Enhanced Pharmacokinetic Bioanalysis of Antibody-drug Conjugates using Hybrid Immunoaffinity Capture and Microflow LC-MS/MS.

The increasing complexity and diversity of antibody-drug conjugates (ADCs) have led to a need for comprehensive and informative bioanalytical methods to enhance pharmacokinetic (PK) understanding. This study aimed to evaluate the feasibility of a hybrid immunoaffinity (IA) capture microflow LC-MS/MS (µLC-MS/MS) method for ADC analysis, utilizing a minimal sample volume for PK assessments in a preclinical study. A robust workflow was established for the quantitative analysis of ADCs by the implementation of solid-phase extraction (SPE) and semi-automation in µLC-MS/MS. Utilizing the µLC-MS/MS approach in conjunction with 1 µL of ADC-dosed mouse plasma sample volume, standard curves of two representative surrogate peptides for total antibody (heavy chain, HC) and intact antibody (light chain, LC) ranged from 1.00 ng/mL (LLOQ) to 5000 ng/mL with correlation coefficients (r2) values of > 0.99. The linear range of the standard curve for payload as a surrogate for the concentration of total ADC was from 0.5 ng/mL (LLOQ) to 2000 ng/mL with high accuracy and precision (< 10% CV at all concentrations). Moreover, a high correlation of concentrations of total antibody between two assay approaches (µLC-MS and ELISA) was achieved with less than 20% difference at all time points, indicating that the two methods are comparable in quantitation of total antibody in plasma samples. The µLC-MS platform demonstrated a greater dynamic range, sensitivity, robustness, and good reproducibility. These findings demonstrated that the cost-effective µLC-MS method can reduce reagent consumption and minimize the use of mice plasma samples while providing more comprehensive information about ADCs being analyzed, including the total antibody, intact antibody, and total ADC.

Simultaneous, Quantitative Characterization of Protein ADP-Ribosylation and Protein Phosphorylation in Macrophages.

The posttranslational modifications (PTMs) ADP-ribosylation and phosphorylation are important regulators of cellular pathways, and while mass spectrometry (MS)-based methods for the study of protein phosphorylation are well developed, protein ADP-ribosylation methodologies are still in a rapidly developing stage. The method described in this chapter uses immobilized metal affinity chromatography (IMAC), a phosphoenrichment matrix, to enrich ADP-ribosylated peptides which have been cleaved down to their phosphoribose attachment sites by a phosphodiesterase, thus isolating the ADP-ribosylated and phosphorylated proteomes simultaneously. To achieve the robust, relative quantification of PTM-level changes we have incorporated dimethyl labeling, a straightforward and economical choice which can be used on lysate from any cell type, including primary tissue. The entire pipeline has been optimized to work in ADP-ribosylation-compatible buffers and with protease-laden lysate from macrophage cells.

Dynamic ADP-Ribosylome, Phosphoproteome, and Interactome in LPS-Activated Macrophages.

We have used mass spectrometry (MS) to characterize protein signaling in lipopolysaccharide (LPS)-stimulated macrophages from human blood, human THP1 cells, mouse bone marrow, and mouse Raw264.7 cells. Protein ADP-ribosylation was truncated down to phosphoribose, allowing for enrichment and identification of the resulting phosphoribosylated peptides alongside phosphopeptides. Size exclusion chromatography-MS (SEC-MS) was used to separate proteoforms by size; protein complexes were then identified by weighted correlation network analysis (WGCNA) based on their correlated movement into or out of SEC fractions following stimulation, presenting an analysis method for SEC-MS that does not rely on established databases. We highlight two modules of interest: one linked to the apoptosis signal-regulating kinase (ASK) signalosome and the other containing poly(ADP-ribose) polymerase 9 (PARP9). Finally, PARP inhibition was used to perturb the characterized systems, demonstrating the importance of ADP-ribosylation for the global interactome. All post-translational modification (PTM) and interactome data have been aggregated into a meta-database of 6729 proteins, with ADP-ribosylation characterized on 2905 proteins and phosphorylation characterized on 2669 proteins. This database-titled MAPCD, for Macrophage ADP-ribosylation, Phosphorylation, and Complex Dynamics-serves as an invaluable resource for studying crosstalk between the ADP-ribosylome, phosphoproteome, and interactome.

ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method.

Protein ADP-ribosylation is a posttranslational modification (PTM) that plays an important role in all major cellular processes, including DNA repair, cellular signaling, and RNA metabolism. Site identification for this PTM has recently become possible through the development of several mass spectrometry-based methods, a critical step in understanding the regulatory role played by mono(ADP-ribose) (MAR), poly(ADP-ribose) (PAR), and the enzymes which make these modifications: poly(ADP-ribose) polymerases (PARPs), best known for their role in DNA repair and as targets for chemotherapeutic PARP inhibitors. Here, we have described our method for enriching and identifying ADP-ribosylation events through the use of a phosphodiesterase to digest protein-conjugated ADP-ribose down to its attachment structure, phosphoribose. We also include here a guide to choosing between collision-induced dissociation (CID)-, higher-energy collisional dissociation (HCD)-, and electron-transfer dissociation (ETD)-based peptide fragmentation for the identification of phosphoribosylated peptides.

ENPP1 processes protein ADP-ribosylation in vitro.

ADP-ribosylation is a conserved post-translational protein modification that plays a role in all major cellular processes, particularly DNA repair, transcription, translation, stress response and cell death. Hence, dysregulation of ADP-ribosylation is linked to the physiopathology of several human diseases including cancers, diabetes and neurodegenerative disorders. Protein ADP-ribosylation can be reversed by the macrodomain-containing proteins PARG, TARG1, MacroD1 and MacroD2, which hydrolyse the ester bond known to link proteins to ADP-ribose as well as consecutive ADP-ribose subunits; targeting this bond can thus result in the complete removal of the protein modification or the conversion of poly(ADP-ribose) to mono(ADP-ribose). Recently, proteins containing the NUDIX domain - namely human NUDT16 and bacterial RppH - have been shown to process in vitro protein ADP-ribosylation through an alternative mechanism, converting it into protein-conjugated ribose-5'-phosphate (R5P, also known as pR). Though this protein modification was recently identified in mammalian tissues, its physiological relevance and the mechanism of generating protein phosphoribosylation are currently unknown. Here, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as the first known mammalian enzyme lacking a NUDIX domain to generate pR from ADP-ribose on modified proteins in vitro. Thus, our data show that at least two enzyme families - Nudix and ENPP/NPP - are able to metabolize protein-conjugated ADP-ribose to pR in vitro, suggesting that pR exists and may be conserved from bacteria to mammals. We also demonstrate the utility of ENPP1 for converting protein-conjugated mono(ADP-ribose) and poly(ADP-ribose) into mass spectrometry-friendly pR tags, thus facilitating the identification of ADP-ribosylation sites.

Nudix hydrolases degrade protein-conjugated ADP-ribose.

ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD(+) to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR.

The Promise of Proteomics for the Study of ADP-Ribosylation.

ADP-ribosylation is a post-translational modification where single units (mono-ADP-ribosylation) or polymeric chains (poly-ADP-ribosylation) of ADP-ribose are conjugated to proteins by ADP-ribosyltransferases. This post-translational modification and the ADP-ribosyltransferases (also known as PARPs) responsible for its synthesis have been found to play a role in nearly all major cellular processes, including DNA repair, transcription, translation, cell signaling, and cell death. Furthermore, dysregulation of ADP-ribosylation has been linked to diseases including cancers, diabetes, neurodegenerative disorders, and heart failure, leading to the development of therapeutic PARP inhibitors, many of which are currently in clinical trials. The study of this therapeutically important modification has recently been bolstered by the application of mass spectrometry-based proteomics, arguably the most powerful tool for the unbiased analysis of protein modifications. Unfortunately, progress has been hampered by the inherent challenges that stem from the physicochemical properties of ADP-ribose, which as a post-translational modification is highly charged, heterogeneous (linear or branched polymers, as well as monomers), labile, and found on a wide range of amino acid acceptors. In this Perspective, we discuss the progress that has been made in addressing these challenges, including the recent breakthroughs in proteomics techniques to identify ADP-ribosylation sites, and future developments to provide a proteome-wide view of the many cellular processes regulated by ADP-ribosylation.

Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells.

Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome.