Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY can handle up to 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic, and tyrosine-specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.

Identification of the Mouse T Cell ADP-Ribosylome Uncovers ARTC2.2 Mediated Regulation of CD73 by ADP-Ribosylation.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

Gas-Phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and Their Implication in Database Searches.

ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and particularly of their acceptor amino acids remains a major challenge. The attachment sites of the modification are difficult to localize by mass spectrometry (MS) because of the labile nature of the linkage and the complex fragmentation pattern of the ADP-ribose in MS/MS experiments. In this study we performed a comprehensive analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine, or lysine residues. In addition to the fragmentation of the peptide backbone, various cleavages of the ADP-ribosylated amino acid side chains were investigated. We focused on gas-phase fragmentations that were specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid, or aspartic acid was the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine was relatively stable. The main cleavage site was the inner bond of the guanidine group, which resulted in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking peptide fragment ions resulting from this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches. Furthermore, the presence of diagnostic ions and of losses of fragments from peptide ions allowed us, in most cases, to distinguish between ADP-ribosylated arginine and serine residues.

Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site.

Despite recent mass spectrometry (MS)-based breakthroughs, comprehensive ADP-ribose (ADPr)-acceptor amino acid identification and ADPr-site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP-ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1-dependent in vivo ADPr-acceptor amino acid. MS analyses of in vitro ADP-ribosylated proteins confirmed tyrosine as an ADPr-acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans-modification of RPS3A is dependent on HPF1. We provide an ADPr-site Localization Spectra Database (ADPr-LSD), which contains 288 high-quality ADPr-modified peptide spectra, to serve as ADPr spectral references for correct ADPr-site localizations.

Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase.

ADP-ribosylation is a posttranslational modification that exists in monomeric and polymeric forms. Whereas the writers (e.g. ARTD1/PARP1) and erasers (e.g. PARG, ARH3) of poly-ADP-ribosylation (PARylation) are relatively well described, the enzymes involved in mono-ADP-ribosylation (MARylation) have been less well investigated. While erasers for the MARylation of glutamate/aspartate and arginine have been identified, the respective enzymes with specificity for serine were missing. Here we report that, in vitro, ARH3 specifically binds and demodifies proteins and peptides that are MARylated. Molecular modeling and site-directed mutagenesis of ARH3 revealed that numerous residues are critical for both the mono- and the poly-ADP-ribosylhydrolase activity of ARH3. Notably, a mass spectrometric approach showed that ARH3-deficient mouse embryonic fibroblasts are characterized by a specific increase in serine-ADP-ribosylation in vivo under untreated conditions as well as following hydrogen peroxide stress. Together, our results establish ARH3 as a serine mono-ADP-ribosylhydrolase and as an important regulator of the basal and stress-induced ADP-ribosylome.

Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography-Tandem Mass Spectrometry.

Protein ADP-ribosylation is a covalent, reversible posttranslational modification (PTM) catalyzed by ADP-ribosyltransferases (ARTs). Proteins can be either mono- or poly-ADP-ribosylated under a variety of physiological and pathological conditions. To understand the functional contribution of protein ADP-ribosylation to normal and disease/stress states, modified protein and corresponding ADP-ribose acceptor site identification is crucial. Since ADP-ribosylation is a transient and relatively low abundant PTM, systematic and accurate identification of ADP-ribose acceptor sites has only recently become feasible. This is due to the development of specific ADP-ribosylated protein/peptide enrichment methodologies, as well as technical advances in high-accuracy liquid chromatography-tandem mass spectrometry (LC-MS/MS). The standardized protocol described here allows the identification of ADP-ribose acceptor sites in in vitro ADP-ribosylated proteins and will, thus, contribute to the functional characterization of this important PTM.

Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography-Tandem Mass Spectrometry.

ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography-high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.

Combining Higher-Energy Collision Dissociation and Electron-Transfer/Higher-Energy Collision Dissociation Fragmentation in a Product-Dependent Manner Confidently Assigns Proteomewide ADP-Ribose Acceptor Sites.

Protein adenosine diphosphate (ADP)-ribosylation is a physiologically and pathologically important post-translational modification. Recent technological advances have improved analysis of this complex modification and have led to the discovery of hundreds of ADP-ribosylated proteins in both cultured cells and mouse tissues. Nevertheless, accurate assignment of the ADP-ribose acceptor site(s) within the modified proteins identified has remained a challenging task. This is mainly due to poor fragmentation of modified peptides. Here, using an Orbitrap Fusion Tribrid mass spectrometer, we present an optimized methodology that not only drastically improves the overall localization scores for ADP-ribosylation acceptor sites but also boosts ADP-ribosylated peptide identifications. First, we systematically compared the efficacy of higher-energy collision dissociation (HCD), electron-transfer dissociation with supplemental collisional activation (ETcaD), and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation methods when determining ADP-ribose acceptor sites within complex cellular samples. We then tested the combination of HCD and EThcD fragmentation, which were employed in a product-dependent manner, and the unique fragmentation properties of the ADP-ribose moiety were used to trigger targeted fragmentation of only the modified peptides. The best results were obtained with a workflow that included initial fast, high-energy HCD (Orbitrap, FT) scans, which produced intense ADP-ribose fragmentation ions. These potentially ADP-ribosylated precursors were then selected and analyzed via subsequent high-resolution HCD and EThcD fragmentation. Using these resulting high-quality spectra, we identified a xxxxxxKSxxxxx modification motif where lysine can serve as an ADP-ribose acceptor site. Due to the appearance of serine within this motif and its close presence to the lysine, further analysis revealed that serine serves as a new ADP-ribose acceptor site across the proteome.

Identification of PARP-Specific ADP-Ribosylation Targets Reveals a Regulatory Function for ADP-Ribosylation in Transcription Elongation.

In a recent issue of Science, Gibson et al. (2016) describe an in vitro chemical genetic approach that maps the specific ADP-ribosylation sites targeted by the ADP-ribosyltransferases PARP-1, PARP-2, and PARP-3 and demonstrate that PARP-1 regulates RNA polymerase II promoter-proximal pausing.

Analysis of Chromatin ADP-Ribosylation at the Genome-wide Level and at Specific Loci by ADPr-ChAP.

Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scales with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation dependent. In combination with deep-sequencing and conventional chromatin immunoprecipitation, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes.