Direct detection of nitrotyrosine-containing proteins using an aniline-based oxidative coupling strategy

Abstract

A convenient two-step method is described for the detection of nitrotyrosine-containing proteins. First, nitrotyrosines are reduced to aminophenols using sodium dithionite. Following this, an oxidative coupling reaction is used to attach anilines bearing fluorescence reporters or affinity probes. Features of this approach include fast reaction times, pmol-level sensitivity, and excellent chemoselectivity.

Fig. 1

Detection of nitrotyrosine-containing proteins using a two-step protocol.

Fig. 2

Oxidative coupling reactions between o-aminophenols and functionalized anilines. The ESI-TOF MS analysis of the reactions of o-aminophenol 1 with fluoresceinamine (2a) or biotin aniline (2b) shows clean conversion to the respective oxidative coupling products (expected [M+H]⁺ m/z = 468.1078 for 3a and 730.3480 for 3b). The reactions were run with 200 μM functionalized anilines, 3 μM 1, and 80 mM ferricyanide in 100 mM phosphate buffer, pH 6.

Fig. 3

Chemoselectivity tests for the oxidative coupling reaction. (a) Two mutants of the MS2 coat protein were used: the wild-type (T19) and a variant containing an aminotyrosine side chain (T19(Y-NH₂), shown). (b) ESI-TOF MS analyses are shown for the MS2 proteins before and after oxidative coupling with functionalized anilines (2a and 2b). Y-NO₂ = nitrotyrosine, Y-NH₂ = aminotyrosine. The reactions were run with 200 μM 2, 10 μM protein (based on monomer), and 80 mM ferricyanide in 100 mM phosphate, pH 6.

Fig. 4

Testing the specificity of the oxidative coupling reaction for o-aminophenol residues on additional proteins. (a) Reactions were run with 200 μM 2a or 2b, 10 μM protein, and 80 mM ferricyanide in 100 mM phosphate buffer, pH 6, for 2 h. (b–d) Following SDS-PAGE, samples labeled with 2a were visualized using fluorescence imaging (shown as an inverted image) and Coomassie stain. Samples labeled with 2b were visualized through Western blotting with anti-biotin-HRP probes. The X labels refer to the tyrosine substituents, as defined in the structure in (a). NH₂ groups in pink indicate protein samples that were purified after dithionite reduction. NH₂ groups in blue indicate samples that were not purified between the steps. The vertical dashed lines indicate where two portions of the same gel were juxtaposed.

Fig. 5

Detection of MS2-T19(Y-NO₂) in cell lysates (24 μg Ramos lysate loaded per lane). Portions of MS2-T19(Y-NO₂) were added to 72 μg of cell lysate in 100 μL of phosphate buffer, pH 6. The samples were reduced with 2 mM sodium dithionite for 20 min, and then treated with 200 μM 2a and 80 mM ferricyanide for 2 h. After ultrafiltration, the reactions were analzed by SDS-PAGE with (a) Coomassie staining and (b) fluorescence detection (inverted image shown). (c) The same procedure was used with 2b, and the samples were visualized via Western blotting with an anti-biotin-HRP probe. (d) For comparison pusposes, MS2-T19Y-NO₂ was also added to lysates and visualized via Western blotting with anti-nitrotyrosine and secondary antibody-HRP.