Secretory Protein Enrichment and Analysis: An Optimized Approach Applied on Cancer Cell Lines Using 2D LC-MS/MS Flaubert Mbeunkui,* Oystein Fodstad, and Lewis K. Pannell Cancer Research Institute, University of South Alabama, Mobile, Alabama 36688 Received November 3, 2005
Reliable methods for profiling secretory proteins are highly desirable for the identification of biomarkers of disease progression. Secreted proteins are often masked by high amounts of protein supplements in the culture medium. We have developed an efficient method for the enrichment and analysis of the secretome of different cancer cell lines, free of essential contaminants. The method is based on the optimization of cell incubation conditions in protein-free medium. Secreted proteins are concentrated and fractionated using a reversed-phase tC2 Sorbent, followed by peptide mass fingerprinting for protein identification. An average of 88 proteins were identified in each cancer cell line, of which more than 76% are known to be secreted, possess a signal peptide or a transmembrane domain. Given the importance of secreted proteins as a source for early detection and diagnosis of disease, this approach may help to discover novel candidate biomarkers with potential clinical significance. Keywords: reversed-phase Sorbent • secretory proteins • cancer cells • mass spectrometry • proteomics
1. Introduction During cancer progression, the microecology of the primary host tissue is always an active participant in the evolution of the tumor. Tumor invasion takes place at the tumor-host boundary, where tumor and stromal cells exchange enzymes and cytokines that alter the extracellular matrix and encourage cell migration.1 Analogous mechanisms are shared by physiological and tumorigenic invasion but the main difference between them is that physiological invasion is regulated whereas tumorigenic invasion is uncontrolled. The appearance of cancer-specific autocrine and paracrine signals often includes the supraphysiological expression of secretory proteins or their receptors.2 Proteins that are secreted from cells into the extracellular microenvironment represent the main class of molecules involved in intercellular communication, cell adhesion, motility and invasion. Investigation of overexpressed proteins in tumor angiogenesis and metastasis is usually focused on surface and integral membrane proteins.3-6 Efficient methods for the identification of proteins secreted by tumor cells are still not available. This is due to technical difficulties that have made a comprehensive analysis quite complicated and led to rather contradictory results. In such studies, cells are commonly cultivated in rich media complemented with serum proteins that are difficult to isolate from the secreted fraction. Protein-free media have been used to study the secreted proteins of the human pathogen Helicobacter pylori, a source of gastric ulcers and cancer.7-9 Cell growth is, however, much slower in such media and cells are prone to spontaneous autolysis resulting in the nonspecific * To whom correspondence should be addressed. Medical Sciences Building, Room 2015, Cancer Research Institute, University of South Alabama, 307 N. University Blvd, Mobile, AL 36688. Tel: (251) 414-8201. Fax: (251) 414-8281. E-mail:
[email protected]. 10.1021/pr050375p CCC: $33.50
2006 American Chemical Society
release of several proteins that make the interpretation of protein profiles difficult. Proteomic analysis of complex protein samples has traditionally involved the resolution of proteins with two-dimensional gel electrophoresis followed by protein identification by mass spectrometry.10,11 The main difficulty with this approach is the precipitation of the solubilized proteins at their isoelectric point. As a result, many researchers have returned to the onedimensional gels3,12 complemented with mass spectrometry for protein identification. This approach is limited by the complexity of protein analysis in each gel band. The shot-gun chromatography/mass spectrometry method13,14 is a good alternative method, in which proteins are first digested with proteases into a more complex peptide mixture which is then analyzed directly by LC-MS/MS. The success of this approach depends on the efficiency of the subcellular protein fraction enrichment; in which case the preparation of an extracellular protein fraction free of cytosolic proteins prior to proteomic analysis is essential to reduce the complexity of the sample and to simplify the analysis. Here, we report a new method for the profiling of secreted proteins, demonstrated with the secretomes of the malignant melanoma cell line WM 266-4, the osteosarcoma cell line OHS and the breast cancer cell line MA11. Culture conditions for minimal autolysis were established, and a concentration and fractionation method for optimal recovery of the extracellular proteins was adapted. The shot-gun chromatography/mass spectrometry approach was used to analyze proteins concentrated, and partially fractionated, with a reversed-phase tC2 Sorbent (Sep-Pak cartridge). This approach provides access to highly enriched secretory proteins for comparison across cell lines and diseases, and represents a significant improvement in the field of secretory proteomics. Journal of Proteome Research 2006, 5, 899-906
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Published on Web 03/10/2006
research articles 2. Materials and Methods Materials. The aggressive osteosarcoma cell line OHS and the breast cancer cell line MA11 were obtained from the Norwegian Radium Hospital (Oslo, Norway). The melanoma cell line WM 266-4 was purchased from the American Type Culture Collection. RPMI 1640 medium was purchased from Mediatech, Inc. (Herndon, VA) and DMEM medium from Invitrogen Corporation (Grand Island, NY). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). Acetonitrile and water (HPLC purified) were purchased from Honeywell Burdick & Jackson (Muskegon, MI). Urea, ammonium bicarbonate, trifluoroacetic acid (TFA), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St Louis, MO). Sequencing grade modified trypsin was purchased from Promega (Madison, WI). The syringe filter 0.45 µm was from Corning Incorporated (Corning, NY). Protein solutions were concentrated with a Savant speed vac concentrator system (Savant Instruments Inc., Holbrook, NY). The reversed-phase tC2 Sorbent (Sep-Pak cartridges) was obtained from Waters (Milford, MA) and the C18 column (0.15 × 150 mm) from Micro-Tech Scientific (Vista, CA). Electrospray tandem mass spectrometry (ESI-MS/MS) was performed with a Q-TOF Ultima API-US mass spectrometer (Waters, Milford, MA), equipped with a nanoflow electrospray. Cell Culture Conditions. WM 266-4 was grown in DMEM medium whereas OHS and MA11 were grown in RPMI 1640 medium, both supplemented with 10% FBS until approaching 60-70% confluency (approximately 2 × 106 cells). WM 266-4, OHS and MA11 cells dishes (10 cm) were washed four times with the corresponding medium without serum, and then incubated in the serum-free medium at 37 °C for different times. The incubation time was ranged from 18 to 42-hours in 6-hour increments. After each incubation time, the conditioned medium was carefully removed, and then filtered using a 0.45 µm syringe filter to remove suspended cells. TFA (0.1%) was immediately added into the conditioned medium that was then stored at -80 °C. The addition of TFA lowered the pH (
