Protein and RNA ADP-ribosylation detection is influenced by sample preparation and reagents used

Abstract

The modification of substrates with ADP-ribose (ADPr) is important in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid–ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts containing ADPr–proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the reactivity of available reagents with diverse ADP-ribosylated protein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross-reactivity with adenylylated RNA, AMPylated proteins, and metabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample preparation methods and have defined the reagents suitable for specific methods and substrates.

Preserving ester-linked modifications reveals glutamate and aspartate mono-ADP-ribosylation by PARP1 and its reversal by PARG

“…We have integrated these protocols with our highly sensitive and specific SpyTag antibodies 17 as well as proteomics technology. Our results are consistent with recent reports showing that boiling the sample causes significant losses of the ADP-ribose signal 20 , 38 . While heating at 60 °C in a non-denaturing lysis buffer, which requires PARP inhibitor to prevent post-lysis ADPr, has been proposed as an alternative to boiling 38 , a key feature of our western blotting protocol is the avoidance of heating samples.…”

“…Importantly, protein extraction and immunoblotting efficiency, assessed via ponceau, PARP1, GAPDH and H3 staining, was not affected by the omission of boiling. Non-denaturing lysis conditions can result in the post-lysis activity of enzymes, including PARP1 11 , 38 , requiring the use of inhibitors. Crucially, 4% SDS at room temperature is sufficient to completely inactivate PARP1 and PARG (Supplementary Fig.…”

Chemoenzymatic and Synthetic Approaches To Investigate Aspartate- and Glutamate-ADP-Ribosylation

“…Previous work has demonstrated that ADPr thermolability and pH stability can vary based on the linkage type . These are critical parameters to consider when preparing and analyzing ADPr samples.…”

Recurrent Loss of Macrodomain Activity in Host Immunity and Viral Proteins

“…In all transfections, the total amount of DNA added was supplemented to 500 ng with an empty cloning vector, pQCXIP (Clontech, Mountain View, CA, USA). Detection of ADP-ribosylation has been shown to be highly dependent on sample conditions [ 38 ], and we have observed that the edges of multiwell plates give less consistent signal than the middle of plates. As a result, only the central eight wells of any given plate were used for transfection.…”