In-Depth Proteomic Quantification of Cell Secretome in Serum- Containing Conditioned Medium

8/16/2019 In-Depth Proteomic Quantification of Cell Secretome in Serum- Containing Conditioned Medium

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In-Depth Proteomic Quantication of Cell Secretome in Serum-Containing 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 ChemicalPhysics, 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 inserum-containing culture media is technically challenging. Most studies have beenperformed under serum-free conditions. However, these conditions might alter thestatus of the cells. Herein, we describe an efficient 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 534secreted proteins from HeLa conditioned media, including 31 cytokines, and growthfactors. This MLEFF strategy was also successfully applied during a comparativesecretome analysis of two human hepatocellular carcinoma cell lines with diff erentially metastatic potentials, enabling the quantication of 61 signicantly changed proteinsinvolved in tumor invasion and metastasis.

T he extracellular microenvironment is closely linked to thephysiological status of cells through interactive commu-nication, including cell recognition, cell−cell signaling,receptor−ligand interactions, and so forth; most of theseprocesses are achieved through secreted proteins, such ascytokines, growth factors, and enzymes.1 These proteins aresecreted or shed into culture medium or body uids,undergoing dynamic changes during cell proliferation, develop-ment, and pathological or environmental stimuli. Therefore, adetailed understanding of the proteomic composition andquantitative changes of the cellular secretome are critical whendescribing various biological processes2 and discoveringpotential disease biomarkers.3

The rapid development of high-resolution mass spectrometry (MS) and shotgun strategies for proteome analysis enablescomprehensive analyses of proteins from given biologicalsamples.4 However, the proteomic proling of cellularsecretomes from serum-containing conditioned media (CMs)remains extremely challenging due to the low abundance of thesecreted proteins (as low as ng mL−1) relative to the complex background of highly abundant serum proteins (∼6 mg mL−1). Alternatively, a serum-free medium is often used over a denedperiod during which secreted proteins are continuously accumulated without serum interference, greatly reducing theproteome complexity and facilitating identication. However,depri ving serum could disturb cell metabolism and prolifer-ation.5 These disturbances may aff ect protein expression andsecretion proles and may even induce cell death, leading to theexperimental biases during qualitative and quantitativesecretome analyses.

To circumvent the above-mentioned problem, JeroenKrijgsveld and co-workers described a novel method combiningthe metabolic labeling of azidohomoalanine (AHA), which is anunnatural amino acid containing an azide group, with pulsedstable isotope labeling through the amino acids during cellculture (pSILAC) labeling to capture and quantif y secretedproteins selectively from serum-containing CMs.6 In thisapproach, AHA was cotranslationally incorporated into newly synthesized proteins. After a copper(I)-catalyzed click cyclo-addition with an alkyne-functionalized agarose resin, the newly synthesized proteins from the CMs could be capturedefficiently. This approach was successfully used for thequantitative secretome analysis of multiple cell lines or ananalysis 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 proteinexpression.8

To obtain real proles of cell secretion, a more directapproach involves a cell secretome analysis from the primary culture supernatant. Some eff orts including protein and/orpeptide fractionation were applied to detect the low-abundancesecreted proteins.9 Due to the wide dynamic range in theabundance of the serum proteins (12 orders of magnitude),10

however, direct fractionation strategies were not eff ective.Consequently, decreasing the complexity of serum proteins

Received: March 8, 2016 Accepted: April 4, 2016Published: April 4, 2016

Article

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© 2016 American Chemical Society 4971 DOI: 10.1021/acs.analchem.6b00910

Anal. Chem. 2016, 88, 4971−4978

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before fractionation is critical.11 Recently, a new protein“equalization” technique (ProteoMiner) has emerged thatcould reduce the dynamic range of protein concentrations by using a combinatorial library of random hexapeptide ligands(206) to capture proteins under capacity-restrained rules. Thismethod demonstrated great advantages in enriching lo w-abundance proteins while removing high-abundance proteins12

and could be used in quantitative proteomic experiments.13

Therefore, the cells should be grown in a serum-containingculture medium to acquire the informative data reecting thereal 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 analysisof serum-containing CMs, and it showed an excellent eff ect inHeLa secretome proling. In addition, this strategy was furtherapplied to in-depth and diff erential secretome analyses of twohuman metastatic HCCs, improving our understanding of tumor invasion and metastasis.

