In-Depth Proteomic Quantification of Cell Secretome in Serum

Apr 4, 2016 - The comprehensive profiling of secreted proteins in serum-containing culture media is technically challenging. Most studies have been pe...
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In-depth Proteomic Quantification 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 Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00910 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 5, 2016

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In-depth Proteomic Quantification 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 Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. ‡ University of Chinese Academy of Sciences, Beijing 100049, China. KEYWORDS: Cell Secretome; Proteomic Quantification; Cancer Metastasis; Secreted Proteins ABSTRACT: Secreted proteins play key roles during cellular communication, proliferation and migration. The comprehensive profiling 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 efficient strategy that avoids the disturbance of serum by combining metabolic labelling, protein 'equalization', protein fractionation and filter-aided sample preparation, called MLEFF, enabling the identification 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 differentially metastatic potentials, enabling the quantification of 61 significantly changed proteins involved in tumour invasion and metastasis.

which is an unnatural amino acid containing an azide group, INTRODUCTION with pulsed stable isotope labelling through the amino acids The extracellular microenvironment is closely linked to the during cell culture (pSILAC) labelling to capture and quantify physiological status of cells through interactive communicasecreted proteins selectively from serum-containing CMs.6 In tion, including cell recognition, cell-cell signalling, receptorthis approach, AHA was cotranslationally incorporated into ligand interactions and so forth; most of these processes are newly synthesized proteins. After a copper (I)-catalysed click achieved through secreted proteins, such as cytokines, growth cycloaddition with an alkyne-functionalized agarose resin, the factors and enzymes.1 These proteins are secreted or shed into newly synthesized proteins from the CMs could be captured culture medium or body fluids, undergoing dynamic changes efficiently. This approach was successfully used for the quanduring cell proliferation, development and pathological or titative secretome analysis of multiple cell lines or an analysis environmental stimuli. Therefore, a detailed understanding of performed under specific stimuli. Although this bioorthogonal the proteomic composition and quantitative changes of the non-canonical amino acid tagging (BONCAT) technique cellular secretome are critical when describing various biologcaused no apparent cytotoxicity,7 the replacement of methioical processes2 and discovering potential disease biomarkers.3 nine by AHA may also induce changes in protein expression.8 The rapid development of high-resolution mass spectromeTo obtain real profiles of cell secretion, a more direct aptry (MS) and shotgun strategies for proteome analysis enables proach involves a cell secretome analysis from the primary comprehensive analyses of proteins from given biological culture supernatant. Some efforts including protein and/or samples.4 However, the proteomic profiling of cellular secrepeptide fractionation were applied to detect the low-abundance tomes from serum-containing conditioned media (CMs) resecreted proteins.9 Due to the wide dynamic range in the mains extremely challenging due to the low abundance of the -1 abundance of the serum proteins (12 orders of magnitude),10 secreted proteins (as low as ng mL ) relative to the complex -1 however, direct fractionation strategies were not effective. background of highly abundant serum proteins (~6 mg mL ). Consequently, decreasing the complexity of serum proteins Alternatively, serum-free medium is often used over a defined before fractionation is critical.11 Recently, a new protein period during which secreted proteins are continuously accu'equalization' technique (ProteoMiner) has emerged that could mulated without serum interference, greatly reducing the proreduce the dynamic range of protein concentrations by using a teome complexity and facilitating identification. However, combinatorial library of random hexapeptide ligands (206) to depriving serum could disturb cell metabolism and prolifera5 capture proteins under capacity-restrained rules. This method tion. These disturbances may affect protein expression and demonstrated great advantages in enriching low-abundance secretion profiles and may even induce cell death, leading to proteins while removing high-abundance proteins12 and could the experimental biases during qualitative and quantitative be used in quantitative proteomic experiments.13 secretome analyses. Therefore, the cells should be grown in a serum-containing To circumvent the above-mentioned problem, Jeroen culture medium to acquire the informative data reflecting the Krijgsveld and co-workers described a novel method combinreal status of cells, and strategies for sensitive, comprehensive ing the metabolic labelling of azidohomoalanine (AHA), ACS Paragon Plus Environment

