Proteomics Analysis of Nasopharyngeal Carcinoma Cell Secretome Using a Hollow Fiber Culture System and Mass Spectrometry Hsin-Yi Wu,†,§ Ying-Hwa Chang,†,§ Yu-Chen Chang,† and Pao-Chi Liao*,†,‡ Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan, and Sustainable Environment Research Center, National Cheng Kung University, Tainan, Taiwan Received August 26, 2008
Secreted proteins, referred to as the secretome, are known to regulate a variety of biological functions and are involved in a multitude of pathological processes. However, some secreted proteins from cell cultures are difficult to detect because of their intrinsic low abundance. They are frequently masked by proteins shed from lysed cells and the substantial amounts of serum proteins used in culture medium. We have proposed an analytical platform for sensitive detection of secreted proteins by utilizing a hollow fiber culture (HFC) system coupled with proteomic approaches. The HFC system enables culture of high-density cells in a small volume where secreted proteins can be accumulated. In addition, cell lysis rates can be greatly reduced, which alleviates the contamination from lysed cells. In this study, nasopharyngeal carcinoma (NPC) cells were utilized to evaluate the efficiency of this system in the collection and analysis of the cell secretome. Cells were adapted to serum-free medium and inoculated into the HFC system. The cell lysis rate in the culture system was estimated to be 0.001-0.022%, as determined by probing four intracellular proteins in the conditioned medium (CM), while a cell lysis rate of 0.32-1.84% was observed in dish cultures. Proteins in the CM were analyzed using SDS-PAGE and liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 134 proteins were identified in 62 gel bands, of which 61% possess a signal peptide and/or a transmembrane domain. In addition, 37% of the identified secretome were classified as extracellular or membrane proteins, whereas 98% of the lysate proteins were identified as intracellular proteins. We suggest that the HFC system may be used to collect secreted proteins efficiently and facilitate comprehensive characterization of cell secretome. Keywords: hollow fiber culture system • cell secretome • nasopharyngeal carcinoma cell • mass spectrometry
Introduction Proteins that cells secrete into the extracellular environment (the secretome) are known to regulate and coordinate many vital biological processes. These proteins are not only components of the extracellular matrix and blood, but are also involved in signal transduction, blood coagulation, immune defense, and carcinogenesis.1 Chronic changes or abnormal secretion of these proteins could be an indication of pathological conditions, and their study could lead to the discovery of suitable targets for therapeutic and biomarker discovery. Particular proteins secreted from cancer cells that enter the circulatory system can be utilized as targets for monitoring or * To whom correspondence should be addressed. Prof. Pao-Chi Liao, Department of Environmental and Occupational Health, National Cheng Kung University College of Medicine, 138 Sheng-Li Road, Tainan 70428, Taiwan. Tel, 886-6-2353535 ext 5566; fax, 886-6-2743748; e-mail, liaopc@ mail.ncku.edu.tw. § These authors contributed equally to this work. † Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University. ‡ Sustainable Environment Research Center, National Cheng Kung University.
380 Journal of Proteome Research 2009, 8, 380–389 Published on Web 11/14/2008
screening for the presence of cancer cells.2,3 However, the complex nature of blood has hampered the identification of biomarkers. Thus, in recent years there has been an elevated interest in studying secreted proteins from cancer cells or tumor tissues to discover new biomarkers by collecting conditioned media (CM).4-12 Compared to proteins that are localized in intracellular compartments, secretome proteins are less often isolated and identified because of their inherent low cellular abundance. One way to study them is to use an appropriate cell culture system that can accommodate large numbers of cells so that analytical sensitivity can be increased. To obtain secreted proteins from large amounts of cells, Sardana et al. utilized a roller bottle flask for cell culture.11 The roller bottle flask offers a large surface area (850 cm2) for cell growth, and in the Sardana et al. study, 66 extracellular candidate tumor markers were identified. Secreted proteins in a roller bottle flask are retained in 400 mL of culture medium that then requires multiple-step concentration (dialysis and lyophilization) or fractionation (e.g., SAX fractionation) before proteomic analysis.13 The hollow fiber culture (HFC) system was developed to 10.1021/pr8006733 CCC: $40.75
2009 American Chemical Society
Cell Secretome Analysis Using an HFC System produce large amount of monoclonal antibodies for biological and clinical applications14-18 and is another device for highdensity cell culture. In this system, cultures can be maintained for a period of time that enables the accumulation of secreted proteins in a relatively small volume. Herein, we propose a novel approach for collecting the cell secretome by using a HFC system followed by liquid chromatography mass spectrometry analysis. The applicability of this strategy to collect and identify the secretome was demonstrated using nasopharyngeal carcinoma (NPC) cells. The limitations of this strategy are also discussed.
