linear
gradient PAGE. The gel images were cropped to similar dimensions and auto-contrasted in Photoshop 7.0.
parison of the 2D gels revealed that the proteomes were
moderately divergent (Fig. 2). Since the two cell populations share a high degree of similarity of transcriptional
profiles (Jeong et al., 2005), this finding implies that the
hydrophobic proteome of these cell populations may not
be proportional to the levels of genetic messages.
Many of the commonly expressed proteins are chaperones Despite the moderate proteomic divergence between
the two 2D gels, we were able to detect 39 shared spots
with the use of an automatic spot-matching module. Thirtyfive spots representing 32 different proteins and 3 posttranslational variants of vimentin (Table 1) were identified
with confidence by MALDI-TOF-MS (Fig. 3). Retrospective analysis of the DNA microarray-based transcriptional
profiles (Jeong et al., 2005) indicated that the gene expression patterns of these molecules were very similar in the
two MSC populations (Table 2), even though their relative
expression intensities varied greatly from gene to gene.
Except for HSPA5 and KNG, whose expression was below
the detection level, and SOD2 whose expression was lower,
the average intensity values were mostly higher in the
MSCs than in the mononuclear cells (MNCs), suggesting
that most if not all molecules could be used as MSC-related
molecular markers.
This subset contains a large number of proteins with chaperone functions, such as heat-shock proteins (HSPs) and
glucose-regulated proteins (GRP): TRA1/HSP96/GRP94,
HSPA5/GRP78, HSPA9B/GRP75, HSPA8/HSP73, HSPD1/
HSP60, P4HB/PDI, GRP58, and HSPB1/HSP27. These
chaperone proteins are known to reside primarily in subcellular compartments such as the endoplasmic reticulum
Ju Ah Jeong et al.
39
Table 1. Identification of shared proteins in the hydrophobic fraction of UCB-derived MSCs.
Spot
No.
Protein Abbr.
name
NCBI access
number
1
2
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
32
33
34
35
MVP
TRA1
GANAB
VCP
GSN
HSPA5
HSPA9B
HSPA8
KRT9
HSPD1
P4HB
GRP58
VIM
TUBB
VIM
VIM
ATP5B
VIM
ENO1
Unnamed
Unnamed
ACTB
ENAH
ANXA2
TPM1
ANXA5
VDAC1
KNG
PGAM1
HSPB1
SOD2
CRYAB
CALM2
MYL6
LGALS1
NP_059447
NP_003290
NP_938148
NP_009057
NP_000168
NP_005338
NP_004125
NP_006588
NP_000217
NP_002147
NP_000909
NP_005304
NP_003371
NP_821133
NP_003371
NP_003371
NP_001677
NP_003371
NP_001419
BAB71275
BAC05002
NP_001092
NP_001008493
NP_004030
NP_001018020
NP_001145
NP_003365
P01042
NP_002620
NP_001531
NP_000627
NP_001876
NP_001734
NP_524149
NP_002296
No. of matched Theoretical Mr
peptides
(kDa)/pI
67 (29)
50 (20)
57 (27)
