title: Monoclonal antibodies against pools of mono- and polyacetylated peptides selectively recognize acetylated lysines within the context of the original antigen.
abstract: Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24-39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.
authors: a Istituto di Biostrutture e Bioimmagini , Consiglio Nazionale delle Ricerche (IBB-CNR) , Napoli , Italy.
authors: Annamaria
authors: A
authors: Sandomenico
authors: a Istituto di Biostrutture e Bioimmagini , Consiglio Nazionale delle Ricerche (IBB-CNR) , Napoli , Italy.
authors: Annalia
authors: A
authors: Focà
authors: b Bioker Multimedica , Napoli , Italy.
authors: Luca
authors: L
authors: Sanguigno
authors: c Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB) , Napoli , Italy.
authors: Andrea
authors: A
authors: Caporale
authors: a Istituto di Biostrutture e Bioimmagini , Consiglio Nazionale delle Ricerche (IBB-CNR) , Napoli , Italy.
authors: Giuseppina
authors: G
authors: Focà
authors: a Istituto di Biostrutture e Bioimmagini , Consiglio Nazionale delle Ricerche (IBB-CNR) , Napoli , Italy.
authors: Angelica
authors: A
authors: Pignalosa
authors: b Bioker Multimedica , Napoli , Italy.
authors: Giusy
authors: G
authors: Corvino
authors: d University of Udine , Department of Medical and Biological Sciences , Udine , Italy.
authors: Angela
authors: A
authors: Caragnano
authors: d University of Udine , Department of Medical and Biological Sciences , Udine , Italy.
authors: Antonio Paolo
authors: AP
authors: Beltrami
authors: d University of Udine , Department of Medical and Biological Sciences , Udine , Italy.
authors: Giulia
authors: G
authors: Antoniali
authors: d University of Udine , Department of Medical and Biological Sciences , Udine , Italy.
authors: Gianluca
authors: G
authors: Tell
authors: e University of Napoli "Federico II," Department of Molecular Medicine and Medical Biotechnology , Napoli , Italy.
authors: Antonio
authors: A
authors: Leonardi
authors: a Istituto di Biostrutture e Bioimmagini , Consiglio Nazionale delle Ricerche (IBB-CNR) , Napoli , Italy.
authors: Menotti
authors: M
authors: Ruvo
mesh_terms: D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000276:immunology*; D011499:Protein Processing, Post-Translational / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008239:Lysine
keywords: APE1/Ref-1; Acetylation; acetylated-APE1; anti-acetyl-peptide monoclonal antibodies; peptide libraries