Materials and Methods NPC Cell Culture in Serum-Free Medium. The nasopharyngeal carcinoma (NPC-TW04) cell line was kindly provided by Dr. J.-S. Yu (Department of Cell and Molecular Biology, Chang Gung University, Tao-Yuan, Taiwan) and was grown in serum medium in 10 cm dishes and incubated in a humidified incubator at 37 °C under 5% CO2. Serum medium contained Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine serum, and 1% antibiotics. After three to five passages during culture in serum medium, cells were adapted gradually to serum-free media. Serum-free medium consists of RPMI 1640 medium, 10% SynQ (serum substitute without Zn, Cell Culture Service, Hamburg, Germany), and 1% antibiotics. During the adaptation, part of the culture medium was substituted with serum-free medium. The percentage of serum medium in the culture medium was gradually reduced from 80%, 50%, 20%, 10%, and 5% (v/v), until the medium was finally totally replaced by serum-free medium. Cell morphology was examined by looking at 100× micrographs (Nikon ECLIPDE TS100). Cells cultured without serum were subcultured three to five times to ensure cell stability. After adaptation, cells were trypsinized and transferred to a hollow fiber culture system. Hollow Fiber Culture (HFC) System Inoculation with NPC Cells. Prior to cell inoculation, it was necessary to equilibrate/preculture the HFC system (C2008, Fibercell system, MD). The HFC system was first circulated with 20 mL of sterile phosphate-buffered saline (PBS) for 24 h, followed by 20 mL of RPMI 1640 medium for another 24 h. Also, before cell inoculation, the HFC system was circulated with 20 mL of serum-free medium for 24 h. NPC cells (∼5 × 107) were suspended in serum-free medium. Cells were inoculated into the extra-capillary space (ECS) of the hollow fiber cartridge using two sterile syringes. The cell-containing syringe was attached to one of the side-ports on the hollow fiber cartridge, while another empty syringe was attached to the other. At the same time, the left and right end-ports were closed. The cell suspension was flushed gently back and forth through the ECS three to five times to ensure a homogeneous distribution of cells. Excess medium flowed into the fibers, while about 15 mL of suspended cells remained trapped in the ECS. To allow cells to attach to the fibers, the hollow fiber cartridge was left undisturbed at 37 °C under 5% CO2 for 30 min. The cartridge was rotated by 180° and incubated for an additional 30 min to allow the remaining suspended cells to attach to the other side of the fibers. A total of 250 mL of serum-free medium was contained in a medium reservoir bottle and circulated at 20 mL/min. The whole HFC system was incubated at 37 °C under 5% CO2 for 5 days. Aliquots of 100 µL of media were aspirated from the reservoir every day for glucose and lactate measurement. The entire CM in the ECS (about 15 mL) containing secreted proteins was aspirated every day, and protein con-
research articles centrations were measured by Bradford assay. The ECS was refilled by an equal volume of fresh serum-free medium. Medium Glucose and Lactate Monitoring. Measurement of daily glucose and lactate concentrations in the reservoir was achieved by GLUC-PAP and LAC kits, respectively (Randox Laboratories, Co. Antrim, U.K.). Serial dilutions of standard glucose were made to obtain solutions of 0, 12.5, 25, 50, and 100 µg/mL in H2O. To 20 mL of standard solutions and sampled media, 200 µL of GLUC-PAP reagents was added and incubated in the dark for 25 min. Their absorbances were measured in triplicate at 500 nm, and a standard calibration curve was established according to the absorbance of the standard solutions. The standard calibration curve for lactate was obtained by preparing standard lactate solutions at 0, 2.5, 5, 10, 20, and 40 µg/mL in H2O. After adding 200 µL of enzyme reagents, the standard solutions and medium samples were incubated in the dark for 10 min. Their respective absorbances were measured in triplicate at 550 nm. The concentrations of glucose and lactate in the medium samples were determined by their absorbances plotted against the external standard calibration curves. The daily quantities of glucose and lactate in the HFC system were derived by multiplying the averages of triplicate samples by the volume of medium in the system. Preparation of Cell Lysate Sample, Conditioned Medium from HFC System, and Dish Medium. The secretome sample was prepared by collecting CM from the ECS of the HFC system. For the comparison of cell lysis rates between the HFC system and dish cultures, 2 × 106 adapted cells were seeded in 10 cm dishes. Ten milliliters of daily dish media was collected, and equal volumes of fresh serum-free media were added. To separate the secreted proteins from the cells, 15 mL of HFC CM and 10 mL of dish media were centrifuged at 1000 rpm for 5 min. Then, the supernatants were centrifuged at 8500g for 2 h to reduce cell debris. The supernatants were concentrated to 1 mL using 5 kDa Amicon Ultra-15 centrifugal filter devices (Millipore, MA). Aliquots of 10 µL concentrated CM were reserved for measuring protein concentration using the Bradford assay (Bio-Rad, CA). CM collected from the first 2 days were combined for further analysis. Cell lysate samples were obtained by disrupting NPC cells. Cells were harvested from dishes with a trypsin-EDTA solution containing 0.25% trypsin and 1 mM EDTA. The dispersed cells were placed into culture tubes and centrifuged at 3000 rpm for 5 min at room temperature. The pellets were washed three times with PBS, resuspended in 1 mL of water, sonicated three times at 20 W for 10 s, and centrifuged at 8500g for 1 h. The supernatant (cell lysate) was collected, and a small aliquot was extracted for the protein assay. Both the HFC CM and cell lysate samples were resolved by SDS-PAGE. SDS-PAGE was performed on an 18 × 18 cm, 8-16% gradient gel with a voltage provided at 100 V for 15 min and 300 V for an additional 4 h. Separated protein bands were visualized using silver staining. MS Analysis of Secreted and Cell Lysate Proteins. Excised gel pieces were washed twice with 50% (v/v) acetonitrile (ACN) in water and 50% (v/v) ACN in 25 mM ammonium bicarbonate. The gel pieces were incubated in 10 mM DTT at 65 °C for 45 min to reduce the proteins prior to being alkylated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate at room temperature for 1 h. To digest proteins, 10 µL of 0.1 mg/mL trypsin digestion buffer (Promega, Madison, WI) in 25 mM ammonium bicarbonate was added to the gel pieces and incubated at 37 °C overnight. At the end of a 16 h overnight incubation, the supernatants were transferred to siliconized 0.5 Journal of Proteome Research • Vol. 8, No. 1, 2009 381
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Figure 1. Scheme diagram of hollow fiber culture (HFC) system and LC-MS/MS analysis. (A) An HFC system consists of a medium reservoir, an oxygen generator, a pump, and a hollow fiber cartridge. (B) The detailed parts of the hollow fiber cartridge. Hollow fibers were sealed in a cartridge and circulated medium was pumped into the cavity of the fiber. Cells were inoculated into the cartridge through the side-ports. (C) Cells were cultured in the extracapillary space (ECS). (D) The conditioned medium collected from ECS was resolved by SDS-PAGE and subjected to LC-MS/MS analysis.