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and unbiased secretome analyses would be highly desirable. Herein, we combined metabolic labelling, protein 'equalization', protein fractionation and filter-aided sample preparation (FASP), called MLEFF strategy, towards the secretomic analysis of serum-containing CMs, and it showed an excellent effect in HeLa secretome profiling. In addition, this strategy was further applied to in-depth and differential secretome analyses of two human metastatic HCCs, improving our understanding of tumour invasion and metastasis. EXPERIMENTAL SECTION Cell culture, metabolic labelling, 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 humidified atmosphere of 5% CO2 at 37ºC in DMEM media (Thermo) supplemented with 10% (v/v) FBS (Gibco) and 1% penicillin/streptomycin (Thermo), respectively. For the ‘medium’ labelling media (Lys-4, Arg6), L-lysine- and L-arginine-depleted SILAC DMEM media (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 1% penicillin/streptomycin mixture. For the ‘heavy’ labelling 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). The MHCC97L cells were grown in the ‘medium’ media, and the MHCC97H cells were grown in the ‘heavy’ media. In addition, HeLa cells were labelled with another ‘heavy’ labelling medium (Lys-6, Arg-10), which used [13C6] L-lysine and [13C6, 15 N4] L-arginine. The SILAC-labelled cells were grown to at least six doubling times to ensure the complete incorporation of the labelled amino acids. Passaging was performed when 80~90% confluency was reached. To prepare the CMs from the HeLa cells, the cells were incubated in complete labelling 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 500 × g and 3,000 × g for 15 min to remove cells and cell debris, respectively. After filtrating through a 0.22 µm filter unit (Millipore, MA), the supernatant was concentrated and desalted with water via Amicon 3 kDa filter 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 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 (2,000 cells/well) and incubated with the above-mentioned media for 1-7 days. After incubation, the cells were supplemented with 20 µL of MTT (5 mg mL-1,

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Sigma) for 4 h at 37 °C. Subsequently, the supernatant was gently removed, and 200 µL 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 modifications. Briefly, the storage solution from ProteoMiner columns containing 50 µL beads was removed by centrifugation (1,000 × g, 30 s), and the beads were washed with 600 µL of 25 mM HEPES (pH 7.5, Sigma) and then with 600 µL water three times. Next, 300 microliters of the concentrated CMs (~60 mg mL-1) 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 µL water five times. The bound proteins were incubated with 300 µL of boiled elution buffer (4% sodium dodecyl sulfate (SDS), 25 mM dithiothreitol (DTT, Sigma)) for 5 min and sequentially eluted with elution buffer and 200 µL 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 manufacturer’s protocol with minor modifications. Briefly, 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 µL of the sample buffer were separated using commercial 8% tris-acetate cartridges (Expedeon, CA), and 10 fractions (~150 µL each) were collected at specified 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 were 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 pre-treatment. FASP pre-treatment. The proteins (~50 µg) from the cells, raw CMs, the equalized CMs and the different 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 filter 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. The concentrates were diluted with 300 µL of 25 mM NH4HCO3 and 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 at 37 °C for 16 h. Subsequently, the digests were obtained through centrifugation and dried in a Speed Vac Concentrator (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 µm, 100 Å, Durashell, China) at a flow rate of 0.5 mL min-1. 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 one2

