Identification and quantification of protein nitration sites in human coronary artery smooth muscle cells in the absence and presence of peroxynitrous acid/peroxynitrite

Peroxynitrous acid/peroxynitrite (ONOOH/ONOO ¡) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, and particularly proteins. Here, we show that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with LC-MS peptide mass mapping providing data on the sites and extents of changes on cellular and extracellular matrix (ECM) proteins. Evidence is presented for selective and specific nitrations at Tyr and Trp on 11 cellular proteins (out of 3668, including 205 ECM species) in the absence of added reagent ONOOH/ONOO ¡, with this being consistent with low-level endogenous nitration. A number of these have key roles in cell signaling/sensing and protein turnover. With added ONOOH/ONOO-, more proteins were modified (84 total; with 129 nitrated Tyr and 23 nitrated Trp, with multiple modifications on some proteins), with this occurring at the same and additional sites to endogenous modification. With low concentrations of ONOOH/ONOO ¡ (50 & mu;M) nitration occurs on specific proteins at particular sites, and is not driven by protein or Tyr/Trp abundance, with modifications detected on some low abundance proteins. However, with higher ONOOH/ONOO ¡ concentrations (500 & mu;M), modification is primarily driven by protein abundance. ECM species are major targets and over-represented in the pool of modified proteins, with fibronectin and thrombospondin-1 being particularly heavily modified (12 sites in each case). Both endogenous and exogenous nitration of cell-and ECM-derived species may have significant effects on cell and protein function, and potentially be involved in the development and exacerbation of diseases such as atherosclerosis.

Automated summary

Peroxynitrous acid/peroxynitrite (ONOOH/ONOO ¡) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, particularly proteins. This study found that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with various modifications occurring on cellular and extracellular matrix proteins. The nitration occurs both endogenously and exogenously, and it may have significant effects on cell and protein function, potentially contributing to the development and exacerbation of diseases such as atherosclerosis.