mL Eppendorf tubes. The remaining peptides in the gel pieces were further extracted by incubation with 20 µL of 50% (v/v) ACN containing 5% (v/v) formic acid for 20 min. The extracted peptides were combined with those in the Eppendorf tubes. Nano-HPLC-MS/MS analysis was performed to identify tryptic peptides. At a flow rate of 0.2 µL/min with a nano-HPLC system (LC Packings, Netherlands), the protein digests were fractionated on a C18 microcapillary column (75 µm i.d. × 15 cm) coupled with an ion trap mass spectrometer (LCQ DECA XP Plus, ThermoFinnigan, San Jose, CA). The mobile phases were 5% (v/v) ACN containing 0.1% (v/v) formic acid (buffer A) and 80% (v/v) ACN containing 0.1% (v/v) formic acid (buffer B). Chromatographic separation of peptides was performed using a 40-min linear gradient from 0 to 60% of buffer B. The eluent were introduced into the mass spectrometer through an electrospray source with the application of a distal 1.6∼1.7 kV spraying voltage. Each cycle of one full scan mass spectrum (m/z 450-2000) was followed by three data dependent MS/ MS spectra. Data Analysis, Database Searching, and Protein Classification. Xcalibur binary (RAW) files were converted into peak list (DTA) files via Bioworks Browser 3.1 (Thermo Finnigan, San Jose, CA). The parameters for DTA creation included a precursor mass tolerence of 1.4 Da, a maximum number of intermediate MS/MS scans of 25 between spectra that had the same precursor masses, a minimum peak value of 12 per MS/MS spectrum, one minimum scan per group, and automatic precursor charge selection. The generated DTA files were concatenated using merge.pl (a Perl script) provided on the 382
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Matrix Science Web site. The resulting peak lists were searched against the Swiss-Prot database using the Mascot search engine (http://www.matrixscience.com, Matrix Science Ltd., U.K.). The search parameters were set as follows: peptide mass tolerance ) 1 Da; MS/MS ion mass tolerance ) 1 Da; enzyme set as trypsin and allowance was set of up to two missed cleavages; variable modifications considered included asparagines and glutamine deamidation, methionine oxidation, and cysteine carboxyamidomethylation; peptide charges were limited to 2+ and 3+ and only human taxonomy was considered. Proteins identified with p-values e0.05 and Mascot scores >35 were considered as promising hits. Subcellular locations of identified proteins were determined based on searches made in the Gene Ontology database. Information of proteins was referred to Human Protein Reference Database, Swiss-Prot database, and published literature. The presence of a signal peptide and/or a transmembrane domain were predicted by SIG-Pred (Signal Peptide Prediction) and TMpred (Prediction of Transmembrane Regions and Orientation), which can be accessed at www. bioinformatics.leeds.ac.uk/prot_analysis/Signal.html and www. ch.embnet.org/software/TMPRED_form.html, respectively. Western Blot. Proteins were separated on gradient gels (1.0 mm × 10 well 4% to 12% NuPAGE Bis-Tris, Invitrogen, Carlsbad, CA) and transferred onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk solutions and Tris-buffered saline with Tween 20 (TBST) (3.0 g/L Tris, 14.4 g/L glycine, 0.5% Tween 20, pH 8.3) at room temperature for 1 h. After washing three times with TBST, the membranes were probed separately with relevant antibodies at 4 °C overnight.
Cell Secretome Analysis Using an HFC System Primary antibodies and concentrations were as follows: antiG3PDH (1:2500, Chemicon), anti-Hsp60 (1:2500, Stressgen Biotechnologies, Victoria, Canada), anti-tubulin (1:5000, Laboratory Vision Corp., Fremont, CA), anti-actin (1:500, Chemicon), anti-integrin beta 4 (1:500, Abcam, U.K.), anti-legumain (1:500, R&D, Minneapolis, MN), anti-laminin subunit alpha-5 (1:500, Abnova, Taiwan), anti-glypican-1 (1:500, Abnova, Taiwan), antilysosomal protective protein (1:500, Abcam, U.K.), and antithrombospondin-1 (1:500, Laboratory Vision Corp., Fremont, CA). After washing again with TBST three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:5000 at room temperature for 1 h. The membranes were washed with TBST three times for a third time, and they were then developed using enhanced chemiluminescence detection.
