Anal Bioanal Chem (2013) 405:55015517

DOI 10.1007/s00216-013-6969-z

ORIGINAL PAPER

Profiling and semiquantitative analysis of the cell surface

proteome in human mesenchymal stem cells
Sang Kwang Lee & Jae Ho Kim & Sung-Soo Kim & Taewook Kang &
Nam Hyun Park & Kyung-Hoon Kwon & Sang Sook Lee & Zee Won Lee &
Hae young Suh-Kim & Kun Cho & Su Yeoung Yun & Ji Young Han &
Jong Shin Yoo & Hyun Joo An & Young Mok Park

Received: 24 October 2012 / Revised: 13 March 2013 / Accepted: 3 April 2013 / Published online: 9 May 2013
# Springer-Verlag Berlin Heidelberg 2013

Abstract Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for
the development of cell therapeutic strategies. MSC surface
protein profiles provide novel biological knowledge
concerning the proliferation and differentiation of these cells,
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-013-6969-z) contains supplementary material,
which is available to authorized users.
S. K. Lee : J. H. Kim : T. Kang : N. H. Park : K.-H. Kwon :
K. Cho : S. Y. Yun : J. Y. Han : J. S. Yoo : Y. M. Park
Mass Spectrometry Research Center, Korea Basic Science Institute,
Ochang 363-887, Republic of Korea
S.-S. Kim : H. young Suh-Kim
Department of Anatomy, Ajou University, School of Medicine,
Suwon 443-749, Republic of Korea
T. Kang : N. H. Park : S. Y. Yun : J. S. Yoo : H. J. An
Graduate Schools of Analytical Science and Technology,
Chungnam National University,
Daejeon 305-764, Republic of Korea
S. S. Lee : Z. W. Lee : Y. M. Park
Biotechnology Fusion Research Team, Korea Basic Science
Institute, Daejeon 305-333, Republic of Korea
Y. M. Park (*)
Mass Spectrometry Research Center, Korea Basic Science Institute,
Ochang 363-883, Republic of Korea
e-mail: ympark@kbsi.re.kr
Present Address:
S. K. Lee
Eulji Medical and Biological Research Institute (EMBRI),
Daejeon 302-799, Republic of Korea
Present Address:
S. S. Lee
Department of Biology, Chungnam National University,
Daejeon 305-764, Republic of Korea

including the potential for identifying therapeutic targets. Basic

fibroblast growth factor (bFGF) affects cell surface proteins,
which are associated with increased growth rate, differentiation
potential, as well as morphological changes of MSCs in vitro.
Cell surface proteins were isolated using a biotinylationmediated method and identified using a combination of onedimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were
cut into 20 bands and digested with trypsin. Each tryptic
fragment was analyzed by liquid chromatography
electrospray ionization tandem mass spectrometry. Proteins
were identified using the Mascot search program and the
International Protein Index human database. Noble MSC surface proteins (n=1,001) were identified from cells cultured
either with (n=857) or without (n=667) bFGF-containing
medium in three independent experiments. The proteins were
classified using FatiGO to elucidate their function. We also
confirmed the proteomics results using Western blotting and
immunofluorescence microscopic analysis. The nature of the
proteins identified makes it clear that MSCs express a wide
variety of signaling molecules, including those related to cell
differentiation. Among the latter proteins, four Ras-related Rab
proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are
implicated in bFGF-induced signal transduction of MSCs.
Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.
Keywords Basic fibroblast growth factor . Mesenchymal
stem cell . Proteome . Surface protein
Introduction
Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells

5502

(MSCs) [1]. MSCs exhibit multipotent differentiation potential, allowing differentiation into a variety of mesodermal
cell types such as osteoblasts, chondrocytes, adipocytes, skeletal muscle cells, and smooth muscle cells [25]. MSCs are
also capable of differentiating into cells of nonmesodermal
origin, such as hepatocytes, glial cells, and neurons [68].
Human MSCs (hMSCs) have been isolated from bone
marrow, periosteum, trabecular bone, adipose tissue,
synovium, skeletal muscle, and deciduous teeth [9]. Several
methods can be used to isolate MSCs on the basis of their
physical and physicochemical characteristics, such as adherence to plastic or other extracellular matrix components. Owing to the ease of isolation and their extensive differentiation
potential, MSCs have clinical potential [10, 11]. The
multipotent differentiation potential of adult MSCs, their capability of growth and differentiation in culture media, and
their highly reduced immunoreactivity after allogenic transfer
make the cells ideal cells for tissue repair and regeneration in
clinical cell therapy applications, including regenerative medicine, cell-based therapy, and tissue engineering [1215].
However, this potential is currently hampered by several
drawbacks associated with bone marrow, which is the
source of MSCs. The drawbacks include high susceptibility
to viral exposure, the necessity of invasive procedures for
marrow collection, the rarity of MSCs in the marrow, and
the age-related significant decrease in cell number, cell
proliferative capacity, and cell differentiation capacity.
Thus, ex vivo expansion of MSCs is necessary prior to their
being used in clinical applications [1618].
Despite these challenges, autologous MSCs may be free
from the complications associated with immune rejection and
teratocarcinoma formation, and their use avoids the ethical
concerns that cloud the use of embryonic stem cells [19].
Fibroblast growth factors (FGFs) are a group of cytokines
that play major regulatory roles in development, wound
healing, hematopoiesis, and tumorigenesis [2023]. To date,
at least 22 FGFs have been identified in vertebrate tissues.
Acidic FGF and basic FGF (bFGF), with molecular masses of
1819 kDa, are especially well characterized and are very
important in human tissues. These two FGFs modulate cellular functions via four distinct high-affinity membrane receptors with an intrinsic tyrosine kinase activity [23]. The FGF
receptor (FGFR) family also consists of four receptor tyrosine
kinases designated FGFR1, FGFR2, FGFR3, and FGFR4
[24]. The acidic FGF and bFGF prototypical members of the
FGF family of growth factors are able to bind FGFR1 and
FGFR2, leading to receptor dimerization/oligomerization, activation of intrinsic receptor tyrosine kinase activity, and the
phosphorylation of specific tyrosine residues in the cytoplasmic tail of the receptor [25]. It was recently reported that bFGF
affects the long-term in vitro expansion of multipotent humanspinal-cord-derived neurospheres in the presence of epidermal
growth factor and ciliary neurotrophic factor [26].

S.K. Lee et al.

MSCs are characterized by their adherence to plastic

surfaces, the presence of a panel of cell surface markers,
and their differentiation capacity in vitro and in vivo
[2730]. Moreover, the phenotypic surface antigen markers,
used alone or in combination, are not specific for MSCs [5,
30]. Their differentiation potential has relied on monitoring
changes in a small number of proteins [31, 32]. Thus,
identification of protein makers that can be used to identify
and differentiate hMSCs from other cell types, as well as to
monitor their differentiation progress, is needed to realize
the full therapeutic potential of hMSCs. Additionally, the
profiles of MSC membrane proteins provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying
therapeutic targets.
Membrane proteins play roles in a variety of fundamental functions in all living organisms. In particular, transmembrane proteins and proteins anchored to the cell
membrane types represent more than 30 % of all proteins
in the human genome [33, 34]. The interactions between
cell surface proteins and soluble factors or insoluble ligands are very influential in regulating MSC functions.
However, the molecular mechanisms involved in these
cellular processes are poorly understood because of the
lack of the knowledge concerning the properties and functions of MSC surface proteins [35].
In this study, we used an alternative approach for selective identification of MSC surface membrane proteins. Firstly, cell surface proteins of intact cells were selectively
labeled with the membrane-impermeable reagent biotin.
Then, biotinylated plasma membrane proteins were enriched
via affinity capture using immobilized avidin [3639]. The
biotinylated proteins were separated by sodium dodecyl
sulfatepolyacrylamide gel electrophoresis (SDS-PAGE)
and identified by mass spectrometry (MS). This strategy
identified 1,001 surface proteins in hMSCs cultured in the
absence or presence of bFGF through three independent
repeats. These proteins were classified using FatiGO [40].
The nature of the proteins identified makes it clear that
MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter
proteins, four Ras-related Rab proteins, laminin-R, and three
14-3-3 proteins that were fractionated from MSCs cultured
on bFGF-containing medium are implicated in bFGFinduced signal transduction of MSCs.

