Development of methods for detection of protein tyrosine nitration via labeling of aminotyrosine-containing proteins

Abstract

Protein tyrosine nitration (PTN) arises from oxidation of tyrosine residues by peroxynitrite anion (ONOO) through a free radical process in biological systems. It can also be catalyzed by myeloperoxidase in the presence of nitrite and hydrogen peroxide (H2O2) in a peroxynitrite-independent pathway. Once tyrosine nitration occurs, the biochemical properties of 3-nitrotyrosine (3NT)-containing proteins can be entirely different from their unmodified forms. Although there are many direct and indirect ways to detect PTN, all of these methods suffer from poor selectivity, low efficiency and multiple derivatization. Here we report several novel analytical approaches to detection and enrichment of 3-aminotyrosine (3AT)-containing proteins, which are important derivatives of 3NT-containing proteins. These methods are applicable to various biological samples and live cells with high detection sensitivity, good selectivity and high efficiency.

Inspired by the serine/threonine ligation method, a series of salicylaldehyde (SAL)-esters 2.50af (Figure 1, left) were designed, and labeling of 3AT via salicylaldehyde (SAL)-ester induced amide coupling was achieved in phosphate buffer. However, in assessment of selectivity, 2.50df were found to have interaction with cysteines. In addition to 3AT itself, two peptides containing a 3AT residue were synthesized and subjected to the labeling reaction by 2.50a-c. The results showed that the products formation was not affected by the position of 3AT residue in peptides. Then, detection of 3AT-containing in bovine serum albumin was performed with 2.50a under physiological conditions, and SAL-ester 2.50a was also found to react with cysteines. Nevertheless, the selectivity of 2.50a could be rescued with cysteine blockade prior to the labeling reaction.

On the basis of the results of 2.50af, improvements were made for even better selectivity and efficiency. SAL-carbamates 3.24 and 3.28 were synthesized after screening a series of SAL-carbamate analogs. With fluorescent SAL-based carbamate 3.24 and alkyne tag 3.28 (Figure 1, right), labeling of 3AT via SAL-carbamate induced urea bond formation under aqueous conditions was achieved. Subsequently, detection and enrichment of 3AT-containing proteins were performed using 3.24 and 3.28, respectively, in various models including single protein, cell lysate of LPS-stimulated macrophages and live cells. For proteomic analysis with 3.28, more than one hundred of 3AT-containing proteins (including histones, enzymes and chaperon proteins) were pulled down after conjugation with biotin-azide.

Figure 1 Structures of the Synthetic Probes for Detection of PTN

In summary, we have developed several methods for detection of PTN via labeling of 3AT-containing proteins. With cysteine blockade, 2.50a can be applied for selective labeling of 3AT-containing proteins. With probes 3.24 and 3.28, detection of PTN can be achieved successfully with high selectivity and efficiency in various biological samples. Notably, proteomic analysis including identification of proteins and investigations on modification sites can be performed with the alkyne tag 3.28.