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

Vascular endothelial cells (ECs) play a key role in physiology by controlling arterial contraction and relaxation, and molecular transport. EC dysfunction is associated with multiple pathologies. Here, we characterize the cellular and extracellular matrix (ECM) proteomes of primary human coronary artery ECs, from multiple donors, and oxidation/nitration products formed on these during cell culture, using liquid chromatography-mass spectrometry. In total ∼9900 proteins were identified in cells from 3 donors, with ∼7000 proteins per donor. Of these ∼5300 were consistently identified, indicating some heterogeneity across the donors, with age a possible cause. Multiple endogenous oxidation products were detected on both ECM and cellular proteins (and particularly endoplasmic reticulum species). In contrast, nitration was mostly detected on cell proteins and particularly cytoskeletal proteins, consistent with intracellular generation of nitrating agents, possibly from endothelial nitric oxide synthase (eNOS) or peroxidase enzymes. The modifications are ascribed to both physiological enzymatic activity (hydroxylation at proline/lysine; predominantly on ECM proteins and especially collagens) and the formation of reactive species (oxidation at tryptophan/tyrosine/histidine; nitration at tryptophan/tyrosine). The identified sites are present on a limited number of peptides (104 oxidized; 23 nitrated) from a modest number of proteins. A small number of proteins were detected with multiple modifications, consistent with these being selective and specific targets. Several nitrated peptides were consistently detected across all donors, and also in human smooth muscle cells suggesting that these are major targets in the vascular proteome. These data provide a 'background' proteome dataset for studies of endothelial dysfunction in disease.