The RiboMaP Spectral Annotation Method Applied to Various ADP-Ribosylome Studies Including INF-γ-Stimulated Human Cells and Mouse Tissues

Figures

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  Figure 1. Inter-study analysis using a single workflow to annotate ADP-ribosyl data. (A) The ADP-ribosyl (ADPr) post-translational modification with its major higher collision energy (HCD) dissociation products (annotated as the m- and p-ions). (B) Schematic depicting sequential dissociation that converts precursor P-ions to fragment p-ions. (C) Workflow for ADP-ribosylome data re-analysis using RiboMaP.

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  Figure 2. Intra- and inter-dataset comparisons of ADP-ribosyl peptide spectral matches. (A) XCorr, the cross-correlation score between a spectrum and database match. (B) Isolation interference, the relative amount of ion current within the isolation window that is not attributed to the isolated precursor. (C) m-series score, the scaled total absolute m-ion intensity normalized by base peak intensity. (D) p-series score, total absolute p-ion intensity normalized by base peak intensity.

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  Figure 3. Applying RiboMaP to increase the number of ADP-ribosyl peptide spectral matches (PSMs). (A) A breakdown of the contribution of individual m-ions to the m-series score. (B) An ADPr PSM identified in two independent studies contains similar fragment ions. UGHD, UDP-glucose 6-dehydrogenase. (C) A spectrum-to-spectrum peak intensity plot from panel B shows that the m3-ion intensity is markedly higher in the QE-HF compared to the Lumos. (D) A comparison between default and RiboMaP-enabled reporting of ADPr peptides. (E) Our current workflow accounts for the majority of ADPr proteins reported previously for the QE-HF liver study (76.6%) and the Lumos THP-1 study (83.4%).

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  Figure 4. A comparison between IFN-γ treatment studies. (A) Common and unique ADPr proteins between human THP-1 cells, and mouse liver or spleen. (B) A linear model plot using R. The gray lines indicate the 95% confidence intervals. (C) Comparisons across studies demonstrate no overlap for IFN-γ effects on the ADP-ribosylomes except for a common sequence in PARP1. (D) PARP1 amino acid sequence determined to be commonly ADP-ribosylated in human THP-1 cells and mouse spleen datasets. The orange amino acids correspond to sequence coverage in THP-1 and mouse PARP1 data. The peptide whose ADPr signal decreased in both THP-1 and mouse spleen data is indicated. (E) The IFN-γ modulated PARP1 ADPr peptide resides within a predicted disordered domain. Both glutamate and serine amino acids were identified (EthcD annotation provided) to be ADP-ribosylated in THP-1 cells (and mouse spleen) (Supplementary Figure S3).