title: Optimized method for identification of the proteomes secreted by cardiac cells.
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.
authors.affiliation: Division of Cardiology, Department of Medicine, School of Medicine, Johns Hopkins Bayview Proteomics Center, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Stastna
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D056148:Chromatography, Reverse-Phase; D017077:Culture Media, Conditioned / Q000737:chemistry*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D061251:Primary Cell Culture; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D053719:Tandem Mass Spectrometry; D014357:Trypsin
chemical_list: D017077:Culture Media, Conditioned; D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin