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.

*** IBB - CNR ***

Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche (IBB-CNR), Napoli, Italy;

Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), Napoli, Italy;

Bioker Multimedica, Napoli, Italy;

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