Article

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In-Depth Proteomic Quantication of Cell Secretome in SerumContaining Conditioned Medium

Yejing Weng,, Zhigang Sui, Yichu Shan, Hao Jiang,, Yuan Zhou, Xudong Zhu,, Zhen Liang,
Lihua Zhang,*, and Yukui Zhang

Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian 116023, China

University of Chinese Academy of Sciences, Beijing 100049, China

S Supporting Information
*

ABSTRACT: Secreted proteins play key roles during cellular communication,

proliferation, and migration. The comprehensive proling of secreted proteins in
serum-containing culture media is technically challenging. Most studies have been
performed under serum-free conditions. However, these conditions might alter the
status of the cells. Herein, we describe an ecient strategy that avoids the disturbance of
serum by combining metabolic labeling, protein equalization, protein fractionation,
and lter-aided sample preparation, called MLEFF, enabling the identication of 534
secreted proteins from HeLa conditioned media, including 31 cytokines, and growth
factors. This MLEFF strategy was also successfully applied during a comparative
secretome analysis of two human hepatocellular carcinoma cell lines with dierentially
metastatic potentials, enabling the quantication of 61 signicantly changed proteins
involved in tumor invasion and metastasis.

To circumvent the above-mentioned problem, Jeroen

Krijgsveld and co-workers described a novel method combining
the metabolic labeling of azidohomoalanine (AHA), which is an
unnatural amino acid containing an azide group, with pulsed
stable isotope labeling through the amino acids during cell
culture (pSILAC) labeling to capture and quantify secreted
proteins selectively from serum-containing CMs.6 In this
approach, AHA was cotranslationally incorporated into newly
synthesized proteins. After a copper(I)-catalyzed click cycloaddition with an alkyne-functionalized agarose resin, the newly
synthesized proteins from the CMs could be captured
eciently. This approach was successfully used for the
quantitative secretome analysis of multiple cell lines or an
analysis performed under specic stimuli. Although this
bioorthogonal noncanonical amino acid tagging (BONCAT)
technique caused no apparent cytotoxicity,7 the replacement of
methionine by AHA may also induce changes in protein
expression.8
To obtain real proles of cell secretion, a more direct
approach involves a cell secretome analysis from the primary
culture supernatant. Some eorts including protein and/or
peptide fractionation were applied to detect the low-abundance
secreted proteins.9 Due to the wide dynamic range in the
abundance of the serum proteins (12 orders of magnitude),10
however, direct fractionation strategies were not eective.
Consequently, decreasing the complexity of serum proteins

he extracellular microenvironment is closely linked to the

physiological status of cells through interactive communication, including cell recognition, cellcell signaling,
receptorligand interactions, and so forth; most of these
processes are achieved through secreted proteins, such as
cytokines, growth factors, and enzymes.1 These proteins are
secreted or shed into culture medium or body uids,
undergoing dynamic changes during cell proliferation, development, and pathological or environmental stimuli. Therefore, a
detailed understanding of the proteomic composition and
quantitative changes of the cellular secretome are critical when
describing various biological processes2 and discovering
potential disease biomarkers.3
The rapid development of high-resolution mass spectrometry
(MS) and shotgun strategies for proteome analysis enables
comprehensive analyses of proteins from given biological
samples.4 However, the proteomic proling of cellular
secretomes from serum-containing conditioned media (CMs)
remains extremely challenging due to the low abundance of the
secreted proteins (as low as ng mL1) relative to the complex
background of highly abundant serum proteins (6 mg mL1).
Alternatively, a serum-free medium is often used over a dened
period during which secreted proteins are continuously
accumulated without serum interference, greatly reducing the
proteome complexity and facilitating identication. However,
depriving serum could disturb cell metabolism and proliferation.5 These disturbances may aect protein expression and
secretion proles and may even induce cell death, leading to the
experimental biases during qualitative and quantitative
secretome analyses.
2016 American Chemical Society

Received: March 8, 2016

Accepted: April 4, 2016
Published: April 4, 2016
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protein concentration to approximately 60 mg mL1 with a 1%