■ EXPERIMENTAL SECTION

Cell Culture, Metabolic Labeling, Cells and MediaPretreatment. The MHCC97H, MHCC97L (HCC cells withhigh and low metastatic potentials, respectively, kindly presented by professor Yinkun Liu, Fudan University), andHeLa 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 (Lys-4, Arg-6), L-lysine- and L-arginine-depleted SILAC DMEMmedia (Thermo) were supplemented with [4,4,5,5-D4] L-lysine(100 μg mL−1 , Thermo), [13C6] L-arginine (100 μg mL

−1 ,Thermo), L-proline (200 μg mL−1 , 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] L-lysine and [13C6] L-arginine were replaced with [13C6 , 15N2] L-lysine (Thermo) and [13C6 ,

15N4] L-arginine (Thermo). TheMHCC97L cells were grown in the “medium” media, and theMHCC97H 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] L-

arginine.The SILAC-labeled cells were grown to at least six doubling

times to ensure the complete incorporation of the labeledamino acids. Passaging was performed when 80−90%conuency was reached. To prepare the CMs from the HeLacells, the cells were incubated in complete labeling medium for24 h. For the MHCC97H and MHCC97L cells, the cells andCMs 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) proteaseinhibitor cocktail (Sigma). Then, cell suspension was ultra-sonicated 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 500 g and 3000 g for15 min to remove cells and cell debris, respectively. Afterltrating through a 0.22 μm lter unit (Millipore, MA), thesupernatant was concentrated and desalted with water via Amicon 3 kDa lter devices (Millipore, MA), increasing the

protein concentration to approximately 60 mg mL−1 with a 1%(v/v) protease inhibitor cocktail additive.

MTT Assay. The cell proliferation was measured via MTTassay. The CMs col lected from the MHCC97L andMHCC97H cells were mixed with equal volumes of freshmedia (50%, v/v), respectively. These two new media wereused to culture MHCC97L cells. The MHCC97L cells wereseeded in 96-well plates (2000 cells/well) and incubated withthe above-mentioned media for 1−7 days. After incubation, thecells were supplemented with 20 μL of MTT (5 mg mL−1 ,Sigma) for 4 h at 37 °C. Subsequently, the supernatant wasgently removed, and 200 μL of DMSO was added to dissolvethe crystals. The absorbance at 490 nm was recorded whileusing 630 nm as the reference with a Microplate reader(BioTek, VT). The data were calculated as the means of eightparallel experiments.

Protein Equalization with ProteoMiner. The concen-trated CMs were processed using published protocols14 withminor modications. Brie y, the storage solution fromProteoMiner columns containing 50 μL beads was removed by centrifugation (1000 g , 30 s), and the beads were washed with 600 μL of 25 mM HEPES (pH 7.5, Sigma) and then with600 μL water three times. Next, 300 μL of the concentratedCMs (∼60 mg mL−1) were transferred to the column andincubated in a rolling incubator (Kylin-Bell Lab Instruments,China) for 2 h at room temperature. Subsequently, theunbound proteins were removed by washing with 600 μL of water ve times. The bound proteins were incubated with 300 μL of boiled elution buff er (4% sodium dodecyl sulfate (SDS),25 mM dithiothreitol (DTT, Sigma)) for 5 min andsequentially eluted with elution buff er and 200 μL of water.Finally, the two eluted samples were combined for furtheranalysis.

