Proteomics analysis of nasopharyngeal carcinoma cell secretome using a hollow fiber culture system and mass spectrometry.
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.
Wu
Hsin-Yi
HY
Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Chang
Ying-Hwa
YH
Chang
Yu-Chen
YC
Liao
Pao-Chi
PC
D014408:Biomarkers, Tumor; D002277:Carcinoma / Q000175:diagnosis / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004867:Equipment Design; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009303:Nasopharyngeal Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013997:Time Factors
D014408:Biomarkers, Tumor; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D011506:Proteins