(v/v) protease inhibitor cocktail additive.
MTT Assay. The cell proliferation was measured via MTT
assay. The CMs collected from the MHCC97L and
MHCC97H cells were mixed with equal volumes of fresh
media (50%, v/v), respectively. These two new media were
used to culture MHCC97L cells. The MHCC97L cells were
seeded in 96-well plates (2000 cells/well) and incubated with
the above-mentioned media for 17 days. After incubation, the
cells were supplemented with 20 L of MTT (5 mg mL1,
Sigma) for 4 h at 37 C. Subsequently, the supernatant was
gently removed, and 200 L of DMSO was added to dissolve
the crystals. The absorbance at 490 nm was recorded while
using 630 nm as the reference with a Microplate reader
(BioTek, VT). The data were calculated as the means of eight
parallel experiments.
Protein Equalization with ProteoMiner. The concentrated CMs were processed using published protocols14 with
minor modications. Briey, the storage solution from
ProteoMiner columns containing 50 L beads was removed
by centrifugation (1000g, 30 s), and the beads were washed
with 600 L of 25 mM HEPES (pH 7.5, Sigma) and then with
600 L water three times. Next, 300 L of the concentrated
CMs (60 mg mL1) were transferred to the column and
incubated in a rolling incubator (Kylin-Bell Lab Instruments,
China) for 2 h at room temperature. Subsequently, the
unbound proteins were removed by washing with 600 L of
water ve times. The bound proteins were incubated with 300
L of boiled elution buer (4% sodium dodecyl sulfate (SDS),
25 mM dithiothreitol (DTT, Sigma)) for 5 min and
sequentially eluted with elution buer and 200 L of water.
Finally, the two eluted samples were combined for further
analysis.
GELFrEE Fractionation. The proteins were separated with
a GELFrEE 8100 Fractionation System (Expedeon, CA)
according to the manufacturers protocol with minor
modications. Briey, 1.2 mg of the equalized proteins were
divided into three equal aliquots, which were loaded into three
channels. In each channel, approximately 0.4 mg of the
equalized proteins in 150 L of the sample buer were
separated using commercial 8% tris-acetate cartridges (Expedeon, CA), and 10 fractions (150 L each) were collected
at specied intervals (57.5, 59.5, 61.5, 64.5, 67.5, 73.5, 85.5,
109.5, 133.5, 160 min). To visualize the separation eciency,
75 L of each fraction from one channel was separated using a
12% polyacrylamide gel and stained with Coomassie Blue. The
same fractions from the other two channels were merged, and
the protein concentration was determined through a BCA assay
for further pretreatment.
FASP Pretreatment. The proteins (50 g) from the cells,
raw CMs, the equalized CMs, and the dierent fractions were
reduced in 20 mM DTT (Sigma) at 56 C for 1.5 h, and the
products were alkylated in 40 mM iodoacetamide (IAA, Sigma)
at room temperature in the dark for 30 min. Next, the proteins
were transferred to 10 kDa lter devices (Sartorius AG,
Germany) and washed with 300 L of 8 M urea in 0.1 M Tris/
HCl (pH 8.5) by centrifugation (14 000g) three times. The
concentrates were diluted with 300 L of 25 mM NH4HCO3
and centrifuged again. After centrifugation, the concentrates
were diluted with 100 L of 25 mM NH4HCO3 containing 1
g of trypsin (Promega), and these mixtures were incubated at
37 C for 16 h. Subsequently, the digests were obtained
through centrifugation and dried in a Speed Vac Concentrator

before fractionation is critical.11 Recently, a new protein

equalization technique (ProteoMiner) has emerged that
could reduce the dynamic range of protein concentrations by
using a combinatorial library of random hexapeptide ligands
(206) to capture proteins under capacity-restrained rules. This
method demonstrated great advantages in enriching lowabundance proteins while removing high-abundance proteins12
and could be used in quantitative proteomic experiments.13
Therefore, the cells should be grown in a serum-containing
culture medium to acquire the informative data reecting the
real status of cells and strategies for sensitive, comprehensive,
and unbiased secretome analyses would be highly desirable.
Herein, we combined metabolic labeling, protein equalization,
protein fractionation, and lter-aided sample preparation
(FASP), called MLEFF strategy, toward the secretomic analysis
of serum-containing CMs, and it showed an excellent eect in
HeLa secretome proling. In addition, this strategy was further
applied to in-depth and dierential secretome analyses of two
human metastatic HCCs, improving our understanding of
tumor invasion and metastasis.