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minute from 5 min to 55 min. Then, fractions with equal collection time interval (5 min) were pooled. In this way, 5 pooled fractions were obtained pending further liquid chromatography coupled with tandem mass spectrometry (LCMS/MS) analysis. LC-MS/MS analysis. The peptides were analysed with a 1D nano-RPLC-MS/MS on a Q-Exactive MS (Thermo Fisher Scientific, CA) coupled with an Ultimate 3000 (Dionex, Germany) nano-LC system. The mobile phases were buffers A (2% acetonitrile, 98% water and 0.1% formic acid) and B (98% acetonitrile, 2% water and 0.1% formic acid). Fusedsilica capillaries (150 µm i.d. × 375 µm o.d.) were obtained from Sino Sumtech (Handan, China). A C18 trap column (150 µm i.d. × 5 cm) was connected to a homemade capillary separation column (75 µm i.d. × 15 cm). Both the trap and separation columns were packed with Daiso C18 particles (5 µm, 100 Å) (Osaka, Japan). To separate the peptides from the HeLa CMs, a short gradient (52 min) was established: 37 min of 6%-25% buffer B, and then 15 min of 25%-35% buffer B with a flow rate of 300 nL min-1. To quantify additional proteins from the MHCC97H and MHCC97L cells and CMs, an 110-min gradient was established, comprised of 90 min of 6%-22% buffer B, and then 20 min of 22%-35% buffer 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 ten 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 1,800 (automatic gain control (AGC) value: 1E6, maximum injection time: 100 ms), and the data were acquired in profile 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-second 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 p.p.m. for the parent ions and at 20 p.p.m. 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 6 amino acids. Carbamidomethylation (C) (+57.0215 Da) was used as the fixed modification. Oxidation (M) (+15.9949 Da) and acetylation (protein N-termini) (+42.0106 Da) were searched as variable modifications. For the peptides from the HeLa CMs, two SILAC-based labels (Lys6, +6.0201 Da) (Arg10, +10.0083 Da) were used as variable modifications. For the peptides from the MHCC97H and MHCC97L cells and CMs, the light 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 identifications were filtered by PD to keep the FDR ≤ 1%. At least one unique peptide was required for each protein identification. 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 non-classical secreted proteins were predicted using a server SecretomeP 2.016 (http://www.cbs.dtu.dk/services/SecretomeP/) with an NNscore > 0.5, but not at a time predicted to contain a signal peptide. The exosome proteins were matched by ExoCarta database17 (http://exocarta.org/). The biological process annotations and protein classifications were performed using PANTHER18 for GO analysis (http://pantherdb.org/).

RESULTS AND DISCUSSION Principle of the MLEFF strategy. An MLEFF strategy combining metabolic labelling, protein equalization, protein fractionation and FASP was firstly used to analyse the secreted proteins from HeLa CMs (Figure 1, qualitative). Stable isotope labelling by amino acids in cell culture (SILAC) has proven its accuracy for the differential study of proteins from cell cultures.19 In our work, this method was used to distinguish the true cellular proteins from foetal bovine serum (FBS) or contaminations. Thus, [13C6] L-lysine and [13C6, 15N4] L-arginine 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 lowmolecular-weight (Mw) region (5). The Y-axis indicates the percentage of the identified CoPros. (c) Number of the CoPros identified by LC-MS/MS in HeLa CMs. The Venn diagram shows the overlap of the two biological replicates with the total number of CoPros identified per replicate. (d) Composition of the secretomes. Among the 585 CoPros identified, 534 (98%) CoPros4

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can be identified within the secretome, including secreted protein, nonclassical secreted protein and extracellular exosome protein. The remaining proteins (10, 2%) remained unassigned.

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 the higher metastatic competency.26 Reports indicate that the tumour-derived secretome can facilitate tumour 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 effect on proliferation (Figure 3a). The results show a significant 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).

Figure 3. MTT proliferation assay of MHCC97L cells cultured in different CM. (a) Workflow of the MTT proliferation assay. The detailed procedures are described in the Methods section. (b) MTT assay results. The asterisks (*P < 0.05 and **P < 0.01) indicate a statistically significant 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 ± s.e.m..

Quantitative analysis of MHCC97L and MHCC97H secretomes. Based on above results, the differences 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 differentially metastatic potentials by restricting quantification 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. One thousand and three hundred CoPros can be detected (1,152 with quantitative information) in the presence of 10% (v/v) FBS (Table S-3), far exceeding the totals