Methods
Materials
SDS-PAGE gels were obtained from Sigma-Aldrich (St. Louis,
MO, USA). Sequencing-grade-modified trypsin was obtained

Proteome analysis of the cell surface proteins in hMSCs

from Promega (Madison, WI, USA). C18 (Aqua, 5 m) column resins were purchased from Phenomenex (Torrance, CA,
USA). Silica capillary tips (20 cm, inner diameter 75 m; tip
inner diameter 8 m) were obtained from Proxeon (Odense,
Denmark).
Cell culture
MSCs were isolated from human bone marrow aspirate as
described previously [41]. Cells were cultured in
Dulbeccos modified Eagles medium containing 10 % fetal
bovine serum, 100 U penicillin, and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), and, in some
experiments, 10 ng/mL bFGF (Dong-A Pharmaceutical,
Youngin, Republic of Korea). To determine the potential
of MSCs to differentiate into adipocytes, osteocytes, and
chondrocytes, differentiation was induced as described
previously [5].
Surface protein isolation
Biotinylation of hMSCs with EZ-link sulfo-NHS-SS-biotin
(Pierce, Rockford, IL, USA) was performed according to the
manufacturers instructions. The hMSCs were grown to 90
95 % confluence in 150-mm-diameter tissue culture plates, the
medium was removed, and the cells were washed twice with
phosphate-buffered saline (PBS). Ten milliliters of PBS
containing 0.25 mg/mL EZ-link sulfo-NHS-SS-biotin was
added to cover the cells, which were incubated at 4 C for
30 min on a rocking platform. The biotinylation reaction was
terminated by addition of 500 L of quenching solution.
Following biotinylation, the cells were harvested by centrifugation (500 g, 3 min) and washed two times with
tris(hydroxymethyl)aminomethane-buffered saline (TBS).
Cells were solubilized in 500 L of lysis buffer (Pierce)
containing mammalian protease inhibitor cocktail (Roche,
Mannheim, Germany). The cells were further disrupted by
brief sonication and incubation for 30 min on ice, with
vortexing every 5 min for 5 s. Solubilized biotinylated membrane proteins were collected by centrifugation at 10,000g for
2 min at 4 C. Clarified supernatant containing biotinylated
membrane proteins was incubated with 500 L of immobilized
NeutrAvidin gel slurry (Pierce), which was prewashed with
wash buffer (Pierce), for 60 min at room temperature with endover-end mixing using a rotator. After incubation with
NeutrAvidin, the surface proteins were eluted from the column
with elution buffer, which was a mixture of 7 M urea, 2 M
thiourea, and 4 % 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate containing a final concentration of 50 mM
dithiothreitol (DTT). For two-dimensional electrophoresis
analysis, through three independent experiments and surface
proteins were normalized using the Bradford protein assay
(Bio-Rad, Hercules, CA, USA).

5503

SDS-PAGE and in-gel digestion

Regular one-dimensional 12 % SDS-PAGE was applied. In
brief, 100 g of membrane proteins eluted as described earlier
was boiled for 5 min, and loaded in a single lane on a 1-mmthick 12 % SDS-PAGE gel (18 cm16 cm). The resolved
proteins were stained with Coomassie brilliant blue R-250.
The highly abundant deep-blue-stained proteins were separately cut from the gel and the areas between them were cut
into equal-sized slices, resulting in a total of 20 bands. Each
gel slice was transferred to a clean microcentrifuge tube. For
in-gel digestion, each gel band was washed twice in 200 L of
30 % methanol for 5 min. After the washing step, 200 L of
50 % acetonitrile containing 10 mM (NH4)HCO3 was added
and vortexed until the Coomassie brilliant blue was completely removed. To reduce the cysteine residues, each gel band
was covered with a 10 mM DTT solution prepared in 100 mM
(NH4)HCO3 for 60 min at 56 C. The DTT solution was
removed, and the excised bands were incubated with 200
L of 55 mM iodoacetamide prepared in 100 mM
(NH4)HCO3 for 40 min in the dark. The iodoacetamide solution was then removed. Washes were performed with 500 L
of distilled water for 5 min. This washing step was repeated
twice, followed by the addition of 100 L of acetonitrile. The
gel bands were then vacuum-dried for 20 min and rehydrated
with 12.5 g/L trypsin in 50 mM (NH4)HCO3 buffer. Digestion was performed by incubation at 37 C overnight.
Following digestion, tryptic peptides were extracted with
100 L of 50 % acetonitrile/5 % trifluoroacetic acid solution
at 25 C for 40 min. The supernatants were collected and dried
by vacuum centrifugation. Trypsin-derived peptides were
stored at 20 C until MS analysis.
Nanoscale liquid chromatography/electrospray ionization
tandem MS and data analysis
All MS/MS experiments for peptide identification were
performed using a nanoscale liquid chromatography
(LC)/MS system consisting of a Surveyor high-performance
LC system and a 7-T Finningan linear ion trap Fourier transform MS system (Thermo Electron, Bremen, Germany)
equipped with a nanoelectrospray ionization source. Ten microliters of each sample was loaded using a Surveyor
autosampler (Thermo Scientific, Pittsburgh, PA, USA) onto
a C18 trap column (inner diameter 300 m, length 5 mm,
particle size 5 m; LC Packings, Sunnyvale, CA, USA) for
desalting and concentration at a flow rate of 20 L/min. The
trapped peptides were then back-flushed and separated on a
homemade 100-mm-long microcapillary column [42] packed
with C18 (particle size 5 m) in 75-m silica tubing (8 m
inner diameter orifice).
The mobile phases, A and B, were composed of 0 % and
80 % acetonitrile, respectively, containing 0.02 % formic

5504

acid and 0.5 % acetic acid. The gradient began at 5 %

mobile phase B for 15 min, was increased to 50 % mobile
phase B for 47 min, to 95 % mobile phase B for 2 min, and
finally, to 95 % mobile phase B for 7 min. The column was
equilibrated with 5 % mobile phase B for 10 min before the
next run. Briefly, the mass spectrometer was operated in the
data-dependent mode to automatically switch between MS
and MS/MS acquisition. Xcalibur was used for each
MS/MS spectrum. Target ions selected for MS/MS were
dynamically excluded for 60 s. MS and MS/MS spectra
were obtained at a heated capillary temperature of 220 C,
an electrospray ionization (ESI) voltage of 2.2 kV, and a
collision gas pressure of 1.3 mTorr; the normalized collision
energy using wide band activation mode was 35 % for
MS/MS. Ion selection thresholds were 500 counts for
MS/MS. An activation q of 0.25 and an activation time of
30 ms was applied in MS/MS acquisitions.
For the database search, the International Protein Index
human database (IPI.HUMAN.v.3.70) was downloaded
from the website of the European Bioinformatics Institute
http://www.ebi.ac.uk/). To assess the false assignment distribution, the reversed sequence database was created from
the International Protein Index human database [43, 44] and
proteins were identified by Mascot version 2.2.2 (Matrix
Science, London, UK).
Peptide tolerance of 50 ppm, MS/MS tolerance of 0.8 Da,
and the carbamidomethyl cysteine as a fixed modification
were assigned as search parameters. One missed cleavage
was allowed, and the variable modifications of methionine
oxidation and carbamidomethyl cysteine were selected. The
required false-positive rate was set to 5 % at the peptide
level and the required data discovery rate was set to 1 % at
the protein level. Also, protein identifications were accepted
only if they contained at least one unique peptide confidently identified multiple assignment peptides.
Gene ontology annotations and biomarker comparison
analysis
The proteins identified in this study were classified by FatiGO
(http://fatigo.bioinfo.cnio.es), where proteins are assigned in
Gene Ontology terms, which rely on a controlled vocabulary
for describing a protein in terms of its molecular function,
biological process, or subcellular localization [40]. Biomarker
comparison analysis was performed using IPA (Ingenuity
Systems, Redwood City, CA, USA).
Western blot analysis
To prepare cell lysates, cells were solubilized in 300 L of lysis
buffer containing 30 mM tris(hydroxymethyl)aminomethane
HCl, pH 7.4, 7 M urea, 2 M thiourea, 4 % 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate, and

S.K. Lee et al.

mammalian protease inhibitor cocktail (Roche). The cells were

further disrupted by brief sonication and incubation for 30 min
on ice, with vortexing every 5 min for 5 s. Solubilized proteins
were collected by centrifugation at 18,000g for 40 min at 4 C.
Clarified supernatant containing proteins was collected. Samples containing equal amounts (20 g) of whole cell lysate
proteins were then subjected to 12 % SDS-PAGE. The resolved
proteins were transferred onto a nitrocellulose membrane. The
membrane was blocked with 0.1 % Tween 20 TBS containing
2 % bovine serum albumin, pH 7.4, overnight. The membrane
was then incubated in 1:200 diluted primary antibody. Antibodies against laminin-R (RPSA; sc-101517), caveolin-1
(CAV1; sc-894), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-47724) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). After it had been
washed in 0.1 % Tween 20 TBS, the membrane was incubated
for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody: anti-mouse IgG-HRP (sc-2005), anti-goat IgGHRP (sc-2020), or anti-rabbit IgG-HRP (A6154) obtained from
Santa Cruz Biotechnology or Sigma-Aldrich. After extensive
washing, we visualized bands by enhanced chemiluminescence
(GE Healthcare, Little Chalfont, UK) according to the manufacturers instructions.
Immunofluorescence microscopy analysis
Cells grown on cover slips were fixed with 3.7 % formaldehyde in PBS (10 min, room temperature), and permeabilized
with 0.1 % Triton X-100 in PBS (5 min, room temperature).
Cells were incubated with goat polyclonal anti-Thy-1 antibody (THY1) (sc-31244) and goat polyclonal anti-Rab 11b
antibody (RAB11B) (sc-26591) for 1 h, washed twice with 1
PBS, and incubated with rabbit anti-goat IgGfluorescein
isothiocyanate (F7367, Sigma-Aldrich) for 30 min. Cells were
washed three times with 1 PBS and mounted on slides using
xylene substitute mountant/histomount solution. Images were
obtained with a confocal microscope (Carl Zeiss, Jena, Germany) with a 40 objective.

