Investigation of local primary structure effects on peroxynitrite-mediated tyrosine nitration using targeted mass spectrometry

Protein-tyrosine nitration (PTN) is a posttranslational modification resulting from cellular nitrosative stress that has been implicated in a wide variety of disease states. Determination of factors that influence selectivity of PTN remains a major challenge due to several issues including low biological levels of PTN, proximity of target sites on a single analyte, and analytical limitations for site-specific quantification of the nitration modification. We report a systematic approach that addresses relevant contributing factors to PTN with particular focus on determining the effect of changing proximal amino acid side chain structure on tyrosine nitration yield. A trend was observed in which nitration yield tends to be greater when the tyrosine residue is surrounded by basic and/or uncharged polar residues compared to nitration levels observed when hydrophobic and acidic residues are proximal to the tyrosine residue. Moreover, an electric dipole effect was observed where a higher degree of charge asymmetry surrounding the tyrosine residue correlates with an increased tyrosine nitration yield in certain cases. The reported data are expected to facilitate site-specific prediction and validation of PTN, especially in cases of potential target residues that share a similar solvent exposure environment and contain elements of known higher-order structure.

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► Site-specific tyrosine nitration (TN) was quantified using an MRM-based assay.
► The linear amino acid sequence surrounding tyrosine affects its nitration susceptibility.
► TN yield increases when the tyrosine is flanked by basic and/or polar uncharged residues.
► Utility of MRM for quantitation of multiple PTMs on same target analyte has been demonstrated.