Results and Discussion A Novel Approach for Cell Secretome Analysis Using a Hollow Fiber Culture (HFC) System. Our strategy takes advantage of a commercially available HFC system to collect and accumulate secreted proteins. The analytical scheme for protein secretion analysis is shown in Figure 1. The HFC system consists of four components: a hollow fiber cartridge, a medium reservoir, a perfusion pump, and an oxygenation system (Figure 1A). Cells are maintained in the cartridge. By means of a perfusion pump, culture medium flows from the reservoir bottle through the oxygenator, where the state of O2 and CO2 in the medium is equilibrated with the atmosphere of the incubator. Medium streams into the fibers (intralumen space, ILS) sealed in the cartridge and recirculates continuously. Figure 1B displays the enlarged form of the hollow fiber cartridge, showing the detailed configuration. A total of 2800 hollow fibers (small capillary filters) are embedded in the cartridge shell. Two ends of the cartridge are closed by polyurethane caps with fiber outlets on them. As a result, while medium is pumped through the cap of the cartridge, it can only pass through the ILS. Cells are inoculated into the culturing cartridge through the side-ports and grow on the outside of the fiber ECS. These fibers provide up to 2100 cm2 of surface area for cell attachment where accommodation of 108∼109 cells is allowed. Such great cell numbers approach tissue-like cell densities.18 Figure 1C illustrates the enlarged cross section of the hollow fiber cartridge as exemplified with three fibers. The hollow fibers with small molecular weight cutoffs of 5 kDa allow for the retention of cells and secreted proteins within the ECS of sizes larger than 5 kDa. In addition, the cutoff filter permits the nutrients in the medium to penetrate into the ECS. On the other hand, cell waste pass through the filter into the ILS and are taken away by medium circulation. Small molecules such as lactate and glucose can easily cross the fiber membrane, thus, allowing the monitoring of cell growth. After inoculation, CM is aspirated for further secretome analysis. For the assessment of the cell death rate in the HFC system, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (Hsp60), tubulin, and actin levels of intracellular and extracellular samples were estimated and functioned as nonsecreted protein markers. Secreted proteins collected from the HFC system were concentrated and separated by SDS-PAGE followed by gel staining. After tryptic digestion of each excised gel bands, the individual peptide mixtures were subjected to LC-MS/MS analysis for the characterization of the secreted proteins (Figure 1D). After database searching, a secreted protein list was generated. Bioinformatics
research articles tools were then utilized to investigate protein functions and subcellular locations. Minimization of Serum Contamination in Conditioned Medium. Cells are normally cultured in serum-supplemented media, which hampers the isolation of secreted proteins from serum proteins. To reduce serum contamination, Zwickl and co-workers incorporated metabolic labeling to discriminate the secretome from residual serum proteins.19 Another commonly used strategy is to use traditional serum-containing media during the first experimental stage and then minimize interference by washing with phosphate buffered saline or serum-free medium several times before incubating cells in the serumfree medium for a period of time prior to collection of accumulate secreted proteins.7-12,20 In several works, only serum-free media was used in the study of secreted proteins.21-23 Sudden changes from serum-containing medium to serum-free medium frequently lead to cell autolysis. Genuine secreted proteins can be concealed and difficult to distinguish from the myriad of proteins in cellular debris. In this study, we adapted nasopharyngeal carcinoma (NPC) cells in serum-free medium by gradually decreasing the proportion of serum supplement. During the adaptation processes, the morphology of the cells at each stage was monitored to avoid undesired cell lysis or morphology changes. Micrographs (100×) were taken when the components of the medium were modified. The morphologies of cells before and after adaptation to serum-free medium are shown in Figure 2A. Neither apparent morphology change nor unwanted cell bursting was observed. Cell numbers were utilized to evaluate whether the cell growth was affected by serum-free culturing. After the third passage, 106 of the original NPC cells and adapted cells were cultured individually for 16 h followed by cell number counting. The average cell numbers for serum cultures and serum-free cultures were 1.86 × 106 and 1.80 × 106, respectively. The differences in cell numbers between cells cultured with and without serum were tested by a two-tail Wilcoxon Signed-rank test using triplicate samples. No significant differences were shown in cell growth rates after culturing in serum-free medium (p ) 0.593 > 0.05). Monitoring of Cell Growth and Protein Secretion in the HFC System. Once NPC cells were accustomed to serum-free medium, they were then cultured in a HFC system prior to the collection of NPC cell secretome. In dish culture, NPC cell division took place every 24 h. As for HFC system, growth of cells was monitored by measuring the daily lactate production and glucose consumption. Initially, 5 × 107 adapted cells were inoculated into the system. As depicted in Figure 2B, glucose consumption on the second day was about 300 mg, twice the amount of the first day. Thus, it was speculated that 108 cells can be reached after the first day. After culturing for 2 days, approximate 2 × 108 cells were obtained. Figure 2B also illustrates that cell numbers reach saturation after the second day. This revealed that about 2 × 108 NPC cells can be maintained in this device. Since we could not at first predict the time point of cell bursting when cell growth came to saturation, all proteins in the ECS were removed every day to prevent the CM sample from being contaminated by abundant intracellular proteins if lysis occurred. Daily CM protein concentrations were measured as shown in Figure 2C, revealing that daily protein concentrations increased and finally reached 200 µg per day on the fifth day. Previous literature has pointed out that protein secretion of tumor cells was observed during the tumor growth phase.24 As a result, the CM sample (pooled Journal of Proteome Research • Vol. 8, No. 1, 2009 383
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Figure 2. Adaptation of NPC cells to serum-free medium and monitoring of cell growth in HFC system. (A) Micrographs (100×) of cell morphology. (B) Daily glucose consumption and lactate concentration in HFC system. (C) Daily protein concentration in the ECS throughout the culture period.