Results
Multipotency of MSCs
To investigate the effects of bFGF on differentiation potential,
hMSCs were cultured with growth medium supplemented
with or without bFGF for six or seven passages, respectively.
Adipogenic, osteogenic, or chondrogenic differentiation was
induced individually for 3 weeks. When undifferentiated
MSCs were incubated in the absence of bFGF, they assumed
an increased spread-out and myoblast-like morphology. When
MSCs were cultured with bFGF, they displayed a consistent
spindle-shape and elongated form (data not shown). When

Proteome analysis of the cell surface proteins in hMSCs

MSCs were cultured with bFGF, the cells were able to differentiate into adipocytes, osteocytes, and chondrocytes. In the
absence of bFGF, these cells were unable to differentiate into
adipocytes. However, the cells still retained the potential to
differentiate into osteocytes and chondrocytes. The results
suggest that bFGF maintains multipotency of MSCs during
culture expansion in vitro.
Surface protein isolation of the hMSCs
The primary aim of this study was to identify and quantify
changes in surface proteins in hMSCs cultured with or without
bFGF. We first enriched surface proteins and membraneassociated proteins using the membrane-impermeable reagent
sulfo-NHS-SS-biotin and separated the proteins using
multidimensional separation strategies [45, 46]. Enriched surface proteins were isolated by biotinavidin interaction,
followed by one-dimensional SDS-PAGE and then digested
by trypsin. Tryptic fragments were separated by reverse-phase
LC prior to characterization by MS/MS. Figure 1 summarizes
this process. To confirm the efficiency of the surface protein
isolation method, the gene list of isolated proteins was submitted to the Database for Annotation, Visualization, and
Integrated Discovery (DAVID, version 6.7, http://david.abcc.
ncifcrf.gov/) [47]. The isolated surface proteins and membrane associated proteins were recovered with recovery rate
of 82.1 % (p<1.6010-120). A list of membrane proteins is
provided in Table S3.
To examine the difference in proteome profiling between
MSCs cultured with or without bFGF, protein samples from
each culture were analyzed by one-dimensional SDS-PAGE
(Fig. 2). Total protein amounts were measured based on equal

Fig. 1 The experimental

method for identification of
surface proteins by surface
biotinylation and nanoscale
liquid chromatography
electrospray ionization tandem
mass spectrometry (nano LC/
ESI-MS/MS). SDS PAGE
sodium dodecyl sulfate
polyacrylamide gel
electrophoresis, emPAI
exponentially modified protein
abundance index

5505

loading amounts of protein (100 ug) in each lane. The observed bands were sliced into 20 bands for each lane, with a
total 40 bands.
ESI-MS/MS analysis of tryptic fragments and data analysis
To develop a reliable proteome identification procedure, we
applied the reversed sequence database to Mascot and selected the high-score peptide sequences with an error rate of
less than 5 %. After MS/MS spectra had been analyzed
using these methods, we were able to identify proteins from
the three independent trials. A list of proteins quantified in
the present study containing detailed exponentially modified
protein abundance index (emPAI) information of the peptides acquired by MS/MS is presented in Table S1.
We identified 1,001 proteins by gathering 857 and 667
proteins from MSCs cultured with and without bFGF, respectively (Fig. 3). Among the 857 proteins identified from MSCs
cultured with bFGF, 179 proteins overlapped among the three
trials. Only 122, 257, and 91 proteins were identified uniquely
in the first, second, and third trials, respectively. Of the 667
proteins identified from MSCs cultured without bFGF, 116
proteins overlapped between the three trials. Only 70, 282,
and 48 proteins were identified uniquely in the first, second,
and third trials, respectively (Fig. 4).
To compare the proteome of MSCs cultured with and
without bFGF-containing medium, the 334 and 144 unique
proteins uniquely identified in the two conditions were classified using FatiGO. As shown in Fig. 5, the unique proteins
were classified and compared by their cellular components,
biological processes, and molecular functions. Consequently,
we found a difference in cellular components, molecular

5506

S.K. Lee et al.

Fig. 4 Venn diagram of overlapping proteins among the three trials

and the number of individual proteins identified with each trial

Differential expression patterns of proteins according

to the medium condition

Fig. 2 One-dimensional electrophoresis of the human mesenchymal

stem cell (hMSC) surface proteins isolated by the biotinavidin
interaction

functions, and biological processes for the proteins identified

in the two groups. For example, differences in the unique
classifications of the parts of the biological process were found
(Table 1). The unique classifications were composed of several
categories, such as response to endogenous stimulus, response
to biotic stimulus, regulation of a molecular function, digestion, response to abiotic stimulus, antigen processing and
presentation, reproductive process, and developmental growth
of MSCs cultured in bFGF-containing medium. In the case of
MSCs cultured in the absence of bFGF, two unique classifications were classified into cytokine production and aging.

For the quantitative analysis, the protein abundance was estimated and compared by the emPAI. This quantification index
is based on the number of sequenced peptides per protein and
is directly proportional to protein content (mol %) [48]. This
index was directly calculated by the Mascot research server.
The difference in the protein content (mol %) in the absence of
bFGF and in the presence of bFGF was calculated, and the
ratio of the averaged emPAI was obtained in two different
grades (Fig. 6). If a protein displayed a higher emPAI value
in the presence of bFGF in all three trials, we classified that
protein as grade 2. If a protein displayed a higher emPAI value
in the presence of bFGF in only two trials, we classified the
protein as grade 1. For proteins expressed more in the absence
of bFGF, we defined grade 1 and grade 2 similarly. If the
protein was identified more in the absence of bFGF in the three
trials, it was classified as grade 2. Grade 1 was reserved for
proteins having a higher emPAI value in the absence of bFGF
in two of the trials. Accordingly, we classified 365 identified
proteins (Table S2). The protein content (mol %) listed was
normalized to the number of total counts estimating the relative
amount of the different proteins within the sample.
Functional classification of identified proteins using Gene
Ontology

Fig. 3 Venn diagram of overlapping proteins between the two different medium conditions. A total of 1,001 unique proteins were identified from the three independent trials. MSC mesenchymal stem cell

We functionally classified 365 proteins having grade more

than 1 or more than 1 using FatiGO. Different proteins
were classified by function, such as signal transduction,
membrane protein, or cell differentiation proteins. As shown
in Table 2, 169 proteins were classified into these three
classes. Five Ras-related Rab proteins that had a plus grade
were found in the cell differentiation class, whereas Rab
proteins that had a minus grade were not found in the cell
differentiation class. Four 14-3-3 family proteins that had a
plus grade were also found in the cell differentiation class.
Thy-1, which had a plus grade, was found in all three

Proteome analysis of the cell surface proteins in hMSCs

5507

Fig. 5 Comparison of unique MSC proteins according to the cellular component (a), molecular function (b), and biological process (c)

classes. Caveolin-1, which had a minus grade, was also

found in all three classes. Additionally, 114 proteins were
classified as membrane proteins. Among these 114 proteins,
87 proteins had a plus grade and the other 27 proteins had a
minus grade. A biomarker comparison analysis was
performed. Three proteinsclathrin heavy chain like 1,
laminin-R (ribosomal protein SA), and semaphorin 7A
were selected as good biomarker protein candidates to select
MSCs having multipotent differentiation potency.

Validation of proteomic profiling data

To confirm the expression level of the proteins identified in the
MSCs cultured in the medium with or without bFGF, Western
blot analysis was performed. The antibodies used in the Western
blot analysis were CAV1, RPSA, and GAPDH. These antibodies
were used in Western blots with an equal amount of protein from
whole cell lysates of hMSCs cultured in the medium with or
without bFGF. Figure 7a illustrates the results. Laminin-R

5508

S.K. Lee et al.