57 (18)
58 (12)
46 (29)
46 (13)
85 (19)
58 (14)
67 (10)
57 (27)
62 (20)
30 (22)
69 (20)
74 (20)
66 (17)
56 (18)
82 (16)
39 (15)
81 (22)
55 (29)
27 (13)
73 (12)
38 (16)
39 (15)
35 (19)
32 (12)
82 (10)
62 (9)
21 (8)
56 (6)
24 (7)
44 (7)
16 (10)
22 (6)
99.66/5.3
92.81/4.8
107.32/5.7
90.16/5.1
86.18/5.9
76.46/5.1
74.02/5.9
71.16/5.4
62.25/5.1
61.25/5.7
57.60/4.8
57.28/6.0
53.74/5.1
50.22/4.8
53.74/5.1
53.74/5.1
56.54/5.3
53.74/5.1
47.58/7.0
47.54/5.0
52.49/5.0
40.22/5.6
63.89/12
38.87/7.7
32.86/4.7
35.86/4.9
30.77/8.8
73.28/6.3
28.93/6.7
22.84/6.0
23.80/6.9
20.14/6.8
16.26/4.1
17.75/4.5
14.99/5.3
Measured Mr
(kDa)/pI
110/5.7
99/4.8
100/6.1
97/5.5
90/5.9
73/5.1
73/5.8
71/5.6
60/5.5
60/5.6
59/4.7
58/6.0
58/5.2
53/4.8
57/5.3
55/4.8
55/5.0
55/5.1
53/7.0
53/4.8
52/4.6
45/5.4
40/4.4
38/7.8
36/4.5
35/5.0
34/9.3
35/4.2
28/7.1
27/5.9
24/7.6
21/7.1
18/3.5
18/4.2
14/5.2
MOWSE Score
1° Sub-cellular
(Pappin et al., 1993)
location*
2.747e+17
5.169e+08
1.514e+16
2.530e+07
1.565e+07
7.595e+14
2.747e+17
1.416e+11
4.191e+07
8.138e+05
3.845e+13
2.972e+09
1.317e+11
2.989e+13
1.012e+09
1.069e+09
8.795e+10
2.705e+08
4.121e+10
5.157e+10
2.207e+13
5.719e+08
3.217e+06
2.122e+09
1.930e+04
1.341e+12
1.688e+08
1.945e+05
3.318e+05
1.203e+04
4.183e+04
2.852e+04
3317
3167
392
CP
ER
ER
ER
CK
ER
MT
CP
CK
MT
ER
ER
CK
CK
CK
CK
MT
CK
CP
CK
CK
CK
CP
CS
CK
CS
MT
ES
CP
CP
MT
CP
CP
CK
ES
CS, cell surface; ES, extracellular space; CP, cytoplasm; ER, endoplasmic reticulum; CK, cytoskeleton; MT, mitochondria.
(ER), mitochondria and cytoplasm, and help the folding
and assembly of other proteins. Interestingly, a number of
recent studies have indicated that they are also expressed
at the cell surface (see below).
Depending on their primary sub-cellular location, the
identified proteins can be grouped into five categories
This subset contains functionally and structurally diverse
proteins, and, depending on their primary sub-cellular location, can be classified into five groups namely, two ex-
tracellular proteins: KNG and LGALS1; two cell surface
proteins, ANXA2 and ANXA5; six ER proteins, TRA1,
GANAB, VCP, HSPA5, P4HB, and GRP58; five mitochondrial proteins: HSPA9B, HSPD1, ATP5B, VDAC1
and SOD2; eight cytoplasmic proteins: MVP, HSPA8,
ENO1, ENAH, PGAM1, HSPB1, CRYAB and CALM2; and
finally nine cytoskeletal proteins: GSN, KRT9, VIM, TUBB,
BAB71275, BAC05002, ACTB, TPM1 and MYL6. However, it should be noted that many of the proteins are known
to localize to more than one sub-cellular compartment.
40
Hydrophobic Fraction Proteomics of UCB-derived MSCs
Table 2. Intensities of gene expression of the shared molecules in UCB-derived MSCs*.
Protein
No.