EXPERIMENTAL SECTION
Cell Culture, Metabolic Labeling, Cells and Media
Pretreatment. The MHCC97H, MHCC97L (HCC cells with
high and low metastatic potentials, respectively, kindly
presented by professor Yinkun Liu, Fudan University), and
HeLa cells (ATCC) were grown in a humidied atmosphere of
5% CO2 at 37 C in DMEM media (Thermo) supplemented
with 10% (v/v) FBS (Gibco) and 1% penicillin/streptomycin
(Thermo), respectively. For the medium labeling media (Lys4, Arg-6), L-lysine- and L-arginine-depleted SILAC DMEM
media (Thermo) were supplemented with [4,4,5,5-D4] L-lysine
(100 g mL1, Thermo), [13C6] L-arginine (100 g mL1,
Thermo), L-proline (200 g mL1, Thermo), 10% dialyzed FBS
(Gibco), and a 1% penicillin/streptomycin mixture. For the
heavy labeling media (Lys-8, Arg-10), only [4,4,5,5-D4] Llysine and [13C6] L-arginine were replaced with [13C6, 15N2] Llysine (Thermo) and [13C6, 15N4] L-arginine (Thermo). The
MHCC97L cells were grown in the medium media, and the
MHCC97H cells were grown in the heavy media. In addition,
HeLa cells were labeled with another heavy labeling medium
(Lys-6, Arg-10), which used [13C6] L-lysine and [13C6, 15N4] Larginine.
The SILAC-labeled cells were grown to at least six doubling
times to ensure the complete incorporation of the labeled
amino acids. Passaging was performed when 8090%
conuency was reached. To prepare the CMs from the HeLa
cells, the cells were incubated in complete labeling medium for
24 h. For the MHCC97H and MHCC97L cells, the cells and
CMs were both harvested after 48 h and were mixed based on
the number of cells.
The collected cells were suspended in 6 M guanidine
hydrochloride (Sigma) supplemented with 1% (v/v) protease
inhibitor cocktail (Sigma). Then, cell suspension was ultrasonicated on ice for 200 s in total (10 s intervals every 10 s),
followed by centrifugation at 20 000 rpm at 4 C for 30 min.
The supernatants were collected, and the protein concentration
was determined by a BCA assay (Beyotime, China).
The collected CMs were centrifuged at 500g and 3000g for
15 min to remove cells and cell debris, respectively. After
ltrating through a 0.22 m lter unit (Millipore, MA), the
supernatant was concentrated and desalted with water via
Amicon 3 kDa lter devices (Millipore, MA), increasing the
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(Thermo, MA). All of the samples were stored at 80 C for
further analysis.
For the peptides from MHCC97L and MHCC97H cells,
they were injected onto an Agilent 2100 HPLC system
(Agilent, CA) with a high pH-stable RP column (4.6 mm 250
mm, 5 m, 100 , Durashell, China) at a ow rate of 0.5 mL
min1. The peptides were eluted with a gradient from 5% to
45% solvent B over 55 min (solvent A: 20 mM ammonium
acetate, pH 10; solvent B: acetonitrile, 20 mM ammonium
acetate, pH 10). In all, 50 fractions were collected every 1 min
from 5 to 55 min. Then, fractions with equal collection time
intervals (5 min) were pooled. In this way, ve pooled fractions
were obtained pending further liquid chromatography coupled
with tandem mass spectrometry (LC-MS/MS) analysis.
LC-MS/MS Analysis. The peptides were analyzed with a 1D
nano-RPLC-MS/MS on a Q-Exactive MS (Thermo Fisher
Scientic, CA) coupled with an Ultimate 3000 (Dionex,
Germany) nano-LC system. The mobile phases were buers
A (2% acetonitrile, 98% water, and 0.1% formic acid) and B
(98% acetonitrile, 2% water, and 0.1% formic acid). Fused-silica
capillaries (150 m i.d. 375 m o.d.) were obtained from
Sino Sumtech (Handan, China). A C18 trap column (150 m
i.d. 5 cm) was connected to a homemade capillary separation
column (75 m i.d. 15 cm). Both the trap and separation
columns were packed with Daiso C18 particles (5 m, 100 ;
Osaka, Japan). To separate the peptides from the HeLa CMs, a
short gradient (52 min) was established: 37 min of 6%25%
buer B and then 15 min of 25%35% buer B with a ow rate
of 300 nL min1. To quantify additional proteins from the
MHCC97H and MHCC97L cells and CMs, a 110 min gradient
was established, comprised of 90 min of 6%22% buer B, and
then 20 min of 22%35% buer B. The spray voltage was 2.5
kV, and the temperature of the ion transfer capillary was set at
275 C. The Q-Exactive MS was operated in positive ion data
dependent mode, and the 10 most intense ions were subjected
to HCD fragmentation with normalized collision energy at
28%. The MS1 scans were performed at a resolution of 70 000
from m/z 300 to 1800 (automatic gain control (AGC) value,
1E6; maximum injection time, 100 ms), and the data were
acquired in prole mode. The MS/MS scans were performed at
a resolution of 17 500 (AGC, 1E5; maximum injection time, 60
ms), and the data were acquired in centroid mode using a 20 s
exclusion window. The unassigned ions or those with a charge
of +1 and >+7 were rejected. One microscan was acquired for
each MS and MS/MS scan. A lock mass correction was also
appended using a background ion (m/z 445.12003).
Database Searching. The raw data were uploaded into
Proteome Discoverer (PD, version 1.4.1.14) with Mascot
(2.3.2) and were searched against the UniProtKB human
complete proteome sequence database (release 2015_04,
42,121 entries). The reverse sequences were appended for an
FDR evaluation. The mass tolerances were set at 7 ppm for the
parent ions and at 20 ppm for the fragments. The peptides were
searched using tryptic cleavage constraints, and a maximum of
two missed cleavages were allowed. The minimal peptide length
was six amino acids. Carbamidomethylation (C) (+57.0215
Da) was used as the xed modication. Oxidation (M;
+15.9949 Da) and acetylation (protein N-termini; +42.0106
Da) were searched as variable modications. For the peptides
from the HeLa CMs, two SILAC-based labels (Lys6, + 6.0201
Da) and (Arg10, + 10.0083 Da) were used as variable
modications. For the peptides from the MHCC97H and
MHCC97L cells and CMs, the medium labels were (Lys4, +