Fig. 5 (continued)

(RPSA) was expressed at higher levels in MSCs cultured with

bFGF than without bFGF, which might indicate that RPSA is
upregulated in the bFGF-induced morphological changes. CAV1
showed abundant expression in MSCs cultured without bFGF.
We also confirmed expression of Thy-1 (THY1) and Rab
11b (RAB11B) using immunofluorescence microscopic analysis. As shown in Fig. 7b, THY1 and RAB11B were
expressed at higher levels in MSCs cultured with bFGF than
without bFGF. In addition to the disparity in expression levels,
there was also a difference in the location of the proteins.
THY1 and RAB11B were predominantly located in the cell
surface membrane regions. The protein expression level confirmed by Western blot and immunofluorescence microscopic
analysis corresponded to the results obtained by onedimensional SDS-PAGE coupled with MS. These results indicate that some of the proteins identified by MS might be
targets for purifying differentiated neuron cells among other
cells, such as astrocytes and oligodendrocytes.

Discussion
MSCs are considered to be suitable candidates for cell-based
therapy owing to their intrinsic capacity to self-renew and

differentiate; there is currently little information available

regarding the mechanisms that govern their self renewal and
differentiation potential [49, 50]. In accordance with previously reported work, the whole cell proteomes of MSCs have
been evaluated via two-dimensional electrophoresis coupled
with MS [19, 51, 52]. Proteomics tools are also valuable in the
elucidation of the underlying molecular mechanisms of differentiation of MSC and their differentiation potential. Further
development of MSC-based therapeutics and regenerative
medicine will depend on our knowledge of MSC-specific
biological properties, most notably self-renewal, differentiation, tissue homing, and mobilization [53].
The objective of this study was to analyze surface
proteomes of MSCs cultured in the presence or absence of
bFGF. bFGF is an important factor in the long-term culture of
MSCs, as it accelerates the speed of cell proliferation and
increases the multidifferentiation potential. If bFGF was not
added to the differentiation medium during long-term subculture with MSCs in vitro, the cells gradually lost their
multidifferentiation potential, especially adipocyte differentiation potential. We identified, however, that the
multidifferentiation potential was maintained for a longer period when the cells were cultured with bFGF-containing medium. Previously we identified differential expression of cell

Proteome analysis of the cell surface proteins in hMSCs

5509

Table 1 Examples of differences in the unique class of biological process

Classification
MSC+bFGF
Response to endogenous stimulus

Response to biotic stimulus

Regulation of a molecular function

Digestion
Response to abiotic stimulus
Antigen processing and presentation
Reproductive process
Developmental growth
MSC - bFGF
Cytokine production
Aging

Protein accession no.

Gene

Protein name

IPI00293464
IPI00293533
IPI00003479
IPI00219097
IPI00219037
IPI00304596
IPI00020127

DDB1
NUP62
MAPK1
HMGB2
H2AFX
NONO
RPA1

LOC100290337 DNA damage-binding protein 1

Nuclear pore glycoprotein p62
Mitogen-activated protein kinase 1
High mobility group protein B2
Histone H2A.x
Non-POU-domain-containing octamer-binding protein
Replication protein A 70-kDa DNA-binding subunit

IPI00018195
IPI00005154
IPI00015947
IPI00555565
IPI00012535
IPI00220578
IPI00018274
IPI00220642
IPI00216319
IPI00414442

MAPK3
SSRP1
DNAJB1
HSP90AB4P
DNAJA1
GNAI3
EGFR
YWHAG
YWHAH
CDK5RAP3

IPI00008380

PPP2CA

IPI00005668
IPI00029733
IPI00180954
IPI00004656

AKR1C2
AKR1C1
CIRBP
B2M

Mitogen-activated protein kinase 3

FACT complex subunit SSRP1
DnaJ homolog subfamily B member 1
Putative heat shock protein 90 kDa alpha, class B member 4
DnaJ homolog subfamily A member 1
Guanine-nucleotide-binding protein G(k) subunit alpha
Isoform 1 of epidermal growth factor receptor
14-3-3 protein gamma
14-3-3 protein eta
Complementary DNA FLJ56403, highly similar to CDK5
regulatory-subunit-associated protein 3
Serine/threonine protein phosphatase 2A catalytic subunit
alpha isoform
Aldo-keto reductase family 1 member C2
Aldo-keto reductase family 1 member C1
Cold-inducible RNA-binding protein
2-Microglobulin

IPI00020021
IPI00456429

DEK
UBA52

Protein DEK
Ubiquitin and ribosomal protein L40 precursor

IPI00410488
IPI00549248

CD276
NPM1

Isoform 1 of CD276 antigen

Isoform 1 of nucleophosmin

bFGF basic fibroblast growth factor, MSC mesenchymal stem cell

surface proteins in hMSCs cultured with or without bFGFcontaining medium by two-dimensional gel electrophoresis
[52]. To examine the comprehensive expression pattern of
the surface proteome in hMSCs, we performed semiquantitative analysis and identified 1,001 unique proteins in hMSCs.
This study also demonstrates the utility of a multidimensional
protein profiling strategy for comprehensive profiling of MSC
surface proteomes. We performed surface protein isolation and
one-dimensional electrophoresis to reduce protein complexity,
and used nanoscale LC/ESI-MS/MS for analysis of tryptic fragments. We analyzed the wide range of proteomes for the hMSCs
using multidimensional protein prefractionation methods, such
as cell surface protein isolation, one-dimensional electrophoresis,
and reverse-phase LC, as it was the most effective method to
identify a large number of proteins [54, 55].
Several signal transduction pathways can be activated by
FGFRs, some of which are implicated in bFGF-induced

neurogenesis and differentiation [56]. The proteins identified

in this work can be separated into three classes related to the
membrane, differentiation, and signal transduction (Table 2).
Sixty-four proteins were classified as cell-differentiation-related
proteins of the 365 proteins that had grade more than 1 or more
than 1 from the MSCs cultured with or without bFGFcontaining medium. Among these 64 proteins, 49 proteins were
detected from the MSCs cultured with bFGF, whereas the other
proteins were detected from the MSCs cultured without bFGF.
Interestingly, among these 49 proteins, five were from the Rasrelated Rab family and four were 14-3-3 related proteins.
Rab proteins are small GTPases that control multiple membrane trafficking events in the cell. The 60 different Rab
GTPases constitute the largest and most diverse group of the
Ras-like small G proteins [57]. Rab proteins control a variety
of important cellular processes, such as endocytosis, trafficking, endosome fusion, and exocytosis. Approximately 12 Rab

5510

S.K. Lee et al.

Fig. 6 Quantification of protein content of MSCs in the absence of

bFGF and in the presence of bFGF by emPAI. Comparisons of protein
content (mol %) in MSCs CAV1 in the absence of bFGF and in the

presence of bFGF for CAV1 (a), RPSA (b), Rab11b (c), YWHAG (d),
YWHAQ (e), and YWHAB (f)

members have now been located on endocytic structures and

several have been implicated in regulating the dynamics of
distinct endocytic processes [52, 53].
The Rab proteins are members of a large GTPase family
with homology to Ras [5861]. One of the most notable
properties of the Rab GTPase family is that individual
isoforms are localized on the surfaces of distinct membranebound organelles [57]. Rab GTPases localize to specific

compartments of both the endocytic and the exocytic pathway

[62]. G-protein-coupled receptors are synthesized and modified in the endoplasmic reticulum, and are then transported to
the Golgi apparatus for additional posttranslational modification before going to the cell surface. Multiple Rab GTPases
comprise several proteins, including Rab1, Rab4, Rab5, Rab7,
and Rab11. These proteins regulate endoplasmic reticulum
Golgi transport as well as the endocytosis and trafficking of G-

Proteome analysis of the cell surface proteins in hMSCs

5511

Table 2 Proteins related to the membrane, cell differentiation, and signal transduction among the proteins which have grade 1 or or grade 1 or
more
Gene

Annotations

Grade

Classification

Gene

Annotations

Grade

Classification

ACAA2
ANPEP
ANXA1
ANXA2
ANXA5
ARCN1
ARF4

IPI00001539
IPI00221224
IPI00218918
IPI00418169
IPI00329801
IPI00298520
IPI00215918

2
2
2
2
2
2
2

M
S, M
S, D, M
M
S, D
M
D, M

LRRC59
MAPK1
MMP14
MRC2
NME1
PHB
PLEC1

1
1
1
1
1
1
1

M
S, D
M
M
S
S, D, M
M

ARPC2

IPI00005161

ATP5B
ATP5O
BZW1

IPI00303476
IPI00007611
IPI00180128, IPI00785096

2
2
2

M
M
S

PLOD2
PLXNB2
PPP2R1A

IPI00396321
IPI00003479
IPI00218398
IPI00005707
IPI00012048
IPI00017334
IPI00398776, IPI00398775,
IPI00186711
IPI00014898, IPI00398779,
IPI00398002
IPI00337495
IPI00853369, IPI00852623
IPI00554737