1
2
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
32
33
34
35
Abbr. name
MVP
TRA1
GANAB
VCP
GSN
HSPA5
HSPA9B
HSPA8
KRT9
HSPD1
P4HB
GRP58
VIM
TUBB
VIM
VIM
ATP5B
VIM
ENO1
Unnamed
Unnamed
ACTB
ENAH
ANXA2
TPM1
ANXA5
VDAC1
KNG
PGAM1
HSPB1
SOD2
CRYAB
CALM2
MYL6
LGALS1
NCBI gene access
number
NM_017458
NM_003299
Absent
NM_007126
NM_000177
NM_005347
NM_004134
NM_006597
NM_000226
NM_002156
NM_000918
NM_005313
NM_003380
NM_001069
Redundant
Redundant
NM_001686
Redundant
NM_001428
Absent
Absent
Absent
Absent
NM_004039
NM_000366
NM_001154
NM_003374
NM_000893
NM_002629
NM_001540
NM_000636
NM_001885
NM_001743
NM_021019
NM_002305
Normalized microarray intensity in UCB-MSC
Donor 1
Average
intensity in MNC
MSC-to-MNC
ratio
Donor 2
Average
13.5
38.3
7.1
36.7
10.3
37.5
3.4
12.9
3.1
2.9
14.4
53.2
< 1.0
26.5
412.7
1.3
1.5
15.1
37.4
729.4
16.6
11.0
32.5
< 1.0
31.8
366.9
2.0
2.1
13.7
45.7
475.9
36.2
12.7
42.9
< 1.0
29.2
389.8
1.7
1.8
14.4
41.6
602.6
26.4
6.1
19.7
< 1.0
21.8
197.5
1.3
< 1.0
1.7
13.3
343.4
10.6
2.1
2.2
1.0
1.3
2.0
1.3
1.8
8.4
3.1
1.8
2.5
78.5
76.6
77.6
64.5
1.2
479.4
425.2
452.3
150.4
3.0
231.0
88.6
76.0
38.4
< 1.0
65.6
301.7
22.9
97.2
244.5
386.2
276.1
304.6
147.5
74.0
41.0
< 1.0
61.0
237.7
6.1
193.8
171.0
418.2
237.3
267.8
118.0
75.0
39.7
< 1.0
63.3
269.7
14.5
145.5
207.7
402.2
256.7
23.7
1.0
17.8
6.5
< 1.0
9.6
26.1
200.1
1.0
60.5
252.5
88.5
11.3
118.0
4.2
6.1
1.0
6.6
10.3
0.1
145.5
3.4
1.6
2.9
* Data are from the DNA microarray-based gene expression profiling study (Jeong et al., 2005).
Discussion
Human MSCs possess versatile differentiation potential
and powerful ex vivo expansion capability, serving as
forerunners of adult stem cells in the field of cell transplantation and therapy. Future development of MSCbased therapeutics and regenerative medicine relies on
our knowledge of MSC-specific biological properties,
such as self-renewal, differentiation, tissue homing and
mobilization. Although candidate molecules responsible
for these properties might be rapidly identified by global
gene expression studies using various high throughput
techniques such as DNA microarrays, SAGE, expressed
sequence tag (EST) scan, and massively parallel signature
sequencing (MPSS), their correct characterization should
be based on a critical evaluation of their expression at the
protein level.
In this study, we attempted to analyze the hydrophobic
proteome of human UCB-derived MSCs. This proteome
contains important proteins of the cell surface and subcellular compartments that may be involved in many crucial functions but are usually overwhelmed by the abun-
Ju Ah Jeong et al.
Fig. 3. Shared protein spots in the hydrophobic proteomes of
UCB-derived MSCs. The identified spots are displayed on a
reference map, with the same numbering as in Table 1. CS, cell
surface; ES, extracellular space; CP, cytoplasm; ER, endoplasmic reticulum; CK, cytoskeleton; MT, mitochondria.
dant signals of housekeeping proteins in ordinary whole
cell proteome analysis. We therefore isolated the hydrophobic fraction from whole UCB-derived MSC extracts
by differential sedimentation and then analyzed its content with the use of 2D GE coupled with MS.
Prior to our proteomic analysis we set out to address
the possibility of inter-individual proteomic variation
within MSCs. For this experiment, we prepared the proteome from the same two UCB-derived MSC populations
as used for our previous global gene expression study
(Jeong et al., 2005). Since the two populations were
proven to be highly similar to each other with respect to
cell characteristics, multilineage potential and gene transcription profile, we expected that their proteomic variation
would be minimal. However, the hydrophobic proteomes of
the two populations turned out to differ somewhat from
each other. This apparent disparity between the genomic
and proteomic contents raises at least two possibilities.
One is that the proteome of MSCs is intrinsically sensitive to functional state so that it becomes more diverse
than its transcriptome. This phenomenon, referred to as
intrinsic stem cell heterogeneity by other investigators,
has been previously observed in the proteome of a UCBderived CD34+ cell population (Zenzmaier et al., 2003;
2005) and is thought to be common to all stem cells. The
second possibility is that both the gene and protein expression profiles of the cells may be constant in early passages, but suddenly diverge during subsequent expansion.
