Abstract
In the past, various studies using different methods have been carried out to analyze proteins secreted by cells. There are several crucial steps that have to be followed to ensure successful secreted proteome detection and identification. Simultaneously with the optimization of the experimental conditions for various cell type culturing and subsequent cell conditioning to obtain conditioned medium with secreted proteins in vitro, the analytical separation methods for fractionation of complex protein mixture and mass spectrometry for protein identification are of high importance. The separation methods primarily used are either gel-based (e.g., 1-DE and 2-DE) or gel-free methods (e.g., liquid chromatography and capillary electrophoresis). Here we outline an optimized protocol for the preparation and analysis of conditioned medium containing proteins secreted by neonatal cardiac myocytes by using reversed-phase liquid chromatography (RPLC) followed by tandem mass spectrometry (LC–MS/MS). Although optimized for neonatal cardiac myocytes, the general steps described in the following chapter can be adapted to other cell types as well.
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Acknowledgement
Supported by the National Heart, Lung, and Blood Institute Proteomic Initiative—contract NHLBI-HV-10-05 (2) (JEV), by NHLBI PO1 HL10026 (Glycoconjungates and Cardiovascular Disease) (JEV), by the AHA grant-in-aid (JEV), and by the Institutional Support RVO: 68081715 (MS).
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Stastna, M., Van Eyk, J.E. (2013). Optimized Method for Identification of the Proteomes Secreted by Cardiac Cells. In: Vivanco, F. (eds) Heart Proteomics. Methods in Molecular Biology, vol 1005. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-386-2_18
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DOI: https://doi.org/10.1007/978-1-62703-386-2_18
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-385-5
Online ISBN: 978-1-62703-386-2
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