GELFrEE Fractionation. The proteins were separated witha GELFrEE 8100 Fractionation System (Expedeon, CA)according to the manufacturer’s protocol with minor

modications. Brie y, 1.2 mg of the equalized proteins weredivided into three equal aliquots, which were loaded into threechannels. In each channel, approximately 0.4 mg of theequalized proteins in 150 μL of the sample buff er wereseparated using commercial 8% tris-acetate cartridges (Ex-pedeon, CA), and 10 fractions (∼150 μL each) were collectedat 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 efficiency,75 μL of each fraction from one channel was separated using a12% polyacrylamide gel and stained with Coomassie Blue. Thesame fractions from the other two channels were merged, andthe protein concentration was determined through a BCA assay for further pretreatment.

FASP Pretreatment. The proteins (∼50 μg) from the cells,

raw CMs, the equalized CMs, and the diff erent fractions werereduced in 20 mM DTT (Sigma) at 56 °C for 1.5 h, and theproducts 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 μL of 8 M urea in 0.1 M Tris/HCl (pH 8.5) by centrifugation (14 000 g ) three times. Theconcentrates were diluted with 300 μL of 25 mM NH4HCO3and centrifuged again. After centrifugation, the concentrates were diluted with 100 μL of 25 mM NH4HCO3 containing 1 μg of trypsin (Promega), and these mixtures were incubated at37 °C for 16 h. Subsequently, the digests were obtainedthrough centrifugation and dried in a Speed Vac Concentrator

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.6b00910 Anal. Chem. 2016, 88, 4971−4978

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(Thermo, MA). All of the samples were stored at −80 °C forfurther 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 × 250mm, 5 μm, 100 Å, Durashell, China) at a ow rate of 0.5 mLmin−1. The peptides were eluted with a gradient from 5% to45% solvent B over 55 min (solvent A: 20 mM ammoniumacetate, pH 10; solvent B: acetonitrile, 20 mM ammoniumacetate, pH 10). In all, 50 fractions were collected every 1 minfrom 5 to 55 min. Then, fractions with equal collection timeintervals (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 1Dnano-RPLC-MS/MS on a Q-Exactive MS (Thermo FisherScientic, CA) coupled with an Ultimate 3000 (Dionex,Germany) nano-LC system. The mobile phases were buff ers A (2% acetonitrile, 98% water, and 0.1% formic acid) and B(98% acetonitrile, 2% water, and 0.1% formic acid). Fused-silicacapillaries (150 μm i.d. × 375 μm o.d.) were obtained fromSino Sumtech (Handan, China). A C18 trap column (150 μmi.d. × 5 cm) was connected to a homemade capillary separationcolumn (75 μm i.d. × 15 cm). Both the trap and separationcolumns were packed with Daiso C18 particles (5 μm, 100 Å;Osaka, Japan). To separate the peptides from the HeLa CMs, ashort gradient (52 min) was established: 37 min of 6%−25% buff er B and then 15 min of 25%−35% buff er B with a ow rateof 300 nL min−1. To quantify additional proteins from theMHCC97H and MHCC97L cells and CMs, a 110 min gradient was established, comprised of 90 min of 6%−22% buff er B, andthen 20 min of 22%−35% buff er B. The spray voltage was 2.5kV, and the temperature of the ion transfer capillary was set at275 °C. The Q-Exactive MS was operated in positive ion datadependent mode, and the 10 most intense ions were subjectedto HCD fragmentation with normalized collision energy at

28%. The MS1 scans were performed at a resolution of 70 000from m/ z 300 to 1800 (automatic gain control (AGC) value,1E6; maximum injection time, 100 ms), and the data wereacquired in prole mode. The MS/MS scans were performed ata resolution of 17 500 (AGC, 1E5; maximum injection time, 60ms), and the data were acquired in centroid mode using a 20 sexclusion window. The unassigned ions or those with a chargeof +1 and >+7 were rejected. One microscan was acquired foreach MS and MS/MS scan. A lock mass correction was alsoappended using a background ion (m/ z 445.12003).