4.0251 Da) and (Arg6, + 6.0201 Da), and the heavy labels were
(Lys8, + 8.0142 Da) and (Arg10, + 10.0083 Da). The peptide
and protein identications were ltered by PD to keep the FDR
1%. At least one unique peptide was required for each
protein identication.
Bioinformatic Analysis. The classical secreted proteins
were searched using Signal or Secreted as keywords in
UniProtKB, and the signal peptide was predicted by the SignalP
4.1 server15 (http://www.cbs.dtu.dk/services/SignalP/). The
nonclassical secreted proteins were predicted using a
SecretomeP 2.016 server (http://www.cbs.dtu.dk/services/
SecretomeP/) with an NN score >0.5, but not at a time
predicted to contain a signal peptide. The exosome proteins
were matched by the ExoCarta database17 (http://exocarta.
org/). The biological process annotations and protein
classications were performed using PANTHER18 for GO
analysis (http://pantherdb.org/).

RESULTS AND DISCUSSION

Principle of the MLEFF Strategy. An MLEFF strategy
combining metabolic labeling, protein equalization, protein
fractionation, and FASP was rst used to analyze the secreted
proteins from HeLa CMs (Figure 1, qualitative). Stable isotope
labeling by amino acids in cell culture (SILAC) has proven its
accuracy for the dierential study of proteins from cell
cultures.19 In our work, this method was used to distinguish
the true cellular proteins from fetal bovine serum (FBS) or
contaminations. Thus, [13C6] L-lysine and [13C6, 15N4] Larginine were used while cultivating HeLa cells, validating their
cell origin.
Subsequently, the performance of the protein equalization
was visualized using SDS-PAGE (Figure S-1). In the untreated
CM lane, the pattern was dominated by serum proteins, such as
albumin and transferrin, and the bands in low-molecular-weight
(Mw) region (<44.3 kDa) were almost invisible. After a
competitive enrichment based on the capacity-restraint
introduced by ProteoMiner beads, the equalized proteins
exhibited uniformly distributed protein bands spanning almost
the entire lane, dramatically decreasing the dynamic range in
protein concentration.
To reduce the complexity of the equalized proteins further,
protein fractionation must be performed before proteolysis.
During our experiment, the equalized proteins from the HeLa
CMs were eluted with a SDS-containing solution. Therefore,
gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), compatible with the upstream equalization process, is
suitable for protein fractionation.20 This technique shares the
principles of SDS-PAGE while increasing the loading capacity
and the protein recovery. The performance of the protein
fractionation process was investigated as follows (Figure S-1):
10 fractions were collected at specied intervals, and the
corresponding bands were well resolved, exhibiting a pattern
based on increasing Mw.
Although the liquid protein fractions dramatically decrease
the sample complexity, residual SDS remained in each fraction,
possibly decreasing the eciency of the trypsin digestion and
hindering the resolving power of reverse phase liquid
chromatography.21 In addition, SDS may lead to ion
suppression or accumulation inside the ion source during LCMS/MS analysis.22 Therefore, the FASP method,23 which could
enable the gel-free processing of biological samples solubilized
with detergents, was used in our experiment to remove SDS
during the subsequent proteomic analysis.
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Figure 2. Secretome analysis of CMs (cultured for 24 h) from HeLa

