Rapid Analysis of ADP-Ribosylation Dynamics and Site-Specificity Using TLC-MALDI

Abstract

Poly(ADP-ribose) polymerases, PARPs, transfer ADP-ribose onto target proteins from nicotinamide adenine dinucleotide (NAD⁺). Current mass spectrometric analytical methods require proteolysis of target proteins, limiting the study of dynamic ADP-ribosylation on contiguous proteins. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates multisite analysis of ADP-ribosylation. We observe divergent ADP-ribosylation dynamics for the catalytic domains of PARPs 14 and 15, with PARP15 modifying more sites on itself (+3-4 ADP-ribose) than the closely related PARP14 protein (+1-2 ADP-ribose)─despite similar numbers of potential modification sites. We identify, for the first time, a minimal peptide fragment (18 amino-acids) that is preferentially modified by PARP14. Finally, we demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARPs 14/15 prefer acidic residues. Our results highlight the utility of MALDI-TOF in the analysis of PARP target modifications and in elucidating the biochemical mechanism governing PARP target selection.

Figure 1

P14 auto-ADP-ribosylates at a single site under physiological conditions. (a) ADP-ribosylation reaction catalyzed by PARP enzymes. (b) TLC-MALDI workflow. (c) P14 was incubated in the presence of increasing concentrations of NAD⁺ and was subjected to TLC-MALDI to visualize the resulting increase in m/z due to ADPr (+541). (d) MS spectra were integrated to determine the relative levels of auto-ADP-ribosylation. (e) Quantification of the results in (b). The bar graphs depict the fraction of the total P14 protein that has been modified at 0, 1, or 2 distinct sites (mean ± S.E.M., n = 3). (f) In vitro P14 ADP-ribosylation assay.

Figure 2

P15 auto-ADP-ribosylates at multiple sites under physiological conditions. (a) P15 was incubated in the presence of increasing concentrations of NAD⁺ and was subjected to TLC-MALDI to visualize the resulting increase in m/z due to ADP-ribosylation (+541). (b) MS spectra were integrated to determine the relative levels of auto-ADP-ribosylation. (c) Quantification of ADPr levels for both P14 and P15. The bar graphs depict the fraction of the total protein that has been modified at 0, 1, 2, 3, or 4 distinct sites (mean ± S.E.M., n = 3). *represents p-value <0.05, two-tailed Student’s t test. The concentration of NAD⁺ is given. (d) In vitro P15 ADP-ribosylation assay.

Figure 3

TLC-MALDI performed with minimal peptide fragments reveals proximal PARP selectivity. (a) P14 was incubated with P14i1 in the presence of increasing concentrations of NAD⁺, and the resulting MS spectra were normalized and plotted against each other to visualize the increase in m/z due to ADPr (+541). The bar graphs depict the fraction of the total peptide that has been modified (mean ± S.E.M., n = 3). The sequence of the P14i1 peptide fragment is provided. (b) P15 is unable to transfer ADPr to the P14i1 peptide. Experiments were performed as in (a) in the presence of 1 mM NAD⁺. (c) Loss of the acidic modification site prevents ADP-ribosylation. Experiments were performed as in (a) in the presence of 1 mM NAD⁺. *represents p-value <0.05, two-tailed Student’s t test.

Figure 4

TLC-MALDI performed following HA treatment confirms acidic residues as the targets of P14 and P15 ADP-ribosylation. (a) HA treatment workflow. (b) P14 was incubated with 400 μM NAD⁺ prior to treatment with HA and was subjected to TLC-MALDI to visualize the resulting increase in m/z due to ADP-ribosylation (+541). The bar graphs depict the fraction of the total protein that has been modified (mean ± S.E.M., n = 3). (c) The same experiment from (a) was performed with P15. (d) P14 was incubated with P14i1 and treated as in (a). The filled circles represent treatment with NAD⁺, HA, or both.