from the first 2 days) was utilized to investigate the secretome of aggressive growing NPC cells. Evaluation of Cell Lysis Rate in HFC System. The secretome is inherently of much lower protein abundance than proteins shed from cell bursting. Even a very small percentage of cell lysis will liberate an amount of protein that far surpasses that of truly secreted proteins. Therefore, it is critical to confirm that proteins collected from the HFC system were mainly from secretion rather than cell lysis. To estimate cell viability, two commonly used laboratory tests, the MTT assay and trypan blue staining, were performed. While the MTT assay was attempted, DMSO (which was used to dissolve the formazan) dissolved fibers and could not be drawn out thoroughly. As for trypan blue staining, we were not able to completely gather all cells; thus, the accurate estimation of dead cell numbers was hindered. Therefore, in this study, G3PDH, tubulin, Hsp60, and actin were used to deduce the cell lysis rate in the HFC system. The existence of those proteins in the CM was an indication of cell leakage.25 To compare the percentage of cell lysis between HFC and dish cultures, the lysed cell number in the dish medium was also assessed. As illustrated in Figure 3A-D, the quantities of intracellular protein of the CM in the HFC and from the dish were examined by Western blotting. The HFC CM was combined from the first 2 days. The standard curve was plotted based on the protein level of cell numbers 384
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Wu et al. versus their corresponding band quantities. The number of lysed cells in the HFC system and dish cultures were then calculated from their band quantities and were plotted against the external standard calibration curve. Using G3PDH as an example, aliquots containing 15 µg of HFC CM proteins and dish CM were loaded (Figure 3A). G3PDH staining revealed about 840 and 6300 lysed cells in the HFC CM and dish CM samples, respectively. HFC CM pooled from the first 2 days contained 250 µg of proteins and dish CM contained 230 µg. Since the average cell number of HFC during these 2 days was 108, it was estimated that 1.4 × 104 cells were lysed. As a result, the cell death rate was estimated to be 0.014% during the first 2 days in the HFC system. As for dish cultures, the average cell number was 107, indicating that 93 000 cells may have lysed and that the lysis rate was 0.97% (nearly 70 times the lysis rate in the HFC system). To provide further confirmation of the cell lysis rate in the HFC system, other cytosolic proteins were examined in a similar manner. As depicted in Figure 3B-D, Western blots revealed that tublin, Hsp60, and actin levels of HFC CM samples indicated that 140, 1800, and 84 cells were lysed, respectively. This suggested the HFC cell lysis rates were 0.002%, 0.022%, and 0.001%, respectively. The tublin, Hsp60, actin levels of dish CM samples resulted in cell lysis numbers of 1400, 16 000, and 5900 cells, corresponding to lysis rates of 0.32%, 1.84%, and 0.68%, respectively. The evaluation of the cell lysis rate of the HFC system and dish culture using four intracellular proteins is summarized in Figure 3E. The cell lysis rate in the HFC system was determined to be 0.001-0.022%, while 0.32-1.84% was the rate in dish culture. This highlights the fact that cell bursting in the HFC system was far decreased when comparing to dish culture. Mbeunkui et al. has controlled cell confluence to reserve enough space for cell growth while simultaneously diminishing the number of cells suspending in the medium.9 On the basis of the study of Mbeunkui et al., one possible explanation of the reduction of cell lysis in the HFC system may arise from the greater surface area offered for cell growth and the dynamic removal of cell waste. The reduction of the contamination of intracellular proteins from the growth medium might allow a much more sensitive identification of secretome. NPC Cell Secretome Characterization. Volume reduction of the CM collected from the HFC system is achieved by ultrafiltration with a 5 kDa molecular weight cutoff centrifuge filter tube. Equal amounts (3 µg) of concentrated CM and cell lysate were resolved on an 8-16% SDS-PAGE gel followed by silver staining (Figure 4). Theoretically, if the CM was contaminated by released intracellular proteins, the protein pattern of HFC CM would be quite similar to that of cell lysate. According to Figure 4, two patterns were remarkably distinctive. To characterize the proteins contained in HFC CM, a total of 62 gel bands detected on SDS-PAGE from CM protein samples were excised. After tryptic digestion, individual peptide mixtures were subjected to LC-MS/MS analysis to characterize the secreted proteins. On the basis of the searching of LC-MS/MS data against Swiss-Prot database, 134 proteins were found to be significant hits. Consequently, a list of the NPC cell secretome was generated (Supporting Table S-1 in Supporting Information). To provide further information, those characterized proteins were classified by their subcellular locations and protein functions according to information in the Gene Ontology database, the Human Protein Reference Database, and the Swiss-Prot database. As displayed in Figure 5A, 27proteins were categorized as extracellular protein (20%) and 23 proteins were
Cell Secretome Analysis Using an HFC System
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Figure 3. Estimation of cell lysis rate in HFC system and dish culture. HFC conditioned medium, dish conditioned medium, and cell lysate samples probed with (A) anti-G3PDH, (B) anti-tubulin, (C) anti-Hsp60, and (D) anti-actin antibodies. Protein levels were evaluated using a calibration curve plotted from cell lysate samples. (E) The bar chart illustrated the evaluated cell lysis rates of HFC system and dish culture conditioned medium using the four intracellular proteins.