from two previous reports (38628 or 61129 proteins), in serumfree CMs from MHCC97H/MHCC97L cells. Of these 1,300 CoPros, 1,011 CoPros could be classified as part of the secretome (classical, non-classical secreted protein and extracellular exosome protein), which includes various growth factors and cytokines involved in tumour proliferation and metastasis, such as Tgfb1, Tgfb2, Gpi, Bmp1, Bmp2, Ccl15, Ccl20, Cxcl15, Cxcl16 and Pf4. In this dataset, 861 CoPros could be reliably quantified 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 significantly 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, Ctss, Ctsv) involved in extracellular matrix disassembly were quantified with high expression, and these proteins were believed to have participated in basement membrane degradation and been implicated in tumour invasion and metastasis.30 While other biological processes, such as regulation of ligase activity, cellular response to stress and homeostatic process were significantly down-regulated in the CMs from MHCC97H. We detected a series of proteasome subunits associated with regulation of ligase activity that exhibited different degrees of reduction: Psma1, Psma2, Psma3, Psma4, Psma5, Psma6, Psma7 and Psmb1, Psmb2, Psmb3, Psmb4, Psmb5, Psmb6, Psmb7, Psmb8. The informative and quantitative nature of these secretome data enable an in-depth biological function analysis that may aid biomarker discovery while helping to explain the mechanisms of tumour growth, proliferation and invasion. Proteins with ratios more than 2 or less than 0.5 (Log2Heavy/Medium > 1 or < -1) and t-test P-value less than 0.05 were considered to be significantly 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 tumour cells.33 Protein NDRG1 (Ndrg1), an important tumour 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 identified in our data play key roles in tumour 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 tumour invasion and metastasis.

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lar matrix proteins, peptide hormones, apolipoproteins, cytokines, growth factors and chemokines. In addition, 476 quantified proteins were overlapped in both the extracellular and intracellular proteomes (Figure 5c). Notably, these proteins show tiny differences at the intracellular level, remaining consistent with our earlier result,36 implying that these intracellular proteins may have limited roles when inducing different cell behaviours. This work presents a comprehensive analysis of the extracellular and intracellular proteins under normal culture conditions without serum starvation. The unsupervised clustering of these quantified proteins shows a more significant change in the extracellular proteins compared than 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 different secretion profiles of these two human metastatic HCCs may contribute significantly 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 different cell behaviours.

Figure 4. Biological analysis of quantified 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 quantified CoPros from triplicate technical replicates. The P-value was determined using a two-sided Student's t-test. Significant regulation of the CoPros was defined 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.

Quantitative comparison between extracellular and intracellular proteomes. Given the significant differences 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 differentially metastatic potentials. In previous studies of metastatic tumour 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 SILAClabelled HCCs were also subjected to quantitative proteomic analyses (Fig 5a, Table S-5). A protein classification analysis revealed a significant difference in the distribution of functionalities between the extracellular and intracellular proteins (Figure 5b). The extracellular proteins dominated in many functionalities, including cell adhesion molecular, extracellu-

Figure 5. Comparison between the extracellular and intracellular proteomes. (a) Schematic illustration of the experimental setup and proteomics workflow. (b) Classification of the proteins within the extracellular (1,300 proteins, blue) and intracellular (3,119 proteins, light blue) proteomes based on PANTHER protein classification. (c) Unsupervised hierarchical clustering of 476 proteins reproducibly quantified in MHCC97H and MHCC97L cell lines from the extracellular and intracellular proteomes.

CONCLUSION In a typical cell culture (1×107 cells, culture for 24 h) experiment, 20~80 µg secreted proteins could be released into the extracellular medium across different cell types.37 Unfortunately, these secreted proteins can be masked by the complex serum protein background (~60 mg). We developed an efficient strategy called MLEFF to overcome this problem. The6

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first step is to distinguish the cellular proteins from the bovine serum background. Only the peptides with SILAC labels are passed for identification. To reduce the dynamic range of the proteins in the CM sample, a protein equalization (ProteoMiner) technique was adopted, enabling the identification of 8.9-fold more proteins than that of the untreated CMs. The equalized samples are compatible with and effective for a subsequent protein fractionation according to molecular weights by 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 quantification because the CMs with different SILAC labels were mixed before any pre-treatment. Many regulated proteins were found closely related to tumour growth, proliferation, invasion and metastasis, as well as worthy of further study during the quantification of HCCs CMs. In addition, we compared the proteomic composition and expression at both the extracellular and intracellular levels, and found more significant differences at the extracellular level. The qualitative and quantitative results reflected the real status of the cell secretions because the culture condition were normal and without any specific stimulus. Because most previous secretome studies are based on serumfree media systems, we expect that our approach will facilitate the study of cell secretomes, particularly for cells that are highly sensitive towards serum starvation.

ASSOCIATED CONTENT Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT 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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