cells by our approach. (a) Cell-originated proteins and peptides
identied in untreated CMs, equalized CMs, and 10 fractions,
respectively. (b) Unique placement and redundancy of protein
fractionation. The x axis represents CoPros identied only in one
fraction (no. 1), or multiple fractions (no. 2, 3, 4, 5, and >5). The y
axis indicates the percentage of the identied CoPros. (c) Number of
the CoPros identied by LC-MS/MS in HeLa CMs. The Venn
diagram shows the overlap of the two biological replicates with the
total number of CoPros identied per replicate. (d) Composition of
the secretomes. Among the 585 CoPros identied, 534 (98%) CoPros
can be identied within the secretome, including secreted protein,
nonclassical secreted protein, and extracellular exosome protein. The
remaining proteins (10, 2%) remained unassigned.

Figure 1. Flowchart for the qualitative and quantitative analyses of the

cell secretome. (a) Acquisition of SILAC-labeled CMs. The HeLa
CMs were used for qualitative analysis; two human metastatic HCC
CMs were pooled for the quantitative analysis. (b) Protein
equalization. The serum-containing CMs were equalized by ProteoMiner beads to reduce the dynamic range of the protein concentration.
(c) Protein fractionation. The equalized proteins were fractionated
through a GELFrEE system based on the protein Mw. (d) FASP and
LC-MS/MS analysis. Each fraction was treated using the FASP
method, followed by LC-MS/MS analysis, enabling an extensive
secretome analysis.

suggesting the eectiveness of fractionation for reducing the

sample complexity.
Furthermore, two biological replicates were performed to
estimate the reproducibility of the entire workow; 585 CoPros
were identied with high overlap (Figure 2c), including 31
cytokines and/or growth factors, 16 receptors, 50 proteases,
and 20 protease inhibitors (Table S-1). In particular, IL-11, a
type of interleukin with a very low expression (ng mL1
range24) was identied in both replicates, demonstrating the
sensitivity of our method under complex serum interference.
Among these 585 CoPros, 294 (50.3%) were identied as
classical secreted proteins marked with the keywords Signal
or Secreted in UniProtKB or predicted by SignalP 4.115
containing a signal peptide (Figure 2d). The numbers and
ratios of the classical secreted proteins identied in HeLa cells
exceeded those in previous studies and our serum-starved
results, which identied 243 (out of 1223 total proteins,
19.9%)25 and 232 (out of 851 total proteins, 27.3%),
respectively. The detailed conditions are listed in Table S-2.
In particular, the higher percentage of classical secreted proteins
could be attributed to the fact that vast majority of HeLa cells
were still alive after the incubation (24 h) in the presence of
FBS. Thus, many cells were intact without the release of
cytoplasmic and nuclear proteins into CMs compared to the
serum-starved results.
Apart from the 294 classical secreted proteins, 138 were
predicted as nonclassical secreted proteins by SecretomeP
2.0,16 and 143 were matched by the ExoCarta exosome
database.17 These extracellular proteins are secreted by cells
through nonclassical or exosome-mediated secretion pathways,
and they are vital components of the cell secretome.
Collectively, these proteins (classical, nonclassical secreted
protein, and extracellular exosome protein) accounted for 98%

Secretome Proling in Serum-Containing HeLa CMs.

