Quantitative analysis of the proteome, and protein oxidative modifications, of primary human coronary artery endothelial cells and associated extracellular matrix

Here, we characterize the cellular and extracellular matrix proteomes of primary human coronary artery endothelial cells, from multiple donors, and oxidation/nitration products formed on these during cell culture. Liquid chromatography-mass spectrometry with or without prior fractionation, and different data acquisition methods were employed. In total ~9900 proteins were identified across 3 donors, with ~7000 proteins per donor. Of these ~5300 were consistently identified, indicating heterogeneity across the donors, with donor age a probable cause. Multiple endogenous oxidations and nitrations were detected. The former are present on both cell and ECM proteins, whilst a majority of the latter were detected on cell proteins, consistent with intracellular generation of nitrating agents, most likely from eNOS or peroxidase enzyme activity. The modifications are ascribed to both physiological enzymatic activity (hydroxylation at proline/lysine, predominantly on ECM proteins and especially collagens) and reactive species (oxidation at tryptophan/tyrosine/histidine; nitration at tryptophan/tyrosine).