1
1
1

M
S, M
S, D, M

CACNA2D1

PRDX2

IPI00027350

CAP1
CD109
CD44
COPE
DCBLD2
DPYSL2
ENG
GNAI2
GPD2
HADHA

IPI00953650, IPI00953262,
IPI00470535
IPI00939159, IPI00008274
IPI00152540,
IPI00297160
IPI00465132, IPI00399319
IPI00419836
IPI00257508
IPI00017567
IPI00926935, IPI00748145
IPI00017895, IPI00719611
IPI00031522,

2
2
2
2
2
2
2
2
2
2

D, M
M
M
M
D, M
S, D
D, M
D
M
M

PRDX4
PSMC5
PTRF
RAB10
RAB11B
RPN1
RPN2
RPSA
SEC23A
SEPT7

IPI00011937
IPI00745502, IPI00023919
IPI00514023
IPI00016513
IPI00020436
IPI00025874
IPI00028635
IPI00411639, IPI00413108
IPI00017375
IPI00033025, IPI00941534

1
1
1
1
1
1
1
1
1
1

D
S
M
D, M
D, M
M
M
D, M
M
S

HIST1H4L

IPI00453473,

S, D

SEPT9

S, M

HSP90AA1
ITGA2
KCTD12
KPNB1
LGALS1
LGALS3
MSN
NT5E
P4HB

IPI00382470
IPI00013744
IPI00060715
IPI00001639
IPI00219219
IPI00465431
IPI00219365, IPI00872814
IPI00009456
IPI00010796

2
2
2
2
2
2
2
2
2

D
D, M
M
S, M
S, D
M
M
M
M

SET
SPTBN1
SQRDL
SSR1
SSR4
STAT1
TGM2
THY1
TLN1

IPI00784808, IPI00784614,
IPI00455033
IPI00072377
IPI00005614, IPI00333015
IPI00009634
IPI00301021
IPI00019385
IPI00030781
IPI00294578
IPI00022892
IPI00298994

1
1
1
1
1
1
1
1
1

S
M
M
M
M
S, D
D, M
S, D, M
S, M

PDIA3
RAB1B
RAB5C
RAB7A
RDX
SERPINE2
SPTAN1
TXNDC5
VCP
YWHAE
YWHAG
YWHAQ
YWHAZ

IPI00893541, IPI00025252
IPI00008964
IPI00016339
IPI00016342
IPI00017367, IPI00903145
IPI00914848, IPI00009890
IPI00843765
IPI00171438
IPI00022774
IPI00000816
IPI00220642
IPI00018146
IPI00021263

2
2
2
2
2
2
2
2
2
2
2
2
2

S, D
D, M
D
D
S, D, M
S
M
S
S, D
D
S, D
D
S, D

TMED10
TNPO1
TUBB2C
TWF2
TXNL1
UACA
VCL
VPS35
ZYX
ACTN1
DBN1
FLNA
FLNB

IPI00028055
IPI00024364
IPI00930130
IPI00550917
IPI00305692
IPI00173359
IPI00307162, IPI00291175
IPI00018931
IPI00926625
IPI00921118, IPI00013508
IPI00003406
IPI00302592
IPI00289334

1
1
1
1
1
1
1
1
1
2
2
2
2

M
S, M
S
M
S, D
S, M
S, M
M
D, M
S, M
S, M
D
S, D, M

5512

S.K. Lee et al.

Table 2 (continued)
Gene

Annotations

Grade

Classification

Gene

Annotations

Grade

Classification

ACAT1
AKAP12
ALDH3A2
ARHGDIA
ATP5A1
ATP5C1
ATP6V1A
CALR
CAND1
CAPN2
CAPZA1
CAPZA2

IPI00030363
IPI00217683
IPI00333619
IPI00003815
IPI00440493
IPI00395769
IPI00007682
IPI00020599
IPI00100160
IPI00289758
IPI00005969
IPI00026182

1
1
1
1
1
1
1
1
1
1
1
1

M
D
M
S, D, M
M
M
M
S, M
S
S
S
S

LIMA1
ACTG1
ACTN4
ALCAM
ALPL
AP2B1
CANX
CAV1
CDH13
CDH2
CLTC
CNN2

IPI00796222, IPI00008918
IPI00021440
IPI00013808
IPI00015102
IPI00419916
IPI00790702, IPI00784156
IPI00020984
IPI00009236
IPI00024046
IPI00290085
IPI00024067
IPI00015262

2
1
1
1
1
1
1
1
1
1
1
1

M
S
S
D, M
M
S, M
M
S, D, M
M
M
M
M

CFL1
CKAP4
COMT
COPB1
COPZ1

IPI00012011
IPI00141318
IPI00011284
IPI00295851
IPI00032851

1
1
1
1
1

S, D
M
M
M
M

COL1A2
EEA1
EHD2
EMD

IPI00304962
IPI00329536
IPI00100980
IPI00032003

1
1
1
1

D
M
M
M

FN1

CRTAP
CTNNA1
CTNNB1
CTNND1
CTSB
CYB5R3

IPI00748502
IPI00215948
IPI00017292
IPI00182469, IPI00219725
IPI00295741
IPI00328415

1
1
1
1
1
1

D
S
S, D, M
D
S
M

HSPA9
HSPB1
IQGAP1
ITGA11
ITGA3
MYH9

IPI00339228, IPI00339224,
IPI00022418
IPI00007765
IPI00025512
IPI00009342
IPI00215613, IPI00941391
IPI00215995
IPI00395772, IPI00019502

1
1
1
1
1
1

S
S
D, M
D, M
S, D, M
S, D, M

DDOST
DDX1
DNAJA2
DNM1L
EEF1D
EHD1
EIF5A
ENPP1
EPHA2
GALNT1

IPI00297084
IPI00293655
IPI00032406
IPI00235412, IPI00037283
IPI00023048, IPI00642971
IPI00017184
IPI00376005
IPI00184311
IPI00021267
IPI00025818

1
1
1
1
1
1
1
1
1
1

M
S
M
M
D
M
S
M
D, M
M

MYO1C
NAPA
NPM1
PDGFRB
PLOD1
RPS14
RRBP1
SLC7A5
SPARC
VDAC1

IPI00743335, IPI00010418
IPI00009253
IPI00549248, IPI00220740
IPI00015902
IPI00027192
IPI00026271
IPI00215743
IPI00008986
IPI00014572
IPI00216308

1
1
1
1
1
1
1
1
1
1

M
S
S, D
D, M
M
S
D, M
M
D
S, D, M

GDI2
GLUD1
HSP90B1
HSPA1A

IPI00031461
IPI00016801
IPI00027230
IPI00304925, IPI00911039,
IPI00845339
IPI00003362
IPI00784154
IPI00743104
IPI00306604
IPI00027505, IPI00922108
IPI00217563
IPI00220327
IPI00217975
IPI00009771

1
1
1
1

D
D
S, M
S

VDAC2

IPI00216026, IPI00024145

1
1
1
1
1
1
1
1
1

S, D, M
S
S, D, M
S, D, M
D, M
D, M
M
M
M

HSPA5
HSPD1
ITGA1
ITGA5
ITGAV
ITGB1
KRT1
LMNB1
LMNB2

D cell differentiation, M membrane, S signal transduction

Proteome analysis of the cell surface proteins in hMSCs

Fig. 7 Validation of proteins identified by one-dimensional SDSPAGE coupled with mass spectrometry. a Western blotting analysis
of CAV1 and RPSA. b Confocal immunofluorescence analysis of
THY1 and RAB11B. The scale bar denotes 20 m

protein-coupled receptors between early, late, and recycling

endosomes and lysosomes [63]. Regulation is effected
through the cyclical binding and hydrolysis of GTP. Among
these Rab proteins, Rab7 is an important regulator of transport
to the late endosome [64].
A similar actin depolymerization/invagination-coupled
process may also be involved in the formation of exosomes
from the limiting membrane of late endosomes. Importantly,
several proteins identified in exosomes are present in macrophage phagosomes: Gi2a, galectin 3, 14-3-3, Alix, syntenin,
Rab7, Rab11, rap1-B, annexin V, hsc70, hsp84, and MFGE8/lactadherin [65].
Rab8 and Rab11 are involved in trafficking proteins from
the Golgi apparatus to plasma membranes [6669]. Rab 8 and
Rab11 are found at the trans-Golgi membrane and the
recycling endosome, a post-Golgi compartment that serves
as a transport intermediate for some cargo en route from the
trans-Golgi network to the plasma membrane [67, 68, 70].
Disruption of either Rab8- or Rab11-specific pathways leads
to the inhibition of recycling-endosome-dependent trans-Golgi network to plasma membrane transport, as well as plasma
membrane recycling [6670].
In parietal epithelial cells, Rab11 controls the cell surface
expression of H+/K+ ATPase by regulating recycling to the
plasma membrane [71]. In a nonpathogenic soil amoeba,
Rab11 was found to be associated with, and to regulate, the
structure and functions of the contractile vacuole system [72].