membrane-bound (17%). Seventy identified proteins were classified as intracellular (52%), whereas 14 proteins were unclassified (10%). Among the NPC cell secretome, 50 proteins (37%) consisting of 27 extracellular proteins and 23 membrane proteins attracted particular interests because they had the potential to have been secreted proteins that would normally go into circulation and become cancer markers or clues to decipher cancer progression. In the studied secretome of this report, the overall percentages of proteins that were extracellular or membrane-bound were 15% and 22-27% for human adipose-derived stem cells and breast cancer cell secretome, respectively.12,26 In addition, the 134 identified proteins could be allotted to a few functional groups including metabolism and energy pathways (24%), protein metabolism (18%), cell growth and/or maintenance (16%), and signal transduction and cellular communication (14%) (Figure 5B). To qualitatively describe the major proteins expressed in NPC cell lysates, 71 of the most intense gel bands from the cell lysate samples were excised and subjected to tryptic digestion and LC-MS/MS analysis. By adapting the criteria used in secretome identification, 216 proteins were considered successful hits. The major identified cell lysate proteins are listed in the Supporting Table S-2 in Supporting Information. The subcellular locations of the lysate proteins were analyzed and elaborated in Figure 5A. Compared to the cellular location classification of proteins identified in the HFC CM, of the characterized 216 major lysate proteins, only 2% were not assigned in the intracellular milieu and only 6% belonged to the membrane. This distribution demonstrates that secreted proteins were concentrated in the HFC system. Limitations of Collecting Secretome by HFC System and Serum-Free Culture. Because MTT assay and trypan blue staining were inapplicable for use with the HFC system, Western blot analysis of released intracellular proteins in the
CM was used to measure the cell death rates. Although there were small amounts of intracellular proteins from lysed cell in the HFC system, cytosolic proteins were detected in the characterized NPC secretome. The existence of these intracellular proteins, even derived from a very small amount of cell lysis in routine cell culture, was identified by high-sensitivity mass spectrometry analysis. Supporting Table S-3 in Supporting Information offers the results of G3PDH, tubulin, Hsp60, and actin in the HFC CM and cell lysate samples. G3PDH and actin were identified both in the CM sample and cell lysate samples. However, they were identified with fewer peptides and lower Mascot scores in the CM than in the lysate. In addition, tubulin and Hsp60 were only identified in cell lysate. The above result corresponded with the lower cell lysis rate in a HFC system, allowing more sensitive detection of authentic secreted proteins. It also implies that reduction of cell lysis should be an important consideration when analyzing cell secretomem. There exists the possibility that fibers themselves may absorb some proteins. This may result in the underestimation of certain released intracellular proteins. To tackle this issue, more than one intracellular protein was inspected and all appeared to have lower abundances in HFC CM than in dish CM. Apart from the limitations listed above, adherence of cells to the ECM and other surfaces may dramatically influence cell differentiation and functionality. The kind of surface chosen in a model system does influence secretion activities. Adaptation of NPC cells to serum-free medium was a means to avoid the serum contamination and reduce unwanted cell bursting. However, the physiology of the adapted cells may change because of this procedure and the obtained secretome may lack those proteins induced by the factors provided by serum. Additionally, it is known that not all cells can endure long-term incubation in serum-free medium and not all cells grow attached. To expand the applicability of this system, future Journal of Proteome Research • Vol. 8, No. 1, 2009 385
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Wu et al. work should focus on the elimination of serum interference while still retaining the ability to inoculate cells with serumsupplemented medium and collect CM from the suspended cell culture. Further, primary cells may show dramatic differences when compared to cultured cells which have been adjusted to specific culture conditions.
Figure 4. SDS-PAGE (8-16% Bis-Tris gel) of secretome obtained from the ECS of the hollow fiber cartridge and cell lysate samples visualized using silver staining. Numbers indicated the bands excised for further LC-MS/MS analysis. 386
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An Example of Application of the Identified NPC Cell Secretome: Suggesting Biomarker Candidates. We have demonstrated the capability of the HFC system to be coupled with LC-MS/MS analysis for the collection and identification of the cell secretome using nasopharyngeal carcinoma (NPC) cells. Many studies have described how the characterization of proteins secreted from cancer cells is potentially applicable to the discovery of diagnostic biomarkers.5,8,10,11,27-32 Here, we intended to propose a procedure for the selection of possible candidates for cancer by utilizing the identified NPC cell secretome as an example. At the first step, probable interference from cell lysis was examined. Although our data inferred that only 0.001-0.022% of cell lysis occurred in the HFC system, release of proteins from lysed cells was unavoidable. We observed that 26 of the 216 intracellular proteins were found in the NPC cell secretome. Detailed information regarding these 26 overlapped proteins is listed in Supporting Table S-4 in Supporting Information. Referring to their subcellular locations, these 26 proteins appear at intracellular areas or membrane areas except for Annexin A2. To avoid possible contamination from major intracellular proteins, these 26 overlapped proteins were excluded from the list of 134 candidates. The remaining 108 proteins were subsequently analyzed for the presence of a signal peptide and/or a transmembrane domain using the SIGPred (Signal Peptide Prediction)33 and TMpred (Prediction of Transmembrane Regions and Orientation)34-38 software to corroborate their secretion. This analysis revealed that 52 of 108 proteins possessed both a signal peptide and a transmembrane domain, whereas 30 proteins contained only a transmembrane domain. These 82 proteins (76%), listed in Supporting Table S-5 in Supporting Information, were destined for secretion through a classical endoplasmic reticulum/Golgidependent pathway.39 The other 26 proteins, containing neither a signal peptide nor a transmembrane domain, were ruled out from the candidate list. Finally, a total of 82 proteins were used for mining of potential targets by screening whether their biological functions were associated with cancer (as exemplified in such properties as adhesion, motility, invasion, metastasis, proliferation, etc.). Moreover, NPC-related phenomena like highly distant metastasis and tumor infiltration of the lymph node were also accepted as properties associated with cancer. Proteins that have been previously identified in a variety of cancer specimens were not included because they were devoid of specificity. Finally, 10 proteins (agrin, laminin subunit alpha5, beta-mannosidase, N-acetylglucosamine-6-sulfatase, legumain, glypican-1, lysosomal protective protein, integrin beta4, thrombospondin-1, and CUB and sushi domain-containing protein 2) were sieved out as candidates (Table 1). Once the protein targets were focused, we verified their existence in the CM prior to further examination. Here, Western blotting was used to verify these 10 proteins. However, we were limited by the availability of commercial antibodies and only laminin subunit alpha-5, legumain, glypican-1, lysosomal protective protein, integrin beta-4, and thrombospondin-1 were analyzed (Figure 6). Our study reveals that 10 candidates are highly correlated with cancer progression. Agrin may play a role in the vascular
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Cell Secretome Analysis Using an HFC System
Figure 5. Subcellular locations and functional classification of the identified proteins. (A) Subcellular locations of the 134 identified NPC secretome and 216 identified cell lysate proteins. (B) Functional classification of the 134 identified proteins according to Gene Ontology database, Human Protein Reference Database, and Swiss-Prot database. Table 1. A Total of 10 Proteins Considered as NPC Biomarker Candidates Swiss-Prot band no. accession no.