Database Searching. The raw data were uploaded intoProteome Discoverer (PD, version 1.4.1.14) with Mascot(2.3.2) and were searched against the UniProtKB humancomplete proteome sequence database (release 2015_04,

42,121 entries). The reverse sequences were appended for anFDR evaluation. The mass tolerances were set at 7 ppm for theparent ions and at 20 ppm for the fragments. The peptides weresearched using tryptic cleavage constraints, and a maximum of two missed cleavages were allowed. The minimal peptide length was six amino acids. Carbamidomethylation (C) (+57.0215Da) was used as the xed modication. Oxidation (M;+15.9949 Da) and acetylation (protein N-termini; +42.0106Da) were searched as variable modications. For the peptidesfrom the HeLa CMs, two SILAC-based labels (Lys6, + 6.0201Da) and (Arg10, + 10.0083 Da) were used as variablemodications. For the peptides from the MHCC97H andMHCC97L 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 peptideand protein identications were ltered by PD to keep the FDR ≤ 1%. At least one unique peptide was required for eachprotein identication.

Bioinformatic Analysis. The classical secreted proteins were searched using “Signal” or “Secreted” as keywords inUniProtK B, and the signal peptide was predicted by the SignalP4.1 server15 (http://www.cbs.dtu.dk/services/SignalP/). Thenonclassical secreted proteins were predicted using aSecretomeP 2.016 server (http://www.cbs.dtu.dk/services/SecretomeP/) with an NN score >0.5, but not at a timepredicted to contain a signal peptide. The exosome proteins were matched by the ExoCarta database17 (http://exocarta.org/). The biological process annotations and proteinclassications were performed using PANTHER 18 for GOanalysis (http://pantherdb.org/).

■ RESULTS AND DISCUSSION

Principle of the MLEFF Strategy. An MLEFF strategy combining metabolic labeling, protein equalization, proteinfractionation, and FASP was rst used to analyze the secretedproteins from HeLa CMs (Figure 1 , qualitative). Stable isotopelabeling by amino acids in cell culture (SILAC) has proven itsaccuracy for the diff erential study of proteins from cellcultures.19 In our work, this method was used to distinguishthe true cellular proteins from fetal bovine serum (FBS) orcontaminations. Thus, [13C6] L-lysine and [

13C6 , 15N4] L-

arginine were used while cultivating HeLa cells, validating theircell origin.

Subsequently, the performance of the protein equalization was visualized using SDS-PAGE (Figure S-1). In the untreatedCM lane, the pattern was dominated by serum proteins, such asalbumin and transferrin, and the bands in low-molecular-weight(Mw) region (


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Secretome Proling in Serum-Containing HeLa CMs.During our proteomic analysis of the HeLa secretome, theproteins from the untreated CMs, equalized CMs, and eachGELFrEE fraction were processed using the FASP method tominimize sample loss and remove the residual SDS (∼0.1%) inliquid phase fractions. Three LC-MS/MS analyses with a shortgradient separation (52 min) led to the identication of only eight cell-originated proteins (CoPros, SILAC-labeled) fromthe untreated CMs. When analyzing the equalized CMs, we

identied 71 CoPros, 8.9-fold more than that of the untreatedCMs; these results suggest a good protein equalization. Whencombining 10 fractions separated by GELFrEE from equalizedCMs, 458 CoPros were identied, 6.5-fold more than that of the equalized CMs (Figure 2a), revealing the power of ourMLEFF strategy for sample preparation during secretomicanalyses achieved by decreasing the complexity of the samples.

To investigate the fractionated proteins further, the proteinMw distribution in each fraction was visualized using violinplots (Figure S-2), revealing an increasing prole analogous tothat of the SDS-PAGE results. In addition, we evaluated thefractionation efficiency (Figure 2 b); more than 75% of theproteins can only be identied in one or two fractions,

suggesting the eff ectiveness of fractionation for reducing thesample complexity.