During our proteomic analysis of the HeLa secretome, the
proteins from the untreated CMs, equalized CMs, and each
GELFrEE fraction were processed using the FASP method to
minimize sample loss and remove the residual SDS (0.1%) in
liquid phase fractions. Three LC-MS/MS analyses with a short
gradient separation (52 min) led to the identication of only
eight cell-originated proteins (CoPros, SILAC-labeled) from
the untreated CMs. When analyzing the equalized CMs, we
identied 71 CoPros, 8.9-fold more than that of the untreated
CMs; these results suggest a good protein equalization. When
combining 10 fractions separated by GELFrEE from equalized
CMs, 458 CoPros were identied, 6.5-fold more than that of
the equalized CMs (Figure 2a), revealing the power of our
MLEFF strategy for sample preparation during secretomic
analyses achieved by decreasing the complexity of the samples.
To investigate the fractionated proteins further, the protein
Mw distribution in each fraction was visualized using violin
plots (Figure S-2), revealing an increasing prole analogous to
that of the SDS-PAGE results. In addition, we evaluated the
fractionation eciency (Figure 2b); more than 75% of the
proteins can only be identied in one or two fractions,
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(Table S-3), far exceeding the totals from two previous reports
(386 28 or 611 29 proteins), in serum-free CMs from
MHCC97H/MHCC97L cells. Of these 1300 CoPros, 1011
CoPros could be classied as part of the secretome (classical,
nonclassical secreted protein, and extracellular exosome
protein), which includes various growth factors and cytokines
involved in tumor proliferation and metastasis, such as Tgfb1,
Tgfb2, Gpi, Bmp1, Bmp2, Ccl15, Ccl20, Cxcl15, Cxcl16, and
Pf4.
In this data set, 861 CoPros could be reliably quantied in at
least two replicates. A gene ontology (GO) analysis suggests
that several biological processes such as translation, extracellular
matrix disassembly, and signal recognition particle (SRP)dependent cotranslational protein targeting members are
signicantly up-regulated in the CMs from MHCC97H (Figure
4a, Table S-4). Among these samples, several metalloproteases
(Adam9, Adam15, Mmp7) and hydrolases (Ctsb, Ctsd, Ctsl,

of all identied proteins. In summary, the above results

demonstrate that our method permits a sensitive and in-depth
secretome analysis reecting the normal growth status of cells
in the presence of bovine serum.
Proliferation Evaluation of MHCC97L. The human
MHCC97L and MHCC97H cells are two clones isolated
from the parent cell line with the same genetic background.
Compared to MHCC97L, MHCC97H exhibits various biological characteristics, such as smaller cell size, faster in vitro and
in vivo growth rates, and higher metastatic competency.26
Reports indicate that the tumor-derived secretome can facilitate
tumor growth and metastasis.27 To compare the bioactivities of
the secretomes derived from these two HCC cell lines, we
performed an MTT assay of MHCC97L cells to investigate the
promotional eect on proliferation (Figure 3a). The results

Figure 3. MTT proliferation assay of MHCC97L cells cultured in

dierent CM. (a) Workow of the MTT proliferation assay. The
detailed procedures are described in the Experimental Section. (b)
MTT assay results. The asterisks (*P < 0.05 and **P < 0.01) indicate
a statistically signicant increase in absorbance when using
MHCC97H CM instead of with MHCC97L CM. All of the results
were reproducible over eight independent experiments and are
reported as the means SEM.

show a signicant increase in the proliferation of MHCC97L

cells when adding the CM from MHCC97H cells, indicating
that a series of low-abundance and important secretory factors
were activated and functionalized in the MHCC97H CM
(Figure 3b).
Quantitative Analysis of MHCC97L and MHCC97H
Secretomes. Based on above results, the dierences in
secretome composition and expression of MHCC97L and
MHCC97H most likely contributed to the diversity in growth
and proliferation. Therefore, we conducted a comparative
proteomic analysis of CMs from two human HCC cell lines
with dierentially metastatic potentials by restricting quantication to the medium (green) and heavy (red) peaks, as shown
in Figure 1 (quantitative). To evaluate the cell secretome in an
unbiased manner, two types of CMs were mixed before sample
preparation. A total of 1300 CoPros can be detected (1152 with
quantitative information) in the presence of 10% (v/v) FBS

