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A site-specific analysis of the ADP-Ribosylome unveils Homogeneous Serine ADP-Ribosylation across wild-type and BRCA-mutant Breast Cancer cell lines

ABSTRACT: ADP-ribosylation (ADPr) signaling plays a crucial role in the DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage – PARP1 – are used as targeted therapies against breast cancers with BRCA1/2 mutations. However, development of resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To better understand the role of ADPr in PARPi sensitivity, we used Liquid Chromatography-Mass Spectrometry (LC-MS) for systems level analysis of the ADP-ribosylome in six breast cancer cell lines with different PARPi sensitivities. We identified 1632 sites on 777 proteins, primarily on serine residues, with a high site overlap of DNA damage-related proteins across all cell lines, demonstrating high conservation in ADPr signaling after DNA damage. We furthermore observed site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants, and unique ADPr sites in PARPi-resistant BRCA mutant cells, which we notably show to have low PARG levels and longer PARP1 ADPr chains.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Holda Anagho

LAB HEAD: Michael Lund Nielsen

PROVIDER: PXD047613 | Pride | 2024-08-02

REPOSITORIES: Pride

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			Action	DRS
	20220218_EXPL7_nLC6_HA_SA_HCC1428_r1_F1.raw	Raw
	20220218_EXPL7_nLC6_HA_SA_HCC1428_r1_F10.raw	Raw
	20220218_EXPL7_nLC6_HA_SA_HCC1428_r1_F11.raw	Raw
	20220218_EXPL7_nLC6_HA_SA_HCC1428_r1_F12.raw	Raw
	20220218_EXPL7_nLC6_HA_SA_HCC1428_r1_F2.raw	Raw

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A Novel Spectral Annotation Strategy Streamlines Reporting of mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-gamma

Project description:Mass spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4-hour exposure to systemic IFN-gamma. HCD collision energy experiments were first performed on the Obitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization.

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In-source CID improves HCD-dependent fragmentation of ADP-ribosyl peptides

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