Furthermore, two biological replicates were performed toestimate the reproducibility of the entire work ow; 585 CoPros were identied with high overlap (Figure 2c), including 31cytokines and/or growth factors, 16 receptors, 50 proteases,and 20 protease inhibitors (Table S-1). In particular, IL-11, atype of interleukin with a very low expression (ng mL−1

range24) was identied in both replicates, demonstrating thesensitivity of our method under complex serum interference.

Among these 585 CoPros, 294 (50.3%) were identied asclassical 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 andratios of the classical secreted proteins identied in HeLa cellsexceeded those in previous studies and our serum-starvedresults, 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 theserum-starved results.

Apart from the 294 classical secreted proteins, 138 werepredicted as nonclassical secreted proteins by SecretomeP2.0,16 and 143 were matched by the ExoCarta exosomedatabase.17 These extracellular proteins are secreted by cellsthrough nonclassical or exosome-mediated secretion pathways,and they are vital components of the cell secretome.Collectively, these proteins (classical, nonclassical secretedprotein, and extracellular exosome protein) accounted for 98%

Figure 1. Flowchart for the qualitative and quantitative analyses of thecell secretome. (a) Acquisition of SILAC-labeled CMs. The HeLaCMs were used for qualitative analysis; two human metastatic HCCCMs were pooled for the quantitative analysis. (b) Proteinequalization. The serum-containing CMs were equalized by Proteo-Miner beads to reduce the dynamic range of the protein concentration.(c) Protein fractionation. The equalized proteins were fractionatedthrough a GELFrEE system based on the protein Mw. (d) FASP and

LC-MS/MS analysis. Each fraction was treated using the FASPmethod, followed by LC-MS/MS analysis, enabling an extensivesecretome analysis.

Figure 2. Secretome analysis of CMs (cultured for 24 h) from HeLacells by our approach. (a) Cell-originated proteins and peptidesidentied in untreated CMs, equalized CMs, and 10 fractions,respectively. (b) Unique placement and redundancy of proteinfractionation. The x axis represents CoPros identied only in onefraction (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 Venndiagram shows the overlap of the two biological replicates with thetotal number of CoPros identied per replicate. (d) Composition of the secretomes. Among the 585 CoPros identied, 534 (98%) CoProscan be identied within the secretome, including secreted protein,nonclassical secreted protein, and extracellular exosome protein. Theremaining proteins (10, 2%) remained unassigned.



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of all identied proteins. In summary, the above resultsdemonstrate that our method permits a sensitive and in-depthsecretome analysis reecting the normal growth status of cellsin the presence of bovine serum.

Proliferation Evaluation of MHCC97L. The humanMHCC97L and MHCC97H cells are two clones isolatedfrom the parent cell line with the same genetic background.Compared to MHCC97L, MHCC97H exhibits various bio-logical characteristics, such as smaller cell size, faster in vitro andin vivo growth rates, and higher metastatic competency.26

Reports indicate that the tumor-derived secretome can facilitatetumor growth and metastasis.27 To compare the bioactivities of the secretomes derived from these two HCC cell lines, weperformed an MTT assay of MHCC97L cells to investigate thepromotional eff ect on proliferation (Figure 3a). The results

show a signicant increase in the proliferation of MHCC97Lcells when adding the CM from MHCC97H cells, indicatingthat a series of low-abundance and important secretory factors were activated and functionalized in the MHCC97H CM

(Figure 3 b).Quantitative Analysis of MHCC97L and MHCC97H

Secretomes. Based on above results, the diff erences insecretome composition and expression of MHCC97L andMHCC97H most likely contributed to the diversity in growthand proliferation. Therefore, we conducted a comparativeproteomic analysis of CMs from two human HCC cell lines with diff erentially metastatic potentials by restricting quanti-cation to the medium (green) and heavy (red) peaks, as shownin Figure 1 (quantitative). To evaluate the cell secretome in anunbiased manner, two types of CMs were mixed before samplepreparation. A total of 1300 CoPros can be detected (1152 withquantitative information) in the presence of 10% (v/v) FBS

