Fig. 3: Short acidic protein digestion with Arg-C Ultra preserves mono-Asp/Glu-ADPr for proteomic analyses. | Nature Communications

Fig. 3: Short acidic protein digestion with Arg-C Ultra preserves mono-Asp/Glu-ADPr for proteomic analyses.

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

Fig. 3

A Schematic overview of standard mass-spectrometry sample preparation workflow (top) or of our updated workflow for the preservation of ester-linked PTMs (bottom). A key feature of our protocol is the short (~3 h) incubation at acidic pH with the proteases Arg-C Ultra and Lys-C, which provide the optimal environment for preservation of ester-linked PTMs while ensuring high digestion efficiency. This schematic was generated in Adobe Illustrator using some graphical elements adapted from Longarini et al.17 B Intensity of Asp/Glu-ADPr peptides, derived from automodified PARP1E988Q, identified with the conventional “Trypsin” or our optimized “Arg-C Ultra + Lys-C” protocols (A). Data is expressed as dot-plot with each dot representing the corresponding intensity of all (n = 56) identified ADPr sites, and the bar representing sample mean, from a representative of 4 independent experiments. c Average abundance of high-confidence (delta score > 40, localization probability ≥ 0.9) mono-ADPr sites identified in GFP-PARP1 from HPF1KO cells. Data is expressed as mean ± SEM of n = 4 independent replicates. D Left: high-resolution High-energy Collisional Dissociation (HCD) fragmentation spectrum of a PARP1 ADPribosylated peptide identified from cells (C). HCD fragmentation of the Asp/Glu-ADPr peptide leads to loss of AMP (∆mass = −347.06 Da) and leaves phosphoribose-H2O (∆mass = 193.998) as a diagnostic fragment that can be used to identify the site of ADPribosylation. Left: corresponding high-resolution ETD spectrum of the same peptide species in which Asp/Glu-ADPr is preserved and can unambiguously assign the modification site. E Schematic depiction of all high-confidence (delta score > 40, localization probability ≥ 0.9) PARP1 Glu- and Ser-ADPr sites identified in cells displayed a linear model of PARP1 (Zn = zinc-finger domain, HD = autoinhibitory domain, ART = catalytic domain). Data from Fig. C and Supplementary Fig. 3g.

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