Fluorogenic and Affinity Derivatization Methods to Enable Proteomic Study of 3-Nitrotyrosine and 3,4-Dihydroxyphenylalanine as Markers of Oxidative Stress
'Paleontological Institute at The University of Kansas'
Abstract
Purpose: Oxidative post-translational modification of protein-bound tyrosine residues can have a significant impact on protein structure and function and thus may be important to physiological and pathological processes. Oxidative stress has been correlated with biological aging and many disease states, including diabetes, atherosclerosis, and neurodegeneration. Proteomic methods targeted to these modifications are important tools for determining which specific modifications may be significant in these conditions. Toward this end, a method designed to fluorogenically label the protein oxidation products 3-nitrotyrosine (3NY) and 3,4-dihydroxyphenylalanine (DOPA) using benzylamine-dependent chemistry is applied to model peptides and proteins, as well as cardiac tissue samples from a rat model for aging. Methods: Peptides or proteins are reacted with excess benzylamine (or a derivative thereof), in the presence of potassium ferricyanide, to fluorescently label DOPA residues by forming 2-phenylbenzoxazole derivatives. To label 3NY residues, the peptides or proteins are first reduced with sodium dithionite to give 3-aminotyrosine, which can undergo a similar reaction with benzylamine and oxidant to give the same products. Products are characterized by fluorescence spectroscopy, high-performance liquid chromatography (HPLC) with UV and fluorescence detection, mass spectrometry, and amino acid analysis. For enrichment by boronate-affinity HPLC, the benzylamine derivative (3R, 4S)-1-(4-(aminomethyl)phenylsulfonyl) pyrrolidine-3,4-diol (APPD) is used as the tagging reagent. Results: Cardiac proteins have been fluorescently labeled and separated, and some putative identifications have been made. A model protein, glycogen phosphorylase b (Ph-b), has been nitrated in vitro and labeled within a matrix of cardiac homogenate, and the products exhibit concentration-dependent fluorescence. The loss of 3NY from nitrated Ph-b upon mixing with cardiac homogenate has been observed and examined. Five model peptides have been labeled with APPD to determine the effect of primary structure on labeling efficiency, fluorescence quantum yield, and molar absorptivity. Conclusions: This method has great potential to aid identification of the protein oxidation products DOPA and 3NY in proteomic studies of tissue samples and can also be adapted for affinity enrichment and relative quantification of these low-abundance species