5513

Cell division, differentiation, and migration are crucial

events for the development of multicellular organisms. During
these processes cells polarize through reorganization of both
external and internal components such as actin, microtubules,
and adhesion receptors [73]. Rab8 modulates polarized membrane transport through reorganization of actin and microtubules, induces the formation of new surface extensions, and
has an important role in directed membrane transport to cell
surfaces [74, 75]. Several reports now point to an important
role of Rab8 in regulating cell morphogenesis and fate. Rab8
regulates a pathway where a specific membrane traffic route
participates in remodeling the cell shape in response to different signals. Rab8 interacts with the germinal center kinase, a
protein involved in tumor necrosis factor signaling [76].
Tumor necrosis factor is a major mediator of inflammatory
responses, and it induces changes in several cellular processes,
such as cell migration, differentiation, necrosis, respiratory
burst, and adhesion [10].
The 14-3-3 proteins are highly conserved, and seven family
members are found in mammals, , , , , , and .
These proteins self-assemble into homodimers and
heterodimers with some family members, and are able to
interact with many different proteins as a result of their specific phosphoserine/phosphothreonine binding activity [77].
The 14-3-3 proteins are able to bind phosphoserine/threonine
residues within a sequence-specific context [7880]. Since the
14-3-3 proteins are able to form stable dimers in which each
subunit is able to bind a separate phosphoserine peptide, it has
been suggested that these proteins may function as adaptors or
scaffolds for the assembly of signaling complexes [79]. In the
case of FGFR1, seven tyrosine residues (Y463, Y583, Y585,
Y653, Y654, Y730, and Y766) in the cytoplasmic domain
have been identified as phosphorylation sites. All seven phosphorylation sites identified in FGFR1 are conserved in FGFR2
[8185]. FGFR2 contains a conserved putative 14-3-3 binding
site at S779, which lies near Y766 in the C-terminal lobe [86].
The 14-3-3 proteins have emerged as important components
of essential biological processes that are regulated by
phosphorylation, such as signal transduction and cell cycle
regulation [77].
In humans, pluripotent stem cells derived from marrow
stroma proliferate ex vivo to form a phenotypically homogeneous population of cells that express several surface markers,
such as THY1 (also known as CD90), CD44, and TFRC (also
known as CD71) [5]. THY1 is a marker for CD34-positive
stem cells, activated endothelial cells, and fetal liver cells [87,
88]. In the adult liver, THY1 is expressed on oval cells but not
on mature hepatocytes [89, 90]. It has also been found on
MSCs generated from rat bone marrow [91].
Caveolins are major defining structural components of
caveolae, a form of lipid raft on the cell membranes. Caveolin
functions as a scaffolding protein that interacts with many
signaling molecules, such as heterotrimeric G proteins, protein

5514

S.K. Lee et al.

kinase C, Shc, SOS, Raf1, and Src family tyrosine kinases,

recruiting them into caveolae [9297]. Previous reports
suggested that upregulation of caveolin-1 is a major attenuator
of proliferative signal transductions that results in cellular
senescence [98, 99].
The laminins (RPSA) consist of a family at least 15 large
trimeric basement membrane proteins. They are made up of ,
, and chains. Laminin-111 is one of the best characterized
laminins and is composed of the 1, 1, and 1 chains [100,
101]. It exists in a cruciform structure, is an important structural component of the basement membrane, and plays a role
in tumor metastasis, cell spreading and attachment, and angiogenesis [102104]. FGF signaling is required for the expression of laminin-1 and collagen-1 (IV), which contribute
to basement membrane assembly and as a consequence to
ectoderm differentiation [105].
These proteins were newly identified compared in 2009
[52] and were classified as proteins related to the membrane,
cell differentiation, and signal transduction. If the sample
complexity is reduced, it could be possible to find comprehensive expression of cell surface proteins with or without
bFGF-containing medium in hMSCs.

Conclusions
In this study, we identified a total of 1,001 proteins in hMSCs.
To increase the confidence in the proteins identified, we used a
reversed sequence database and strict criteria such as multiple
hit proteins and grades. This work has shown that a combination of technologies is required for reducing sample complexity. We also provide evidence of different surface proteins and
surface-protein-associated proteins in MSCs cultured in a
medium with or without bFGF. Some proteins such as laminin
and Ras-related Rab proteins are related to bFGF-induced
signal transduction of MSCs. Consequently, our results also
provide insight into the understanding of the surface proteome
of hMSCs.
Acknowledgments This work was supported by grants to Y.M.P.
from the Korea Institute of Science & Technology Evaluation and
Planning (20062004605) and from the Korea Basic Science Institute
(G30124).
Study approval The Institutional Review Board of Ajou University
Medical Center (Suwon, Republic of Korea) approved this study.

Competing interests

The authors claim no competing interests.

Authors' contributions S.K.L. conducted preliminary experiments

and wrote the final manuscript. J.H.K., T.W.K., K.H.K., N.H.P., S.Y.Y.,
H.J.A., and J.S.Y. conducted the proteomic analysis. S.S.K. and H.S.
performed the cell culture and prepared the samples. S.S.L. and Z.W.L.
conducted the confocal microscopy analysis. Y.M.P. conceived the

study, obtained grant funding, and participated in experimental design

and coordination. All authors read and approved the final manuscript.

References
1. Prockop DJ (1997) Marrow stromal cells as stem cells for
nonhematopoietic tissues. Science 276:7174
2. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD,
Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M et
al (2002) Pluripotency of mesenchymal stem cells derived from
adult marrow. Nature 418:4149
3. Krause DS (2002) Plasticity of marrow-derived stem cells. Gene
Ther 9:754758
4. Phinney DG, Kopen G, Isaacson RL, Prockop DJ (1999) Plastic
adherent stromal cells from the bone marrow of commonly used
strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 72:570585
5. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,
Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR
(1999) Multilineage potential of adult human mesenchymal stem
cells. Science 284:143147
6. Brazelton TR, Rossi FM, Keshet GI, Blau HM (2000) From
marrow to brain: expression of neuronal phenotypes in adult
mice. Science 290:17751779
7. Hermann A, Gastl R, Liebau S, Popa MO, Fiedler J, Boehm BO,
Maisel M, Lerche H, Schwarz J, Brenner R, Storch A (2004)
Efficient generation of neural stem cell-like cells from adult
human bone marrow stromal cells. J Cell Sci 117:44114422
8. Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat
and human bone marrow stromal cells differentiate into neurons.
J Neurosci Res 61:364370
9. Barry FP, Murphy JM (2004) Mesenchymal stem cells: clinical
applications and biological characterization. Int J Biochem Cell
Biol 36:568584
10. Beyaert R, Fiers W (1994) Molecular mechanisms of tumor
necrosis factor-induced cytotoxicity. What we do understand
and what we do not. FEBS Lett 340:916
11. Aggarwal S, Pittenger MF (2005) Human mesenchymal stem cells
modulate allogeneic immune cell responses. Blood 105:18151822
12. Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall
RY, Muul L, Hofmann T (2002) Isolated allogeneic bone
marrow-derived mesenchymal cells engraft and stimulate growth
in children with osteogenesis imperfecta: implications for cell
therapy of bone. Proc Natl Acad Sci USA 99:89328937
13. Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL,
Neel M, Sussman M, Orchard P, Marx JC, Pyeritz RE, Brenner
MK (1999) Transplantability and therapeutic effects of bone
marrow-derived mesenchymal cells in children with osteogenesis
imperfecta. Nat Med 5:309313
14. Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W
(2002) Allogeneic mesenchymal stem cell infusion for treatment
of metachromatic leukodystrophy (MLD) and Hurler syndrome
(MPS-IH). Bone Marrow Transplant 30:215222
15. Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM,
Mukhachev V, Lavroukov A, Kon E, Marcacci M (2001) Repair
of large bone defects with the use of autologous bone marrow
stromal cells. N Engl J Med 344:385386
16. Amos TA, Gordon MY (1995) Sources of human hematopoietic
stem cells for transplantation a review. Cell Transplant 4:547
569
17. Mueller SM, Glowacki J (2001) Age-related decline in the osteogenic potential of human bone marrow cells cultured in threedimensional collagen sponges. J Cell Biochem 82:583590