protein name
no. of Mascot matched score peptides
S1
O00468
Agrin [Precursor]
160
12
S2
O15230
Laminin subunit alpha-5 [Precursor]
561
30
S7 S12
O00462 P15586
94 130
5 8
S20 S23 S23
Q99538 P35052 P10619
98 67 198
5 1 5
S35 S37 S42
P16144 P07996 Q7Z408
Beta-mannosidase [Precursor] N-acetylglucosamine-6-sulfatase [Precursor] Legumain [Precursor] Glypican-1 [Precursor] Lysosomal protective protein [Precursor] Integrin beta-4 [Precursor] Thrombospondin-1 [Precursor] CUB and sushi domaincontaining protein 2
46 77 38
3 3 2
a
subcellular location
signal transmembrane previously peptide domain reporteda
Secreted protein; extracellular space; extracellular matrix Secreted protein; extracellular space; extracellular matrix; basement membrane Lysosome Lysosome
Yes
Yes
Yes
Yes
Yes Yes
Yes Yes
Yes Yes
Lysosome extracellular space, membrane endoplasmic reticulum, lysosome membrane extracellular unclassified
Yes Yes Yes
Yes Yes Yes
Yes Yes
Yes Yes No
Yes Yes Yes
proteins that have been reported as secreted proteins of NPC cells in a previous work.8
and ductular proliferation characteristic of cirrhosis, and may promote tumor progression by supporting stromal cell growth and neoangiogenesis in the HCC.40 Laminin subunit alpha-5 is known to mediate the attachment, migration, and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components. Tumor
infiltration of the lymph nodes is a common syndrome in the early stage of NPC, resulting in metastasis of the lymph node. The level of laminin subunit alpha-5 was found to correlate with lymph node metastasis in nonsmall cell lung cancers,41 implying that the Laminin subunit alpha-5 may be a potential biomarker for NPC. β-Mannosidase is an exoglycosidase inJournal of Proteome Research • Vol. 8, No. 1, 2009 387
research articles
Wu et al.
Figure 6. Western blot analysis of the 6 selected NPC biomarker candidates.
volved in the degradation of N-linked oligosaccharide moieties of glycoproteins. Changes in glycosylation are known to occur early in tumor progression.42 A recent study has shown the differential level of β-mannosidase in human esophageal cancer, and demonstrates its significant role in cancer development and progression.43 It has previously been shown that the overexpression of N-acetylglucosamine-6-sulfatase, a sugarprocessing protein, represents an excess of N-acetylglucosamineinvolvedcarbohydratemetabolismduringcancerprogression.44,45 Legumain promotes cell migration and its overexpression is associated with enhanced tissue invasion and metastasis.46 Glypican-1 has been described to have differential expression between breast cancer and normal breast tissue, which suggests that glypican-1 may contribute to disease progression in malignancy.29,47 According to the study reported by Kozlowski et al., lysosomal protective proteins may play roles in malignant transformation and metastatic dissemination of malignant melanoma.48 Integrin beta-4 is a receptor for laminin and plays a critical role in the hemidesmosome of epithelial cells. It is also involved in cell communication, signal transduction, migration, and adhesion. A previous study has shown that the expression of the integrin beta-4 gene was suppressed in malignant NPC cells. Hence, this protein may be utilized as a screening biomarker for detecting early stage NPC.49 Thrombospondin-1 is an adhesive glycoprotein that mediates cellto-cell and cell-to-matrix interactions. It is also a multifunctional matricellular glycoprotein involved in several mechanisms critical to the formation and progression of solid tumors such as cell adhesion, proliferation, migration, invasion, and angiogenesis.50 CUB and sushi domain-containing protein 2 have been described in the database. These proteins are normally only detected in head and neck cancer cells. During the selection of potential candidates, proteins found in both the CM and the cell lysate, those proteins lacking a signal peptide or a transmembrane domain were excluded from the list. The exclusion of intracellular proteins from the list may risk losing authentic secreted proteins. For example, Annexin A2, identified both in CM and cell lysate, was excluded from the candidate list. However, its secretion has been reported to be relevant to breast cancer, osteousarcoma, lung cancer, and bladder cancer.5,9,51 In addition, proteins without a signal 388
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peptide and/or a transmembrane domain were unlikely to be liberated from lysed cells since they were not the major constituent proteins in the NPC cell lysate. Although many studies have also ruled out those nonsecreted proteins from the secretome, there is a possibility that these proteins were secreted proteins. It is hypothesized that proteins possessing no signal peptide and/or transmembrane domain may be secreted by means of a nonclassical secretory route whose mechanism has not been clearly ascertained.9,31,52 Recently, Tanudji et al. has reported a nonclassical secretory route using green florescent proteins.53 Additionally, neither interleukin1β nor galectins follow the classical secretory pathway. Interleukin-1β is exported through an ATP-binding cassette system54,55 and galectins are secreted via a vesicle-bedding mechanism.56,57 Thus, to avoid losing relevant findings, identified proteins without a signal peptide and/or a transmembrane domain should be subjected to further investigation. Since the absence of a signal peptide and/or a transmembrane domain cannot define a protein as a nonsecreted one, a more direct analysis of specimens containing secreted proteins would be required to reliably define the actual cell/tissue secretome, rather than only depend on their software prediction.39
Conclusion A strategy for comprehensive analysis of the secretome using a HFC system for collecting secreted proteins and protein identification by LC-MS/MS analysis was developed and demonstrated with the NPC cell line. A low cell lysis rate was observed in the HFC system, which allows this approach to identify specific proteins secreted from cancer cells and to identify novel biomarkers in the future. Although a significant amount of work is required to verify the candidate biomarkers, characterized secretomes would greatly increase our knowledge of secreted proteins and their roles in cancer initiation and progression.