Figure 4. Biological analysis of quantied CoPros in two human

metastatic HCCs. (a) Biological process analysis based on GO. Each
violin plot shows a kernel density distribution of the log2 protein ratio.
The box plots show the median and the span from 25th to 75th
percentile. For each cluster, the enriched biological process terms are
shown with the hypergeometric P value based on a PANTHER overrepresentation test using the Bonferroni correction for multiple
testing. (b) Volcano plot of the quantied CoPros from triplicate
technical replicates. The P value was determined using a two-sided
Students t-test. Signicant regulation of the CoPros was dened as
log2 (Heavy/Medium) > 1 or < 1 where P < 0.05. The unregulated,
up-regulated, and down-regulated CoPros are shown in blue, red, and
green, respectively.
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Ctss, Ctsv) involved in extracellular matrix disassembly were
quantied with high expression, and these proteins were
believed to have participated in basement membrane
degradation and been implicated in tumor invasion and
metastasis.30 While other biological processes, such as
regulation of ligase activity, cellular response to stress, and
homeostatic process were signicantly down-regulated in the
CMs from MHCC97H. We detected a series of proteasome
subunits associated with regulation of ligase activity that
exhibited dierent degrees of reduction: Psma1, Psma2,
Psma3, Psma4, Psma5, Psma6, and Psma7 and Psmb1,
Psmb2, Psmb3, Psmb4, Psmb5, Psmb6, Psmb7, and Psmb8.
The informative and quantitative nature of these secretome
data enables an in-depth biological function analysis that may
aid biomarker discovery while helping to explain the
mechanisms of tumor growth, proliferation, and invasion.
Proteins with ratios more than 2 or less than 0.5
(Log2Heavy/Medium >1 or < 1) and t-test P values less
than 0.05 were considered to be signicantly secreted. On this
basis, we found that the levels of 43 and 18 CoPros were
elevated and decreased in the MHCC97H CMs versus those of
MHCC97L, respectively (Figure 4b), including several
cytokines, growth factors, and proteases. Apolipoprotein E
(Apoe), a secreted protein with a key role in lipid binding and
transport, showed the largest increase (14.2-fold) in
MHCC97H CMs; this protein has already demonstrated
overexpression in various cancers, including HCC.31 Granulins
(Grn), a pluripotent growth factor, was up-regulated 2.3-fold in
MHCC97H CMs. The overexpression of granulins implies that
the growth and invasion of HCC are promoted.32 In addition,
some members of the Cathepsin family, such as Cathepsin B
(Ctsb, 2.1-fold), Cathepsin D (Ctsd, 2.3-fold), and Cathepsin S
(Ctss, 3.1-fold), were also up-regulated in the MHCC97H
CMs. These proteins were involved in the disassembly and
organization of the extracellular matrix, which are key steps
during the migration and invasion of tumor cells.33 Protein
NDRG1 (Ndrg1), an important tumor metastasis suppressor in
many cell types,34 showed the largest decrease (0.23-fold) in
the MHCC97H CMs. Although reports indicate that many of
the regulated proteins identied in our data play key roles in
tumor metastasis, most of these studies were performed based
on intracellular proteins. From an extracellular perspective, our
results contain abundant information based on quantitative
proteomics, improving our understanding of tumor invasion
and metastasis.
Quantitative Comparison between Extracellular and
Intracellular Proteomes. Given the signicant dierences
between MHCC97L and MHCC97H regarding cell proliferation and invasion and the shared genetic background of these
two cells, we deduced that both the extracellular and
intracellular proteins played key roles in generating dierentially metastatic potentials. In previous studies of metastatic
tumor cells, most of the attention was focused on either
intracellular or extracellular proteins; few studies35 combined
both. Unfortunately, the above extracellular proteins were
obtained under serum-free conditions, which might perturb the
real status of the cells.
The intracellular proteins extracted from these two SILAClabeled HCCs were also subjected to quantitative proteomic
analyses (Figure 5a, Table S-5). A protein classication analysis
revealed a signicant dierence in the distribution of
functionalities between the extracellular and intracellular
proteins (Figure 5b). The extracellular proteins dominated in

