Toward a Quantitative Understanding of Poly(ADP‐ribosyl)ation Through Mass Spectrometry

Abstract

Post‐translational protein modification provides real‐time modulation of protein form and function. Poly(ADP‐ribosyl)ation is a post‐translational modification (PTM) that has been implicated in processes as diverse as chromatin remodeling, the DNA damage response, and mammalian cell death. More than 200 proteins have been implicated as targets of poly(ADP‐ribosyl)ation, predominantly through the actions of the nuclear enzyme PARP‐1. However, there is very little data to support the existence of a covalent linkage between modified proteins and poly(ADP‐ribose) polymers, indicating that this association may be non‐covalent. While mass spectrometry offers the ability to directly identify sites of post‐translational modification, no published “ADP‐ribosylated proteome” datasets are presently available. In an effort to provide the means to generate such a dataset and elucidate the nature of the protein linkage in this important PTM, we have embarked upon an effort to utilize mass spectrometry for its evaluation. As an initial series of investigations, both histones and PARP‐1 were poly(ADP‐ribosyl)ated using an in vitro system. The products of these reactions were analyzed by mass spectrometric techniques. The results of these investigations will be reported.