Detection and quantification of PAR and PARP activity

Identification of protein interaction partners should help understanding the PARP function in the DNA repair and cellular processes. Moreover, the quantification of PAR as well as the determination of PARP activity would be extremely useful for drug development. All methods used to determine ADP-ribose polymers in intact cells and tissues must be very sensitive and selective, since the content of ADP-ribose polymers is very low relative to other adenine-containing nuclear polymers such as DNA and RNA. In particular, the

39 quantitative analysis of basal PAR levels in vivo is a challenging task that requires specific and highly sensitive methods. While the quantification of ADP-ribose polymers is still not easy, a number of chemical and immunological methods are now available for the investigation of ADP-ribose polymer metabolism. Moreover, it is highly important to be able to determine PARP activity in vitro and in vivo, to establish high-throughput assays for screening PARP inhibitors.

1.6.1. Chromatographic methods

The first milestone to determine ADP-ribose polymer content in cultured cells and animal tissues was achieved by Jacobson et al. [25]. Dihydroxyboryl Bio-Rex (DHBB) affinity chromatography for isolating PAR was used, followed by compound derivatization and LC-based detection, which provided an accurate quantification. These resins have been used in combination with both chemical and immunological detection methods to quantify ADP-ribose polymer residues in vivo. Unfortunately, routine usage was hampered by the amount of cellular material required, radioactive labeling or derivatization of the analytes, and the overall time-consuming procedure.

Another approach allowing the quantification of total polymer residues is based on the conversion of the nucleotides to highly fluorescent 1, N⁶-etheno derivatives. The mixture can be separated by reverse-phase HPLC and quantified at the picomole level [165]. Other approaches were based on treating cells with [³H]adenine as a radioactive precursor to NAD⁺ [166]. Although leading to precise results, these techniques were prone to overestimate PAR levels, due to artificial DNA damage induced by the radioactive isotopes and subsequent collateral PARP activation.

The combination of boronate chromatography with high-resolution anion-exchange HPLC has allowed the preparation and separation of oligomers up to the 50-mer and multibranched polymers [167]. Another chromatographic method involving two-dimensional thin-layer chromatography on cellulose plates has been used to separate and quantify the three diagnostic nucletides of poly(ADP-ribose) generated by phosphodiesterase digestion: AMP, PR-AMP, and (PR)2AMP.

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1.6.2. Immunodetection of PAR

Most of the current tools to quantify PAR rely on antibodies, which are widely used in basic research as well as preclinical and clinical studies [168, 169]. Four types of antibodies have been generated: anti-poly(ADP-ribose) sera [170], anti-PR-AMP sera [171], antibodies specific for 5′AMP [172] and antibodies against an analogue of ADP-ribose [173]. The first raised antibodies against poly(ADP-ribose) did not bind poly(A) or other related nucleotides, nor yeast RNA or calf thymus DNA, but bound poly(ADP-ribose) and, to a lesser degree, ADP-ribose and PR-AMP [174]. However, a cross-reaction with double stranded RNA, poly(A)-poly(U), or poly(I)-poly(C) duplexes was observed.

Furthermore, the reactivity of these antibodies against poly(ADP-ribose) was found to be dependent on the polymer size, binding smaller polymers less efficiently than larger polymers. A radioimmunoassay involving this antibody has been used to detect poly(ADP-ribose) in vivo, but these methods were semi-quantitative [175]. For semi-quantitative determinations, prior fractionation of the sample according to polymer size was necessary.

In the early 1980s, the first monoclonal antibodies to poly(ADP-ribose) were reported to have a greater potential for quantitative immunoassays for ADP-ribose polymers, since monoclonal antibodies recognize specific antigenic determinants [176].

Characterization of two monoclonal antibody preparations demonstrated that one of them recognized the linear structure of ADP-ribose polymers, while the second one recognized additional structures including the branched portions of the polymers. Specific antibodies have proved to be very useful for the cytological detection of ADP-ribose polymers within individual cells [10].

Antibodies highly specific for poly(ADP-ribose) can be easily visualized by coupling them to dyes such as fluorescein isothiocyanate (FITC) or by using FITC-labeled anti-IgG antibody. Cross-reactivity of the antibodies with DNA is of particular concern, because DNA is present in nuclei in much higher concentration than poly(ADP-ribose). Most antibodies do not exhibit cross-reactivity with DNA, RNA, poly(A), ADP-ribose or NAD when examined by double-immunodiffusion and membrane-binding assays [177]. However, since

41 the reactivity of the antibody varies significantly with the chain length of the polymer, the fluorescence intensity observed in immunohistochemistry is therefore a function of both the amount and size of polymer [10, 176]. Such approaches are also limited with regard to sensitivity and linearity of quantification. For example, one of the most widely used anti-PAR antibodies, i.e., 10H, preferentially binds long PAR chains over short ones, thus leading to a potential underestimation of PAR levels and lack of linearity in quantification [110]. Furthermore, immuno-based methods lack chemical specificity and do not use internal standards to control for losses during sample preparation. Isotope dilution mass spectrometry is the current standard method used to quantify nucleosides derived from DNA and RNA, including enzymatic modifications and damage products [178]; however, similar methods to quantify cellular PAR have not been described yet.