(Table S-3), far e xceeding the totals from two previous reports(38628 or 61129 proteins), in serum-free CMs fromMHCC97H/MHCC97L cells. Of these 1300 CoPros, 1011CoPros could be classied as part of the secretome (classical,nonclassical secreted protein, and extracellular exosomeprotein), which includes various growth factors and cytokinesinvolved in tumor proliferation and metastasis, such as Tgfb1,

Tgfb2, Gpi, Bmp1, Bmp2, Ccl15, Ccl20, Cxcl15, Cxcl16, andPf4.In this data set, 861 CoPros could be reliably quantied in at

least two replicates. A gene ontology (GO) analysis suggeststhat several biological processes such as translation, extracellularmatrix disassembly, and signal recognition particle (SRP)-dependent cotranslational protein targeting members aresignicantly up-regulated in the CMs from MHCC97H (Figure4a, Table S-4). Among these samples, several metalloproteases(Adam9, Adam15, Mmp7) and hydrolases (Ctsb, Ctsd, Ctsl,

Figure 3. MTT proliferation assay of MHCC97L cells cultured indiff erent CM. (a) Work ow of the MTT proliferation assay. Thedetailed procedures are described in the Experimental Section. (b)MTT assay results. The asterisks (* P < 0.05 and ** P < 0.01) indicatea statistically signicant increase in absorbance when usingMHCC97H CM instead of with MHCC97L CM. All of the results

were reproducible over eight independent experiments and arereported as the means ± SEM.

Figure 4. Biological analysis of quantied CoPros in two humanmetastatic 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 75thpercentile. For each cluster, the enriched biological process terms areshown with the hypergeometric P value based on a PANTHER over-representation test using the Bonferroni correction for multipletesting. (b) Volcano plot of the quantied CoPros from triplicatetechnical replicates. The P value was determined using a two-sidedStudent’s t -test. Signicant regulation of the CoPros was dened aslog2 (Heavy/Medium) > 1 or < −1 where P < 0.05. The unregulated,up-regulated, and down-regulated CoPros are shown in blue, red, andgreen, respectively.



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Ctss, Ctsv) involved in extracellular matrix disassembly werequantied with high expression, and these proteins were bel ieved to have partic ipated in basement membranedegradation and been implicated in tumor invasion andmetastasis.30 While other biological processes, such asregulation of ligase activity, cellular response to stress, andhomeostatic process were signicantly down-regulated in theCMs from MHCC97H. We detected a series of proteasomesubunits associated with regulation of ligase activity thatexhibited diff erent 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 secretomedata enables an in-depth biological function analysis that may aid biomarker discovery while helping to explain themechanisms 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 lessthan 0.05 were considered to be signicantly secreted. On this basis, we found that the levels of 43 and 18 CoPros wereelevated and decreased in the MHCC97H CMs versus those of MHCC97L, respectively (Figure 4 b), including severalcytokines, growth factors, and proteases. Apolipoprotein E(Apoe), a secreted protein with a key role in lipid binding andtransport, showed the largest increase (14.2-fold) inMHCC97H CMs; this protein has already demonstratedoverexpression in various cancers, including HCC.31 Granulins(Grn), a pluripotent growth factor, was up-regulated 2.3-fold inMHCC97H CMs. The overexpression of granulins implies thatthe 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 MHCC97HCMs. These proteins were involved in the disassembly andorganization of the extracellular matrix, which are k ey stepsduring the migration and invasion of tumor cells.33 Protein

NDRG1 (Ndrg1), an important tumor metastasis suppressor inmany cell types,34 showed the largest decrease (0.23-fold) inthe MHCC97H CMs. Although reports indicate that many of the regulated proteins identied in our data play key roles intumor metastasis, most of these studies were performed basedon intracellular proteins. From an extracellular perspective, ourresults contain abundant information based on quantitativeproteomics, improving our understanding of tumor invasionand metastasis.

