An Advanced Strategy for Comprehensive Profiling of ADP-ribosylation Sites Using Mass Spectrometry-based Proteomics*

Ivo A Hendriks,Sara C Larsen,Michael L Nielsen

doi:10.1074/mcp.tir119.001315

Abstract

ADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for comprehensive profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. Although ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered >11,000 unique ADP-ribosylated peptides mapping to >7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. High-resolution MS/MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADP-ribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADP-ribosylation linked to the mitochondrion.

Highlights

ADP-ribosylation is an emerging reversible post-translational modification (PTM)1 catalyzed by a large and diversified group of enzymes known as ADP-ribosyltransferases (ARTs)
Crosstalk Between Serine ADP-ribosylation and Phosphorylation—With ADP-ribosylation and phosphorylation both intricately involved in regulation of cellular signaling, and able to modify a similar range of amino acid residues, we investigated our expansive repertoire of ADP-ribosylation sites for crosstalk between the two PTMs
Like what we observed for ADPribosylated peptides in general, we found that electron transfer disassociation with supplemental higher-collisional disassociation (EThcD) was more successful at localizing both PTMs compared with pure electron transfer dissociation (ETD), all experimental conditions identified and localized dozens of co-modified peptides

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—HeLa cells (CCL-2) were acquired from the American Type Culture Collection, and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/ml; Gibco) at 37 °C and 5% CO2. As not all peptides dissolved at alkaline pH after being purified at acidic pH, samples were cleared by centrifugation in a swing-out centrifuge at room temperature, for 30 min at 4250 ϫ g. Purification of ADP-ribosylated Peptides—After overnight incubation with PARG, samples were cleared from mild precipitation by centrifugation in a swing-out centrifuge at 4 °C, for 30 min at 4250 ϫ g. ADP-ribosylated peptides were eluted off the beads using two bead volumes of ice-cold elution buffer (0.15% TFA). Dried purified ADP-ribosylated peptides were dissolved by addition of 10 ␮l 0.1% formic acid, allowed to stand without agitation for 5 min, gently tapped to aid final dissolution, briefly centrifuged to move samples to the bottom of the tubes, and stored at Ϫ20 °C until mass spectrometric measurement. The online STRING database (version 10.5) was used for generation of protein interaction networks [47], and Cytoscape (version 3.7.0) was used for manual annotation and visualization of the STRING networks [48]

RESULTS

ADPr studies

H Other studies

G GOCC: FACT complex

DISCUSSION