Acknowledgment. This study was supported by grants NSC-96-2628-M006-005 from National Science Council, DOH97TD-G-111-019 from Department of Health, Executive Yuan, and National Cheng Kung University Project of Promoting
research articles
Cell Secretome Analysis Using an HFC System Academic Excellence & Developing World Class Research Centers from Ministry of Education of Taiwan.
Supporting Information Available: Supplementary Table S-1 shows the omplete list of 134 proteins identified from 62 gel bands of secrectome fraction and Supplementary Table S-2 shows the complete list of 216 proteins identified from 71 most abundant gel bands of cell lysate fraction. Supplementary Table S-3 presents the identification of G3PDH, tubulin, Hsp60, and actin in HFC CM and cell lysate samples; Supplementary Table S-4 lists 26 proteins identified both in cell lysate and secretome sample. Supplementary Table S-5 enumerates the 82 proteins with a signal peptide and/or a transmembrane domain. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ladunga, I. Curr. Opin. Biotechnol. 2000, 11, 13–18. (2) Liotta, L. A.; Ferrari, M.; Petricoin, E. Nature 2003, 425, 905. (3) Etzioni, R.; Urban, N.; Ramsey, S.; McIntosh, M.; Schwartz, S.; Reid, B.; Radich, J.; Anderson, G.; Hartwell, L. Nat. Rev. Cancer 2003, 3, 243–252. (4) Celis, J. E.; Gromov, P.; Cabezon, T.; Moreira, J. M.; Ambartsumian, N.; Sandelin, K.; Rank, F.; Gromova, I. Mol. Cell. Proteomics 2004, 3, 327–344. (5) Lin, C. Y.; Tsui, K. H.; Yu, C. C.; Yeh, C. W.; Chang, P. L.; Yung, B. Y. Proteomics 2006, 6, 4381–4389. (6) Alvarez-Llamas, G.; Szalowska, E.; de Vries, M. P.; Weening, D.; Landman, K.; Hoek, A.; Wolffenbuttel, B. H.; Roelofsen, H.; Vonk, R. J. Mol. Cell. Proteomics 2007, 6, 589–600. (7) Sato, K.; Sasaki, K.; Akiyama, Y.; Yamaguchi, K. Cancer Lett. 2001, 20, 153–159. (8) Wu, C. C.; Chien, K. Y.; Tsang, N. M.; Chang, K. P.; Hao, S. P.; Tsao, C. H.; Chang, Y. S.; Yu, J. S. Proteomics 2005, 5, 3173–3182. (9) Mbeunkui, F.; Fodstad, O.; Pannell, L. K. J. Proteome Res. 2006, 5, 899–906. (10) Mbeunkui, F.; Metge, B. J.; Shevde, L. A.; Pannell, L. K. J. Proteome Res. 2007, 6, 2993–3002. (11) Sardana, G.; Marshall, J.; Diamandis, E. P. Clin Chem. 2007, 53, 429–437. (12) Kulasingam, V.; Diamandis, E. P. Mol. Cell. Proteomics 2007, 25. (13) Chevallet, M.; Diemer, H.; Van Dorssealer, A.; Villiers, C.; Rabilloud, T. Proteomics 2007, 7, 1757–1770. (14) Lipman, N. S.; Jackson, L. R. Res. Immunol. 1998, 149, 571–576. (15) Yazaki, P. J.; Shively, L.; Clark, C.; Cheung, C. W.; Le, W.; Szpikowska, B.; Shively, J. E.; Raubitschek, A. A.; Wu, A. M. J. Immunol. Methods 2001, 253, 195–208. (16) Jacobin, M. J.; Santarelli, X.; Laroche-Traineau, J.; Clofent-Sanchez, G. Hum. Antibodies 2004, 13, 69–79. (17) Leong, M.; Babbitt, W.; Vyas, G. Biologicals 2007, 35, 227–233. (18) Gloeckner, H.; Lemke, H. D. Biotechnol. Prog. 2001, 17, 828–831. (19) Zwickl, H.; Traxler, E.; Staettner, S.; Parzefall, W.; Grasl-Kraupp, B.; Karner, J.; Schulte-Hermann, R.; Gerner, C. Electrophoresis 2005, 26, 2779–2285. (20) Pellitteri-Hahn, M. C.; Warren, M. C.; Didier, D. N.; Winkler, E. L.; Mirza, S. P.; Greene, A. S.; Olivier, M. J. Proteome Res. 2006, 5, 2861–2864. (21) Albertson, N.; Wenngren, I.; Sjo¨stro¨m, J. E. J. Clin. Microbiol. 1998, 36, 1232–1235. (22) Vanet, A.; Labigne, A. Infect. Immun. 1998, 66, 1023–1027. (23) Marcus, E. A.; Scott, D. R. Helicobacter 2001, 6, 93–99. (24) Ochsenbein, A. F. Cancer Gene Ther. 2002, 9, 1043–1055. (25) Ferguson, R. E.; Carroll, H. P.; Harris, A.; Maher, E. R.; Selby, P. J.; Banks, R. E. Proteomics 2005, 5, 566–571.
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