Development of a mass-spectrometry based method for the identification of the in vivo whole blood and plasma ADP-ribosylomes

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modifications of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove the modification after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we established that plasma phosphodiesterases and ADP-ribosylhydrolases rapidly demodified in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a novel mass-spectrometry based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.