title: Analytical constraints for the analysis of human cell line secretomes by shotgun proteomics.
abstract: Human cell line secretome represents a valuable source of therapeutic targets and candidate biomarkers. Secreted proteins found in biological fluids or culture media are by essence highly diluted. Secretome investigation with proteomic approaches is hardly compatible with the high content of proteins found in complete cell culture media. Therefore, many studies are currently done with media containing few or no protein. Such conditions may perturb cell metabolism and proliferation. Here, we compared seventeen different compositions of culture media for the human bronchial epithelial BEAS-2B cell line. Cell viability, proliferation rate and initial protein charge were systematically compared. We have shown that an important difficulty for the proteomic analysis is due to the presence of detergents such as Pluronic F-68 which hinders peptide mass spectrometry. The high glucose containing DMEM medium which is free of proteins was shown to preserve a good viability and proliferation of cells. With this conditioning medium, we identified 81 extracellular proteins in the secretome of BEAS-2B cells. Moreover, to illustrate this approach, we exposed BEAS-2B cells to a low toxic dose of CoCl(2,) and found 24 extracellular proteins modulated by cobalt. This study highlights the possible contribution of such proteomic approach in the field of toxicology.
authors.affiliation: CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, F-30207, France. veronique.malard@cea.fr
authors.forename: Véronique
authors.initials: V
authors.lastname: Malard
authors.forename: Laetitia
authors.initials: L
authors.lastname: Chardan
authors.forename: Stamatiki
authors.initials: S
authors.lastname: Roussi
authors.forename: Carine
authors.initials: C
authors.lastname: Darolles
authors.forename: Nicole
authors.initials: N
authors.lastname: Sage
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Gaillard
authors.forename: Jean
authors.initials: J
authors.lastname: Armengaud
mesh_terms: D016587:Antimutagenic Agents / Q000009:adverse effects / Q000494:pharmacology; D001980:Bronchi / Q000378:metabolism*; D002460:Cell Line; D003035:Cobalt / Q000009:adverse effects / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000032:analysis; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D020545:Respiratory Mucosa / Q000378:metabolism*
chemical_list: D016587:Antimutagenic Agents; D017077:Culture Media, Conditioned; D020543:Proteome; D003035:Cobalt; C018021:cobaltous chloride