Proteome analysis of the cell surface proteins in hMSCs

18. Stenderup K, Justesen J, Clausen C, Kassem M (2003) Aging is
associated with decreased maximal life span and accelerated
senescence of bone marrow stromal cells. Bone 33:919926
19. Sun HJ, Bahk YY, Choi YR, Shim JH, Han SH, Lee JW (2006) A
proteomic analysis during serial subculture and osteogenic differentiation of human mesenchymal stem cell. J Orthop Res 24:2059
2071
20. Akita S, Akino K, Tanaka K, Anraku K, Hirano A (2008) A basic
fibroblast growth factor improves lower extremity wound healing
with a porcine-derived skin substitute. J Trauma 64:809815
21. Douwes Dekker PB, Kuipers-Dijkshoorn NJ, Baelde HJ, van der
Mey AG, Hogendoorn PC, Cornelisse CJ (2007) Basic fibroblast
growth factor and fibroblastic growth factor receptor-1 may contribute to head and neck paraganglioma development by an
autocrine or paracrine mechanism. Hum Pathol 38:7985
22. Kashiwakura I, Takahashi TA (2005) Fibroblast growth factor
and ex vivo expansion of hematopoietic progenitor cells. Leuk
Lymphoma 46:329333
23. Ornitz DM, Itoh N (2001) Fibroblast growth factors. Genome
Biol 2:reviews3005.3001reviews3005.3012
24. Naski MC, Ornitz DM (1998) FGF signaling in skeletal development. Front Biosci 3:d781d794
25. Eswarakumar VP, Lax I, Schlessinger J (2005) Cellular signaling
by fibroblast growth factor receptors. Cytokine Growth Factor
Rev 16:139149
26. Akesson E, Piao JH, Samuelsson EB, Holmberg L, Kjaeldgaard
A, Falci S, Sundstrom E, Seiger A (2007) Long-term culture and
neuronal survival after intraspinal transplantation of human spinal
cord-derived neurospheres. Physiol Behav 92:6066
27. Gronthos S, Franklin DM, Leddy HA, Robey PG, Storms RW,
Gimble JM (2001) Surface protein characterization of human
adipose tissue-derived stromal cells. J Cell Physiol 189:5463
28. Haynesworth SE, Baber MA, Caplan AI (1992) Cell surface
antigens on human marrow-derived mesenchymal cells are
detected by monoclonal antibodies. Bone 13:6980
29. Tocci A, Forte L (2003) Mesenchymal stem cell: use and perspectives. Hematol J 4:9296
30. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U,
Krause U, Blake J, Schwager C, Eckstein V, Ansorge W, Ho AD
(2005) Comparative characteristics of mesenchymal stem cells
from human bone marrow, adipose tissue, and umbilical cord
blood. Exp Hematol 33:14021416
31. Abdallah BM, Jensen CH, Gutierrez G, Leslie RG, Jensen TG,
Kassem M (2004) Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1. J Bone Miner Res 19:841852
32. Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L,
Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS
(1990) Progressive development of the rat osteoblast phenotype
in vitro: reciprocal relationships in expression of genes associated
with osteoblast proliferation and differentiation during formation
of the bone extracellular matrix. J Cell Physiol 143:420430
33. Paulsen IT, Sliwinski MK, Nelissen B, Goffeau A, Saier MH Jr
(1998) Unified inventory of established and putative transporters
encoded within the complete genome of Saccharomyces
cerevisiae. FEBS Lett 430:116125
34. Wallin E, von Heijne G (1998) Genome-wide analysis of integral
membrane proteins from eubacterial, archaean, and eukaryotic
organisms. Protein Sci 7:10291038
35. Nunomura K, Nagano K, Itagaki C, Taoka M, Okamura N,
Yamauchi Y, Sugano S, Takahashi N, Izumi T, Isobe T (2005) Cell
surface labeling and mass spectrometry reveal diversity of cell
surface markers and signaling molecules expressed in
undifferentiated mouse embryonic stem cells. Mol Cell Proteomics
4:19681976
36. Chen WN, Yu LR, Strittmatter EF, Thrall BD, Camp DG 2nd,
Smith RD (2003) Detection of in situ labeled cell surface proteins

5515

37.

38.

39.
40.

41.

42.

43.

44.

45.

46.

47.

48.

49.
50.

51.

52.

53.

54.

by mass spectrometry: application to the membrane subproteome

of human mammary epithelial cells. Proteomics 3:16471651
Sabarth N, Lamer S, Zimny-Arndt U, Jungblut PR, Meyer TF,
Bumann D (2002) Identification of surface proteins of Helicobacter
pylori by selective biotinylation, affinity purification, and twodimensional gel electrophoresis. J Biol Chem 277:2789627902
Zhang W, Zhou G, Zhao Y, White MA (2003) Affinity enrichment of plasma membrane for proteomics analysis. Electrophoresis 24:28552863
Zhao Y, Zhang W, Kho Y (2004) Proteomic analysis of integral
plasma membrane proteins. Anal Chem 76:18171823
Al-Shahrour F, Diaz-Uriarte R, Dopazo J (2004) FatiGO: a web
tool for finding significant associations of Gene Ontology terms
with groups of genes. Bioinformatics 20:578580
Kim SS, Choi JM, Kim JW, Ham DS, Ghil SH, Kim MK, KimKwon Y, Hong SY, Ahn SC, Kim SU et al (2005) cAMP induces
neuronal differentiation of mesenchymal stem cells via activation of
extracellular signal-regulated kinase/MAPK. Neuroreport 16:1357
1361
Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR,
Garvik BM, Yates JR 3rd (1999) Direct analysis of protein
complexes using mass spectrometry. Nat Biotechnol 17:676682
Moore RE, Young MK, Lee TD (2002) Qscore: an algorithm for
evaluating SEQUEST database search results. J Am Soc Mass
Spectrom 13:378386
Wu SL, Choudhary G, Ramstrom M, Bergquist J, Hancock WS
(2003) Evaluation of shotgun sequencing for proteomic analysis
of human plasma using HPLC coupled with either ion trap or
Fourier transform mass spectrometry. J Proteome Res 2:383393
Elschenbroich S, Kim Y, Medin JA, Kislinger T (2010) Isolation
of cell surface proteins for mass spectrometry-based proteomics.
Expert Rev Proteomics 7(1):141154
Kemper K, Sprick MR, de Bree M, Scopelliti A, Vermeulen L,
Hoek M, Zeilstra J, Pals ST, Mehmet H, Stassi G, Medema JP
(2010) The AC133 epitope, but not the CD133 protein, is lost
upon cancer stem cell differentiation. Cancer Res 70(2):719729
Mastroleo F, Leroy B, Van Houdt R, s Heeren C, Mergeay M,
Hendrickx L, Wattiez R (2009) Shotgun proteome analysis of
Rhodospirillum rubrum S1H: integrating data from gel-free and
gel-based peptides fractionation methods. J Proteome Res
8(5):25302541
Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J,
Mann M (2005) Exponentially modified protein abundance index
(emPAI) for estimation of absolute protein amount in proteomics by
the number of sequenced peptides per protein. Mol Cell Proteomics
4:12651272
Deans RJ, Moseley AB (2000) Mesenchymal stem cells: biology
and potential clinical uses. Exp Hematol 28:875884
Ohgushi H, Caplan AI (1999) Stem cell technology and
bioceramics: from cell to gene engineering. J Biomed Mater
Res 48:913927
Wang D, Park JS, Chu JS, Krakowski A, Luo K, Chen DJ, Li S
(2004) Proteomic profiling of bone marrow mesenchymal stem
cells upon transforming growth factor beta1 stimulation. J Biol
Chem 279:4372543734
Lee SK, Kim Y, Kim SS, Lee JH, Cho K, Lee SS, Lee ZW, Kwon
KH, Kim YH, Suh-Kim H et al (2009) Differential expression of
cell surface proteins in human bone marrow mesenchymal stem
cells cultured with or without basic fibroblast growth factor
containing medium. Proteomics 9:43894405
Jeong JA, Lee Y, Lee W, Jung S, Lee DS, Jeong N, Lee HS, Bae
Y, Jeon CJ, Kim H (2006) Proteomic analysis of the hydrophobic
fraction of mesenchymal stem cells derived from human umbilical cord blood. Mol Cells 22:3643
Gautier V, Mouton-Barbosa E, Bouyssi D, Delcourt N, Beau M,
Girard JP, Cayrol C, Burlet-Schiltz O, Monsarrat B, Gonzalez de

5516

55.

56.

57.
58.

59.
60.
61.
62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

S.K. Lee et al.

