RESUMO
ADP-ribosylation (ADPr) signaling plays a crucial role in DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage, poly(ADP-ribose) polymerase 1 (PARP1), are used to treat patients with breast cancer harboring BRCA1/2 mutations. However, resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To understand the role of ADPr in PARPi sensitivity, we use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze ADPr in six breast cancer cell lines exhibiting different PARPi sensitivities. We identify 1,632 sites on 777 proteins across all cell lines, primarily on serine residues, with site-specific overlap of targeted residues across DNA-damage-related proteins across all cell lines, demonstrating high conservation of serine ADPr-signaling networks upon DNA damage. Furthermore, we observe site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants and unique ADPr sites in PARPi-resistant BRCA-mutant HCC1937 cells, which have low poly(ADP-ribose) glycohydrolase (PARG) levels and longer ADPr chains on PARP1.
Assuntos
ADP-Ribosilação , Proteína BRCA1 , Neoplasias da Mama , Dano ao DNA , Serina , Humanos , Feminino , Linhagem Celular Tumoral , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Serina/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.
Assuntos
Adenosina Difosfato Ribose , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Adenosina Difosfato Ribose/química , ADP-Ribosilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Peptídeos/químicaRESUMO
ADP-ribosylation is a post-translational modification of proteins that has required the development of specific technical approaches for the full definition of its physiological roles and regulation. The identification of the enzymes and specific substrates of this reaction is an instrumental step toward these aims. Here we describe a method for the separation of ADP-ribosylated proteins based on the use of the ADP-ribose-binding macro domain of the thermophilic protein Af1521, coupled to mass spectrometry analysis for protein identification. This method foresees the coupling of the macro domain to resin, an affinity-based pull-down assay, coupled to a specificity step resulting from the clearing of cell lysates with a mutated macro domain unable to bind ADP-ribose. By this method both mono- and poly-ADP-ribosylated proteins have been identified.
Assuntos
Adenosina Difosfato Ribose/genética , Técnicas In Vitro/métodos , Proteínas/genética , Proteômica/métodos , ADP Ribose Transferases/genética , ADP-Ribosilação , Humanos , Processamento de Proteína Pós-Traducional/genética , Espectrometria de Massas em TandemRESUMO
ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography-high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.