Figure 5. Comparison between the extracellular and intracellular

proteomes. (a) Schematic illustration of the experimental setup and
proteomics workow. (b) Classication of the proteins within the
extracellular (1,300 proteins, blue) and intracellular (3,119 proteins,
light blue) proteomes based on PANTHER protein classication. (c)
Unsupervised hierarchical clustering of 476 proteins reproducibly
quantied in MHCC97H and MHCC97L cell lines from the
extracellular and intracellular proteomes.

many functionalities, including cell adhesion molecular,

extracellular matrix proteins, peptide hormones, apolipoproteins, cytokines, growth factors, and chemokines.
In addition, 476 quantied proteins were overlapped in both
the extracellular and intracellular proteomes (Figure 5c).
Notably, these proteins show tiny dierences at the intracellular
level, remaining consistent with our earlier result,36 implying
that these intracellular proteins may have limited roles when
inducing dierent cell behaviors. This work presents a
comprehensive analysis of the extracellular and intracellular
proteins under normal culture conditions without serum
starvation. The unsupervised clustering of these quantied
proteins shows a more signicant change in the extracellular
proteins compared to the corresponding intracellular ones. A
large portion of the changed proteins are primarily involved in
cell adhesion, ligase activity regulation, and extracellular matrix
disassembly. Therefore, the dierent secretion proles of these
two human metastatic HCCs may contribute signicantly to
cell growth, proliferation, and invasion. The intimate
connections between the extracellular proteins (secretome)
and the intracellular proteins compose the complicated and
precise mechanisms that regulate dierent cell behaviors.

CONCLUSION
In a typical cell culture (1 107 cells, culture for 24 h)
experiment, 2080 g of secreted proteins could be released
into the extracellular medium across dierent cell types.37
Unfortunately, these secreted proteins can be masked by the
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Analytical Chemistry

complex serum protein background (60 mg). We developed

an ecient strategy called MLEFF to overcome this problem.
The rst step is to distinguish the cellular proteins from the
bovine serum background. Only the peptides with SILAC labels
are passed for identication. To reduce the dynamic range of
the proteins in the CM sample, a protein equalization
(ProteoMiner) technique was adopted, enabling the identication of 8.9-fold more proteins than that of the untreated
CMs. The equalized samples are compatible with and eective
for a subsequent protein fractionation according to molecular
weights by the GELFrEE system, which decreases the
complexity of the samples further and provides more
opportunities for identifying proteins with low abundance.
The advantages of our approach lay in the integrated process of
protein equalization, fractionation, and FASP-based digestion,
which enables the sensitive, high-throughput, reproducible,
unbiased secretome analysis.
Then, we demonstrated the successful application of MLEFF
in the comprehensive and quantitative secretome analysis from
two human metastatic HCCs. This approach showed a great
advantage in accurate quantication because the CMs with
dierent SILAC labels were mixed before any pretreatment.
Many regulated proteins were found closely related to tumor
growth, proliferation, invasion, and metastasis, as well as being
worthy of further study during the quantication of HCC CMs.
In addition, we compared the proteomic composition and
expression at both the extracellular and intracellular levels and
found more signicant dierences at the extracellular level. The
qualitative and quantitative results reected the real status of
the cell secretions because the culture conditions were normal
and without any specic stimulus. Because most previous
secretome studies are based on serum-free media systems, we
expect that our approach will facilitate the study of cell
secretomes, particularly for cells that are highly sensitive toward
serum starvation.

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ASSOCIATED CONTENT

S Supporting Information
*

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.analchem.6b00910.
SDS-PAGE proling of untreated CM, equalized CM,
and 10 fractions separated by GELFrEE system; violin
plots of Mw distribution of identied proteins in dierent
fractions (PDF)
Supplementary Data Set 1 (XLSX)
Supplementary Data Set 2 (XLSX)
Supplementary Data Set 3 (XLSX)
Supplementary Data Set 4 (XLSX)
Supplementary Data Set 5 (XLSX)

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: lihuazhang@dicp.ac.cn.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
This work was supported by National Basic Research Program
of China (2012CB910604), National Natural Science Foundation (21190043), and The Creative Research Group Project by
NSFC (21321064).
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