Peredo A (2012) Label-free quantification and shotgun analysis
of complex proteomes by one-dimensional SDS-PAGE/NanoLCMS: evaluation for the large scale analysis of inflammatory
human endothelial cells. Mol Cell Proteomics 11(8):527539
Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R (2000)
Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci USA 97(17):9390
9395
Reuss B, von Bohlen und Halbach O (2003) Fibroblast growth
factors and their receptors in the central nervous system. Cell
Tissue Res 313:139157
Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107117
Ferro-Novick S, Novick P (1993) The role of GTP-binding proteins in transport along the exocytic pathway. Annu Rev Cell Biol
9:575599
Novick P, Brennwald P (1993) Friends and family: the role of the
Rab GTPases in vesicular traffic. Cell 75:597601
Pfeffer SR (1994) Rab GTPases: master regulators of membrane
trafficking. Curr Opin Cell Biol 6:522526
Zerial M, Stenmark H (1993) Rab GTPases in vesicular transport.
Curr Opin Cell Biol 5:613620
Sonnichsen B, De Renzis S, Nielsen E, Rietdorf J, Zerial M
(2000) Distinct membrane domains on endosomes in the
recycling pathway visualized by multicolor imaging of Rab4,
Rab5, and Rab11. J Cell Biol 149:901914
Bhattacharya M, Babwah AV, Ferguson SS (2004) Small GTPbinding protein-coupled receptors. Biochem Soc Trans 32:1040
1044
Feng Y, Press B, Wandinger-Ness A (1995) Rab 7: an important
regulator of late endocytic membrane traffic. J Cell Biol
131:14351452
Garin J, Diez R, Kieffer S, Dermine JF, Duclos S, Gagnon E,
Sadoul R, Rondeau C, Desjardins M (2001) The phagosome
proteome: insight into phagosome functions. J Cell Biol
152:165180
Ang AL, Folsch H, Koivisto UM, Pypaert M, Mellman I (2003)
The Rab8 GTPase selectively regulates AP-1B-dependent
basolateral transport in polarized Madin-Darby canine kidney
cells. J Cell Biol 163:339350
Chen W, Feng Y, Chen D, Wandinger-Ness A (1998) Rab11 is
required for trans-golgi network-to-plasma membrane transport
and a preferential target for GDP dissociation inhibitor. Mol Biol
Cell 9:32413257
Li Y, Luo L, Schubert M, Wagner RR, Kang CY (1993) Viral
liposomes released from insect cells infected with recombinant
baculovirus expressing the matrix protein of vesicular stomatitis
virus. J Virol 67:44154420
Zhang W, Yang H, Kong X, Mohapatra S, San Juan-Vergara H,
Hellermann G, Behera S, Singam R, Lockey RF, Mohapatra SS
(2005) Inhibition of respiratory syncytial virus infection with
intranasal siRNA nanoparticles targeting the viral NS1 gene.
Nat Med 11:5662
Ang AL, Taguchi T, Francis S, Folsch H, Murrells LJ, Pypaert M,
Warren G, Mellman I (2004) Recycling endosomes can serve as
intermediates during transport from the Golgi to the plasma
membrane of MDCK cells. J Cell Biol 167:531543
Chen YA, Scales SJ, Patel SM, Doung YC, Scheller RH (1999)
SNARE complex formation is triggered by Ca2+ and drives
membrane fusion. Cell 97:165174
Harris E, Yoshida K, Cardelli J, Bush J (2001) Rab11-like GTPase
associates with and regulates the structure and function of the
contractile vacuole system in dictyostelium. J Cell Sci 114:3035
3045
Drubin DG, Nelson WJ (1996) Origins of cell polarity. Cell 84:335
344

74. Peranen J, Auvinen P, Virta H, Wepf R, Simons K (1996) Rab8

promotes polarized membrane transport through reorganization
of actin and microtubules in fibroblasts. J Cell Biol 135:153167
75. Peranen J, Furuhjelm J (2001) Expression, purification, and
properties of Rab8 function in actin cortical skeleton organization and polarized transport. Methods Enzymol 329:188
196
76. Ren M, Zeng J, De Lemos-Chiarandini C, Rosenfeld M, Adesnik
M, Sabatini DD (1996) In its active form, the GTP-binding
protein rab8 interacts with a stress-activated protein kinase. Proc
Natl Acad Sci U S A 93:51515155
77. Morrison DK (2009) The 14-3-3 proteins: integrators of diverse
signaling cues that impact cell fate and cancer development.
Trends Cell Biol 19:1623
78. Aitken A (2006) 14-3-3 proteins: a historic overview. Semin
Cancer Biol 16:162172
79. Mackintosh C (2004) Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes.
Biochem J 381:329342
80. Yaffe MB, Elia AE (2001) Phosphoserine/threonine-binding domains. Curr Opin Cell Biol 13:131138
81. Furdui CM, Lew ED, Schlessinger J, Anderson KS (2006) Autophosphorylation of FGFR1 kinase is mediated by a sequential
and precisely ordered reaction. Mol Cell 21:711717
8 2 . H i n s b y A M , O l s e n J V, M a n n M ( 2 0 0 4 ) Ty r o s i n e
phosphoproteomics of fibroblast growth factor signaling: a role
for insulin receptor substrate-4. J Biol Chem 279:4643846447
83. Lundin L, Ronnstrand L, Cross M, Hellberg C, Lindahl U,
Claesson-Welsh L (2003) Differential tyrosine phosphorylation
of fibroblast growth factor (FGF) receptor-1 and receptor proximal signal transduction in response to FGF-2 and heparin. Exp
Cell Res 287:190198
84. Mohammadi M, Dikic I, Sorokin A, Burgess WH, Jaye M,
Schlessinger J (1996) Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation
of their importance in receptor activation and signal transduction.
Mol Cell Biol 16:977989
85. Mohammadi M, Dionne CA, Li W, Li N, Spivak T, Honegger AM,
Jaye M, Schlessinger J (1992) Point mutation in FGF receptor
eliminates phosphatidylinositol hydrolysis without affecting
mitogenesis. Nature 358:681684
86. Lonic A, Barry EF, Quach C, Kobe B, Saunders N, Guthridge
MA (2008) Fibroblast growth factor receptor 2 phosphorylation
on serine 779 couples to 14-3-3 and regulates cell survival and
proliferation. Mol Cell Biol 28:33723385
87. Fiegel HC, Kluth J, Lioznov MV, Holzhuter S, Fehse B, Zander
AR, Kluth D (2003) Hepatic lineages isolated from developing
rat liver show different ways of maturation. Biochem Biophys
Res Commun 305:4653
88. Fiegel HC, Park JJ, Lioznov MV, Martin A, Jaeschke-Melli S,
Kaufmann PM, Fehse B, Zander AR, Kluth D (2003) Characterization of cell types during rat liver development. Hepatology
37:148154
89. Petersen BE, Goff JP, Greenberger JS, Michalopoulos GK (1998)
Hepatic oval cells express the hematopoietic stem cell marker
Thy-1 in the rat. Hepatology 27:433445
90. Thorgeirsson SS (1996) Hepatic stem cells in liver regeneration.
FASEB J 10:12491256
91. Javazon EH, Colter DC, Schwarz EJ, Prockop DJ (2001) Rat
marrow stromal cells are more sensitive to plating density and
expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells 19:219225
92. Anderson RG (1998) The caveolae membrane system. Annu Rev
Biochem 67:199225
93. Brown DA, London E (1998) Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14:111136

Proteome analysis of the cell surface proteins in hMSCs

94. Galbiati F, Razani B, Lisanti MP (2001) Emerging themes in lipid
rafts and caveolae. Cell 106:403411
95. Okamoto T, Schlegel A, Scherer PE, Lisanti MP (1998) Caveolins,
a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J Biol Chem
273:54195422
96. Simons K, Toomre D (2000) Lipid rafts and signal transduction.
Nat Rev Mol Cell Biol 1:3139
97. Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA,
Scherer PE, Okamoto T, Lisanti MP (1999) Caveolins, liquidordered domains, and signal transduction. Mol Cell Biol
19:72897304
98. Park WY, Park JS, Cho KA, Kim DI, Ko YG, Seo JS, Park SC
(2000) Up-regulation of caveolin attenuates epidermal growth
factor signaling in senescent cells. J Biol Chem 275:20847
20852
99. Volonte D, Zhang K, Lisanti MP, Galbiati F (2002) Expression of
caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts. Mol Biol Cell 13:25022517

5517
100. Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R,
Edgar D, Ekblom P, Engel J, Engvall E, Hohenester E, Jones JC
et al (2005) A simplified laminin nomenclature. Matrix Biol
24:326332
101. Ekblom P, Lonai P, Talts JF (2003) Expression and biological role
of laminin-1. Matrix Biol 22:3547
102. Kibbey MC, Grant DS, Kleinman HK (1992) Role of the SIKVAV
site of laminin in promotion of angiogenesis and tumor growth: an
in vivo Matrigel model. J Natl Cancer Inst 84:16331638
103. Kleinman HK, Weeks BS, Schnaper HW, Kibbey MC,
Yamamura K, Grant DS (1993) The laminins: a family of basement membrane glycoproteins important in cell differentiation
and tumor metastases. Vitam Horm 47:161186
104. Nurcombe V (1992) Laminin in neural development. Pharmacol
Ther 56:247264
105. Li X, Chen Y, Scheele S, Arman E, Haffner-Krausz R, Ekblom P,
Lonai P (2001) Fibroblast growth factor signaling and basement
membrane assembly are connected during epithelial morphogenesis of the embryoid body. J Cell Biol 153:811822