22 - Identifying Nitration Sites in Extracellular Matrix Protein Laminin by LC-MS/MS

Abstract

Reactive nitrogen species (RNS), such as peroxynitrous acid (ONOOH), produced by activated leukocytes and other cells, can nitrate Tyr and Trp residues on proteins to give 3-nitroTyr and 6-nitroTrp. These long-lived chemically-stable post-translational modifications are used as biomarkers of damage, and may contribute to human pathologies. Extracellular matrix (ECM) proteins are highly abundant, long-lived, contain few alternative targets for RNS (Cys and Met), and are exposed to high RNS fluxes. ECM proteins are therefore likely to accumulate nitration products. Evidence has been presented for tyrosine nitration in human atherosclerotic lesions, and we have shown that this co-localizes with the major ECM protein laminin. Furthermore, nitration alters laminin self-assembly and polymerization, and decreases cell binding. Study aim To identify sites of nitration in laminin using tandem mass spectrometry to understand how and why nitration alters laminin structure and function. Results Using standard shotgun proteomics 101 nitration sites have been identified in murine laminin-111 treated with high (500-fold) molar excesses of ONOOH in vitro. These sites include those involved in cell attachment, domains involved in laminin polymerization, and binding sites of other ECM proteins. 20 nitration sites were identified with low (10-fold molar excess) ONOOH concentrations, including Y145 in the laminin γ1 chain that is located in a cell-binding motif, consistent with reduced endothelial cell binding to modified laminin. Conclusions These data indicate that laminin is readily nitrated by low levels of ONOOH, and that this occurs in functionally important domains, consistent with the observed biological changes. Laminin nitration appears to be a sensitive marker of damage, and may contribute to human disease through decreased cell adhesion and altered ECM structure and assembly.