41
This possibility is relevant because the genomic and proteomic profiles of the cells were analyzed at different passage numbers, i.e., between 3rd and 5th passages, and at
the 8th passage, respectively.
Cell surface proteins are of particular interest because
they not only execute various important functions, such as
induction of intracellular signaling, environment adaptation and disease pathogenesis, but also provide potential
targets for drug development. Moreover, the discovery of
new surface proteins, particularly of MSCs, may lead to
improvements in the protocols for their isolation, which
have so far relied mainly on various physicochemical
properties. Two annexin molecules, albeit classified as
surface proteins in this study, do not belong to this category because they are loosely associated with the cytosolic face of the cell membrane.
However, several lines of investigation suggest that
many other proteins listed here are surface proteins. A
recent proteomic investigation of various cancer cell types
showed that a number of chaperone proteins, such as
HSPA5, HSPA9B, HSPA8, HSPD1, P4HB, GRP58, and
HSPB1, were highly expressed on the cell surface (Shin et
al., 2003). This finding was further corroborated by recent
reports that the cell membrane housed these chaperones.
For instance, HSPA8 was found to be one of the components of the Dengue Virus receptor complex (Reyes-Del
Valle et al., 2005) and was also abundant on the surface
of embryonic stem cells (Son et al., 2005). In addition,
HSPD1 appeared to be a specific receptor for lipopolysaccharide on the cell membrane (Habich et al., 2005).
Moreover, GRP58 is a frequently found on the plasma
membrane (Guo et al., 2002). It is likely that another
chaperone protein, TRA1, is also expressed on the cell
surface because its N-terminal domain interacts with the
surface receptor CD91 (Binder et al., 2000). Coincidently
and surprisingly, all of these chaperones are on our protein list, suggesting that they may be expressed on the cell
membrane of UCB-derived MSCs. However, it remains
unclear how they are incorporated into the hydrophobic
lipid bilayer structure since they lack any transmembrane
domain or motif.
There are also several reports that two mitochondrial
proteins, ATB5B and VDAC1, are surface proteins. The
former is a subunit of the mitochondrial ATP synthase
complex that has been reported to be present on the cell
surface (Bae et al., 2004), while the latter, previously
known as a mitochondrial outer membrane channel protein, has been found in the plasma membrane (Bathori et
al., 1999; Thinnes and Reymann, 1997), and to have
NADH-ferricyanide reductase activity (Baker et al., 2004).
These two proteins contain transmembrane α-helical and
16-stranded β-barrel domains, respectively, so that they,
uniquely in our list of proteins, fall into the class of integral membrane proteins. This paucity of integral membrane proteins in the hydrophobic proteome suggests that
42
Hydrophobic Fraction Proteomics of UCB-derived MSCs
the strategy used here may not be effective in detecting
this class of proteins, since they are not only expressed in
limited amounts but are also poorly resolved on 2D gels.
A better approach may require rigorous fractionation of
plasma membranes (Pasquali et al., 1999) and/or a biotinylation-based molecular tagging and capturing strategy
(Shin et al., 2003).
Finally, our protein list contains a number of newly
discovered proteins: KRT9, P4HB, ENAH, VDAC1,
KNG, SOD2, CRYAB, CALM2, MYL6, and LGALS1. It
is very likely that the presence of these molecules was not
evident in previous proteomic analyses because they were
overwhelmed by the abundant expression signals of other
proteins. Consistent with this, transcripts of ENOI, MYL6,
CRYAB and LGLALS1, were abundant in this cell population (Jeong et al., 2005). This result helps to expand the
proteomic inventory used to define UCB-derived MSCs at
the molecular level.
Acknowledgments We thank Dr. Suk Am Kim and Ms. Eun
Hye Cho (Proteomtech Inc., Korea) for technical assistance.
This research was supported by a grant (SC3200) from the Stem
Cell Research Center of the 21st Century Frontier Research
Program funded by the Ministry of Science and Technology,
Republic of Korea.
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