Method to study stoichiometry of protein post-translational modification.

Abstract

We report a new method to study stoichiometry of protein post-translational modification (PTM) via manipulating small-molecule labels. Such labels can simultaneously bind with all the modified amino acids in a target protein, resulting in a large signal. However, if the small-molecule labels are previously attached with a macromolecule comparable to the target protein in size, binding of one such label will prevent subsequent binding of more labels of its kind; thus, only one of the modified amino acids in the target protein can be bound with the label, resulting in a small signal. Therefore, the stoichiometric number of PTM can be determined by a ratio of the two signals. The proposed method can analyze nitration of several essential regulatory proteins of cellular life, all of which are found to have an integer stoichiometric number indicating regulated site-specific nitration. These results validate the efficacy of our method to provide detailed information on PTMs with biological significance.