1.6.3. Quantification of Cellular Poly(ADP-ribosyl)ation by Stable Isotope Dilution Mass Spectrometry

Recently, new method based on HPLC-coupled tandem mass spectrometry (LC-MS/MS) was employed for the quantification of PARylation in cells and tissues [179]. Using this method it was possible to determine PAR levels in various biological systems, revealing cell type- and tissue-specific differences in the PARylation dynamics. An accurate and sensitive bioanalytical approach based on isotope dilution mass spectrometry was also developed in order to quantify steady-state and stress-induced PAR levels in cells and tissues and to characterize pharmacological properties of PARP inhibitors. In contrast to existing PAR-detection techniques, the LC−MS/MS method uses authentic isotope-labeled standards, providing unequivocal chemical specificity to quantify cellular PAR in absolute terms with femtomol sensitivity [179].

Using this method, it was possible to quantify poly(ADP-ribosyl)ation in solid tissues and to identify tissue-dependent associations between PARP-1 expression and PAR levels in ex vivo pharmacodynamic studies in human lymphocytes as well as in a series of different mouse organs. This study demonstrates that mass spectrometric quantification of cellular

poly(ADP-42

ribosyl)ation has a broad range of applications in basic research as well as in drug development [179].

1.6.4. Mass spectrometric characterization of ADP-ribose

Apart from the quantification of PAR and the ability to identify PARP activity, tools for the determination of interaction partners to understand the complete role of PARP in organisms are of high importance. Gagné et al.

performed the first large scale proteomic identification of the PAR binding proteins [180, 181]. This mass spectrometric study confirmed the implications of PARylation in several pathways and led to the identification of new target proteins. Furthermore, Jiang et al. used 6-alkyne-NAD with a biotin affinity tag to identify 79 proteins as potential PARP-1 substrates [182]. Another analytical challenge is to identify the modifications/binding sites on a protein.

Unfortunately, traditional approaches such as radioactive labeleling or antibodies cannot be used to localize the ADP ribose moiety on a specific amino-acid residue. Up to now, many studies to identify the modification sites have been performed. While ¹H- and ¹³C-nuclear magnetic resonance (NMR) has been applied to overcome the limitations of other techniques, this method has the disadvantage that it requires relatively pure sample and has insufficient sensitivity to detect minor physiological amounts of modified proteins. Mass spectrometry has been previously attempted to directly interrogate the structure of ADP-ribosylated peptides with modest success. Goodlett and co-workers [183] compared the fragmentation of an ADP-ribosylated peptide using MS fragmentation technique like collisionally activated/induced dissociation (CAD/CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD). The application of CAD and IRMPD fragmentation methods might lead to the identification of ADP-ribose modifications. Fragmentation of the ADP-ribose moiety also occurs. But, neither CAD nor IRMPD fragmentation provided sufficient sequence coverage of the ADP-ribosylated peptide to localize the modification. ECD, in contrast to CAD and IRMPD, showed higher potential for localizing the ADP- ribose moiety on a specific residue. A two-step procedure using CAD/IRMPD followed by ECD was recommended for the

43 analysis of ADP-ribosylated peptides. In another approach, a quadrupole tandem mass spectrometer was recently applied for characterizing of ADP-ribosylated peptides, where precursor ion scans are first used to search for an ADP-ribosylated arginine carbodiimide marker ion (resulting from fragmentation of the ADP-ribosylated peptide), indicative for Arg-ADP-ribosylated peptides [184]. In order to identify the modification site, an additional two-step fragmentation of this ADP-ribosylated carbodiimide peptide has to be performed. Unfortunately, none of these approaches did not provide a sensitive and robust high-throughput, large-scale experiments for the detection of ADP-ribosylated peptides [185]. The application of electron-transfer dissociation ETD greatly simplifies the mass spectrometric sequence analysis of ADP-ribosylated peptides, compared to the conventional CAD approach [186]. The advantage of ETD fragmentation of ADP-ribosylated peptides is that it allows the fragmentation of highly charged peptides. Moreover, it should be useful for high-throughput large scale studies on the unique class of modified peptides.

Furthermore, a simpler MS alternative using nanoflow LC-MS/MS with ETD was proposed to directly detect the ADP-ribose moiety, covalently attached to the peptide [51, 185, 186]. A crucial aspect is that the intramolecular radical induced mechanism of ETD does not lead to the cleavege of the labile ADP-ribose group. In contrast to other reports, Messner et al. observed partial fragmentation of the ADP-ribose at the phosphodiester bond by application of ETD [186].

However, conventional CID mass spectrometry is cleaving ADP-ribose modification off from the peptide during fragmentation. The commonly used CID fragmentation, instead of ETD fragmentation, seems to be the reason why many efforts to identifiy ADP-ribosylated residues failed in the past. The ETD facilitates the characterization of ADP-ribosylated proteins by allowing sensitive and high-throughput large-scale sequencing experiments on peptides and is therefore supposed to be a promising technique for defining in vivo substrates [186].

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