TY - JOUR

T1 - Identification and quantification of protein nitration sites in human coronary artery smooth muscle cells in the absence and presence of peroxynitrous acid/peroxynitrite

AU - Xu, Shuqi

AU - Chuang, Christine Y.

AU - Hawkins, Clare L.

AU - Hägglund, Per

AU - Davies, Michael J.

N1 - Publisher Copyright: © 2023 The Authors

PY - 2023

Y1 - 2023

N2 - 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 μM) 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 μM), 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.

AB - 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 μM) 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 μM), 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.

KW - 3-Nitrotyrosine

KW - Extracellular matrix

KW - LC-MS

KW - Nitration

KW - Oxidation

KW - Peroxynitrite

KW - Proteomics

KW - Smooth muscle cell

UR - http://www.scopus.com/inward/record.url?scp=85164293490&partnerID=8YFLogxK

U2 - 10.1016/j.redox.2023.102799

DO - 10.1016/j.redox.2023.102799

M3 - Journal article

C2 - 37413764

AN - SCOPUS:85164293490

VL - 64

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

M1 - 102799

ER -