Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Systems-wide analysis of ADP-ribosylation in human cells using quantitative mass spectrometry (#14)

Sara C. Larsen 1 , Ivo A. Hendriks 1 , Alexandra Stripp 1 , Michael L. Nielsen 1
  1. university of Copenhagen, Copenhagen, COPENHAGEN, Denmark

ADP-ribosylation is a reversible posttranslational modification involved in a range of cellular processes. Although ADP-ribosylation was described more than 50 years ago, identification of proteins modified with ADP-ribosylation have remained notoriously difficult due to their low abundance and high structural heterogeneity. Previous approaches using chemical approaches or engineered cell culture systems either lack efficiency or sensitivity for systems-wide studies of ADP-ribosylation in complex biological samples. Alleviating these limitations we describe a proteomic strategy for comprehensive mapping of the human ADP-ribosylome, using quantitative high-resolution mass spectrometry in combination with our Af1521 enrichment approach and augmented ETD/EThcD fragmentation.

To benchmark our methodology, we characterized the ADP-ribosylation response in human cells exposed to oxidative stress yielding quantitative identification of >7.000 ADP-ribosylation sites residing on >2.200 nuclear proteins. Our data demonstrate that ADP-ribosylation catalyzed by PARP1/PARP2 is able to modify more than one-third of all nuclear proteins, confirming that ADP-ribosylation is a widespread modification with a regulatory scope comparable to other extensive posttranslational modifications. We find that serine residues are the major induced target of ADP-ribosylation during oxidative stress (>6.300), and our data provides novel insights into extensive and site-specific crosstalk between serine ADP-ribosylation and phosphorylation.

We additionally investigated the crosstalk between ADP-ribosyaltion and SUMOylation as both modifications occurs in the nucleus of cells leading to extensive chain-like modification. However, despite that SUMO and ADP-ribosylation being nuclear modifications targeting same biological processes, the extent of proteins and biological pathways regulated by their individual crosstalk remains enigmatic. Hence we applied our proteomics methodologies for systems-wide analysis, and describe a regulatory crosstalk between SUMO conjugates and ADP-ribosylation during oxidative stress.

By applying our analytical strategy for the detection and quantification of ADP-ribosylation patterns under various cellular perturbations and in tissue samples, we showcase the potential of the described methodology for both basic research and diagnostic purposes.