Reanalysis of published high-quality proteomics datasets
For the reanalysis of published high-quality proteomics datasets, public raw files were analyzed with MaxQuant proteomics suite of algorithms (version 1.5.3.17) (Cox and Mann, 2008), using the integrated search engine Andromeda (Cox et al., 2011).
Data from the published proteomics study of peptides enriched with an ADPr-binding macrodomain (Martello et al., 2016) were searched against the human proteome database (downloaded 09.10.2015 from UniProt) with the following parameters: the maximum allowed mass deviation was set to 4.5 ppm for precursor ions and 20 ppm for fragment ions; the minimum peptide length was set to 6 amino acids and the maximum number of missed cleavages was set to 5 with the maximum charge state 7. Variable modifications included acetylation (Protein N-term), Oxidation (M) and ADPr (DEKRSTCYNQHM). The variable modification ADPr allowed for neutral losses of adenine (m/z 136.0618); adenosine with loss of water (m/z 250.0935); AMP (m/z 348.0704); ADP (m/z 428.0367) and ADP-ribose (m/z 542.0684). FTMS top peaks per 100 Da were set to 20. We employed the annotated mass spectrometry (MS)/MS spectra generated by MaxQuant as the basis for our manual validation of spectra. To consider a peptide as modified on Tyr, we required the presence of fragment ions with either the intact ADP-ribose or phosphoribose (resulting from the loss of AMP) pointing to ADPr on Tyr. Unmodified “native” sequence ions were not considered as evidence for localization since it is impossible to distinguish between an original lack of modification and complete loss of ADPr during fragmentation. Two additional pieces of evidence supporting Tyr modification could also be observed in lower mass regions of these spectra. First, a peak matching the immonium ion of modified Tyr (+ ADPr – AMPloss) could be observed (albeit weakly) in these spectra at 330.0742 Da (+1). The native (unmodified) Tyr immonium ion (136.0762) was also generally very weak (∼5%) in comparison to the immediately neighboring Adenine peak (136.0623) in these spectra, as opposed to those of peptides containing Tyr but with ADPR on serine. The significance of this ratio as support of Tyr modification can only be fully assessed with larger numbers of ETD-verified peptide spectra.
For the cellular ADPr characterization with HCD and EThcD study (Bilan et al., 2017), variable modifications included oxidation (M), acetylation (Protein N-term and K) and ADPr (DEKRSTYCMNQHM). For confident identification of ADPr sites, we considered only ETD MS/MS spectra and required a minimum Andromeda score of 100, mass deviation smaller than 3 ppm after MaxQuant recalibration and a localization score above 0.9. In addition, we manually validated all the representative spectra by requiring extensive coverage of the peptide backbone fragment ions. For localization we required the clear presence of multiple high-intensity fragment ions pinpointing the modification site.
For the cellular MS analysis of endogenous histones study (Leidecker et al., 2016), variable modifications included oxidation (M), acetylation (Protein N-term and K), methylation (KR), dimethylation (K), trimethylation (K) and ADPr (DEKRSTYCMNQHM). Similarly, we considered only ETD MS/MS spectra and required a minimum Andromeda score of 100, mass deviation smaller than 3 ppm after MaxQuant recalibration and a localization score above 0.9.