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Cited by 5PARP inhibition in cancer therapyMass Spectrometry Techniques and ApplicationsAdvanced Proteomics Techniques and Applications

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Using TLC-MALDI-TOF to Interrogate In Vitro Peptidyl Proximal Preferences of PARP14 and Glycohydrolase Specificity

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Javed, Zeeshan, Nguyen, Hannah H., Harker, Kiana K., Mohr, Christian M., Vano, Pia, Wallace, Sean R., Silvers, Clarissa, Sim, Colin, Turumella, Soumya, Flinn, Ally, Moritz, Anthony, Carter-O’Connell, Ian·2023·Molecules
DOI 10.3390/molecules28166061PMC PMC10459978

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Abstract

The transfer of ADP–ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human diphtheria toxin-like ADP-ribosyltransferases (ARTDs; previously referred to as poly-ADP–ribose polymerases or PARPs) and the removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about the sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the in vitro analysis of proximal factors that guide ARTD target selection. We identify a minimal 5-mer peptide sequence that is necessary and sufficient to drive glutamate/aspartate targeting using PARP14 while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is pH and temperature dependent, sequence independent, and occurs within hours. Finally, we use the ADPr–peptides to highlight differential activities within the glycohydrolase family and their sequence preferences. Our results highlight (1) the utility of MALDI-TOF in analyzing proximal ARTD–substrate interactions and (2) the importance of peptide sequences in governing ADPr transfer and removal.

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Figures

Figure 1 Overview of the experimental approach. (a) ADP-ribosylation (step 1) and hydrolysis reactions (step 2). (b) TLC-MALDI workflow.
Figure 1 Overview of the experimental approach. (a) ADP-ribosylation (step 1) and hydrolysis reactions (step 2). (b) TLC-MALDI workflow.CC BY · PubMed Central
Figure 2 Identification of a minimal P14 selective peptide sequence. (a) Peptide truncations used in this study (full sequences in Table S1). The E acceptor is indicated. (b) P14c and the indicated peptide were incubated in the presence of NAD+ and subjected to TLC-MALDI to visualize the resulting increase in m/z due to MARylation (+541 Da). The dashed line represents the intensity observed for ADP-ribosylation of P14p1. (c) MS spectra for P14c treatment were integrated to determine the relative levels of ADP-ribosylation. The bar graphs depict the fraction of the total peptide that was modified (mean ± S.E.M., n = 3). (d) MS spectra from P14-∆553 treatment were integrated as in (c). The bar graphs depict the fraction of the total peptide that was modified (mean ± S.E.M., n = 3). * represents p-value < 0.05 and ** represents p-value < 0.01, two-tailed Student’s t test, n.s. represents a non-significant difference.
Figure 2 Identification of a minimal P14 selective peptide sequence. (a) Peptide truncations used in this study (full sequences in Table S1). The E acceptor is indicated. (b) P14c and the indicated peptide were incubated in the presence of NAD+ and subjected to TLC-MALDI to visualize the resulting increase in m/z due to MARylation (+541 Da). The dashed line represents the intensity observed for ADP-ribosylation of P14p1. (c) MS spectra for P14c treatment were integrated to determine the relative levels of ADP-ribosylation. The bar graphs depict the fraction of the total peptide that was modified (mean ± S.E.M., n = 3). (d) MS spectra from P14-∆553 treatment were integrated as in (c). The bar graphs depict the fraction of the total peptide that was modified (mean ± S.E.M., n = 3). * represents p-value < 0.05 and ** represents p-value < 0.01, two-tailed Student’s t test, n.s. represents a non-significant difference.CC BY · PubMed Central
Figure 3 P14 preferentially MARylates a basic–K/R–D/E proximal sequence. (a) Alanine (A) substituted peptides used in this study. (b) P14c and the indicated peptide were incubated in the presence of NAD+ and subjected to TLC-MALDI to visualize the resulting increase in m/z due to MARylation (+541 Da). The dashed line represents the intensity observed for ADP-ribosylation of P14p6 and the inset highlights the +MAR spectra. (c) MS spectra were integrated to determine the relative levels of ADP-ribosylation. The bar graphs depict the fraction of the total peptide that was modified (mean ± S.E.M., n = 3). * represents p-value < 0.05 and ** represents p-value < 0.01, two-tailed Student’s t test, n.s. represents a non-significant difference. (d) Methionine (M) and lysine (K) substituted peptides used in this study. (e) P14p13 and P14p14 MS spectra normalized as in (b). (f) Relative levels of P14p13 and P14 p12 MARylation analyzed as in (c). (g) Shuffled peptides with the R-E signature slid towards the C-terminus. (h) P14p15 and P14p16 MS spectra normalized as in (b). (i) Relative levels of P14p15 and P14p16 MARylation analyzed as in (c).
Figure 3 P14 preferentially MARylates a basic–K/R–D/E proximal sequence. (a) Alanine (A) substituted peptides used in this study. (b) P14c and the indicated peptide were incubated in the presence of NAD+ and subjected to TLC-MALDI to visualize the resulting increase in m/z due to MARylation (+541 Da). The dashed line represents the intensity observed for ADP-ribosylation of P14p6 and the inset highlights the +MAR spectra. (c) MS spectra were integrated to determine the relative levels of ADP-ribosylation. The bar graphs depict the fraction of the total peptide that was modified (mean ± S.E.M., n = 3). * represents p-value < 0.05 and ** represents p-value < 0.01, two-tailed Student’s t test, n.s. represents a non-significant difference. (d) Methionine (M) and lysine (K) substituted peptides used in this study. (e) P14p13 and P14p14 MS spectra normalized as in (b). (f) Relative levels of P14p13 and P14 p12 MARylation analyzed as in (c). (g) Shuffled peptides with the R-E signature slid towards the C-terminus. (h) P14p15 and P14p16 MS spectra normalized as in (b). (i) Relative levels of P14p15 and P14p16 MARylation analyzed as in (c).CC BY · PubMed Central
Figure 4 The MAR–peptide bond is hydrolyzed under mild conditions. (a) A synthesized MAR–P14p6 peptide was equilibrated at 37 °C, and pH 6.0 (blue), pH 7.0 (purple), or pH 8.0 (green) and MAR hydrolysis was monitored at the indicated times using TLC-MALDI. The unmodified and modified peaks are shown for comparison. (b) MS spectra were integrated to determine the relative levels of MAR hydrolysis and fit to a pseudo first-order rate expression to determine the half-life of the modification (mean ± S.E.M., n = 3). (c) Incubation at room temperature stabilized the MAR–peptide bond. Experiments were performed as in (a) at 25 °C. (d) Determination of MARylation half-lives at 25 °C. (e) Incubation on ice effectively halts MAR hydrolysis in mild acid. Experiments were performed as in (a) at 4 °C. (f) Determination of MARylation half-lives at 4 °C.
Figure 4 The MAR–peptide bond is hydrolyzed under mild conditions. (a) A synthesized MAR–P14p6 peptide was equilibrated at 37 °C, and pH 6.0 (blue), pH 7.0 (purple), or pH 8.0 (green) and MAR hydrolysis was monitored at the indicated times using TLC-MALDI. The unmodified and modified peaks are shown for comparison. (b) MS spectra were integrated to determine the relative levels of MAR hydrolysis and fit to a pseudo first-order rate expression to determine the half-life of the modification (mean ± S.E.M., n = 3). (c) Incubation at room temperature stabilized the MAR–peptide bond. Experiments were performed as in (a) at 25 °C. (d) Determination of MARylation half-lives at 25 °C. (e) Incubation on ice effectively halts MAR hydrolysis in mild acid. Experiments were performed as in (a) at 4 °C. (f) Determination of MARylation half-lives at 4 °C.CC BY · PubMed Central
Figure 5 ADPr glycohydrolases display differential preferences for P14 selective sequence motifs. (a) Synthesized MAR–P14p6 or MAR–P14p9 peptides (dashed lines) were incubated in the presence of either macroD1 (mD1), macroD2 (mD2), or TARG1 and subjected to TLC-MALDI to determine the relative levels of hydrolysis. Non-enzymatic controls (solid line) were utilized to ascertain the levels of background hydrolysis. The unmodified and modified peaks are shown for comparison. (b) MS spectra were integrated to determine the relative levels of MAR hydrolysis. The bar graphs depict the amount of MAR removal (mean ± S.E.M., n = 3). ** represents p-value < 0.01, two-tailed Student’s t test, * represents a p-value < 0.05, and n.s. represents a non-significant difference.
Figure 5 ADPr glycohydrolases display differential preferences for P14 selective sequence motifs. (a) Synthesized MAR–P14p6 or MAR–P14p9 peptides (dashed lines) were incubated in the presence of either macroD1 (mD1), macroD2 (mD2), or TARG1 and subjected to TLC-MALDI to determine the relative levels of hydrolysis. Non-enzymatic controls (solid line) were utilized to ascertain the levels of background hydrolysis. The unmodified and modified peaks are shown for comparison. (b) MS spectra were integrated to determine the relative levels of MAR hydrolysis. The bar graphs depict the amount of MAR removal (mean ± S.E.M., n = 3). ** represents p-value < 0.01, two-tailed Student’s t test, * represents a p-value < 0.05, and n.s. represents a non-significant difference.CC BY · PubMed Central

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Metadata

Authors
Javed, Z., Nguyen, H. H., Harker, K. K., Mohr, C. M., Vano, P., Wallace, S. R., Silvers, C., Sim, C., Turumella, S., Flinn, A., Moritz, A., & Carter-O’Connell, I.
Affiliations
Santa Clara University (US)
Corresponding author
Carter-O’Connell, Ian
ORCID
Venue
Molecules
Publisher
MDPI AG
Published
2023-08-15
Volume
28
Issue
16
Pages
6061
ISSN
1420-3049 (ISSN-L)
DOI
10.3390/molecules28166061
PMC
PMC10459978
OpenAlex
W4385835449
Cited by
5
References
48
Citations by year
2026: 3 · 2025: 1 · 2024: 1
FWCI
0.61
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecules (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health (NIH 1R15GM139150)
  • Santa Clara University (NIH 1R15GM139150)
Topics
  • Health Sciences › Medicine › Oncology › PARP inhibition in cancer therapy
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
Keywords
PARP14, ADP-ribosyltransferase, ADP-ribose, NAD+, MALDI-TOF mass spectrometry, poly(ADP-ribosyl)ation

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CoChat. (2026). Citation check: Using TLC-MALDI-TOF to Interrogate In Vitro Peptidyl Proximal Preferences of PARP14 and Glycohydrolase Specificity. https://app.cochat.ai/papers/10.3390/molecules28166061
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Javed, Z., Nguyen, H. H., Harker, K. K., Mohr, C. M., Vano, P., Wallace, S. R., Silvers, C., Sim, C., Turumella, S., Flinn, A., Moritz, A., & Carter-O’Connell, I. (2023). Using TLC-MALDI-TOF to Interrogate In Vitro Peptidyl Proximal Preferences of PARP14 and Glycohydrolase Specificity. Molecules. https://doi.org/10.3390/molecules28166061

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