TY - JOUR T1 - Protein and RNA ADP-ribosylation detection is influenced by sample preparation and reagents used JF - Life Science Alliance JO - Life Sci. Alliance DO - 10.26508/lsa.202201455 VL - 6 IS - 1 SP - e202201455 AU - Lisa Weixler AU - Nonso Josephat Ikenga AU - Jim Voorneveld AU - Gülcan Aydin AU - Timo MHR Bolte AU - Jeffrey Momoh AU - Mareike Bütepage AU - Alexandra Golzmann AU - Bernhard Lüscher AU - Dmitri V Filippov AU - Roko Žaja AU - Karla LH Feijs Y1 - 2023/01/01 UR - https://www.life-science-alliance.org/content/6/1/e202201455.abstract N2 - 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. ER -