Quantitative Comparison between Extracellular andIntracellular Proteomes. Given the signicant diff erences between MHCC97L and MHCC97H regarding cell prolifer-ation and invasion and the shared genetic background of thesetwo cells, we deduced that both the extracellular and

intracellular proteins played key roles in generating diff er-entially metastatic potentials. In previous studies of metastatictumor cells, most of the attention was focused on eitherintracellular or extracellular proteins; few studies35 combined both. Unfortunately, the above extracellular proteins wereobtained under serum-free conditions, which might perturb thereal status of the cells.

The intracellular proteins extracted from these two SILAC-labeled HCCs were also subjected to quantitative proteomicanalyses (Figure 5a, Table S-5). A protein classication analysisrevealed a signicant diff erence in the distribution of functionalities between the extracellular and intracellularproteins (Figure 5 b). The extracellular proteins dominated in

many functionalities, including cell adhesion molecular,

extracellular matrix proteins, peptide hormones, apolipopro-teins, cytokines, growth factors, and chemokines.

In addition, 476 quantied proteins were overlapped in boththe extracellular and intracellular proteomes (Figure 5c).Notably, these proteins show tiny diff erences at the intracellularlevel, remaining consistent with our earlier result,36 implyingthat these intracellular proteins may have limited roles wheninducing diff erent cell behaviors. This work presents acomprehensive analysis of the extracellular and intracellularproteins under normal culture conditions without serumstarvation. The unsupervised clustering of these quantiedproteins shows a more signicant change in the extracellularproteins 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 diff erent secretion proles of thesetwo human metastatic HCCs may contribute signicantly tocell growth, proliferation, and invasion. The intimateconnections between the extracellular proteins (secretome)and the intracellular proteins compose the complicated andprecise mechanisms that regulate diff erent cell behaviors.

■ CONCLUSIONIn a typical cell culture (1 × 107 cells, culture for 24 h)experiment, 20−80 μg of secreted proteins could be releasedinto the extracellular medium across diff erent cell types.37

Unfortunately, these secreted proteins can be masked by the

Figure 5. Comparison between the extracellular and intracellularproteomes. (a) Schematic illustration of the experimental setup andproteomics work ow. (b) Classication of the proteins within theextracellular (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 theextracellular and intracellular proteomes.



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complex serum protein background (∼60 mg). We developedan efficient 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 labelsare passed for identication. To reduce the dynamic range of the proteins in the CM sample, a protein equalization(ProteoMiner) technique was adopted, enabling the identi-cation of 8.9-fold more proteins than that of the untreatedCMs. The equalized samples are compatible with and eff ectivefor a subsequent protein fractionation according to molecular weights by the GELFrEE system, which decreases thecomplexity of the samples further and provides moreopportunities 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 MLEFFin the comprehensive and quantitative secretome analysis fromtwo human metastatic HCCs. This approach showed a greatadvantage in accurate quantication because the CMs withdiff erent SILAC labels were mixed before any pretreatment.

Many regulated proteins were found closely related to tumorgrowth, 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 andexpression at both the extracellular and intracellular levels andfound more signicant diff erences at the extracellular level. Thequalitative and quantitative results reected the real status of the cell secretions because the culture conditions were normaland without any specic stimulus. Because most previoussecretome studies are based on serum-free media systems, weexpect that our approach will facilitate the study of cellsecretomes, particularly for cells that are highly sensitive towardserum starvation.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on the ACS Publi cat ions website at DOI: 10.1021/acs.anal-chem.6b00910.

SDS-PAGE proling of untreated CM, equalized CM,and 10 fractions separated by GELFrEE system; violinplots of Mw distribution of identied proteins in diff erentfractions (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 )

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected].

NotesThe authors declare no competing nancial interest.

■ ACKNOWLEDGMENTSThis work was supported by National Basic Research Programof China (2012CB910604), National Natural Science Founda-tion (21190043), and The Creative Research Group Project by NSFC (21321064).

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In-Depth Proteomic Quantification of Cell Secretome in Serum- Containing Conditioned Medium

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