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.

Evaluation of combined use of Oxyma and HATU in aggregating peptide sequences

“…For Aib-Enkephalin, we observed an increase from 51% to about 74% and a remarkable increase of yield in the Aib-Aib coupling. Also in the Aβ [26][27][28][29][30][31][32][33][34][35] , which are both predicted to be hot spots of aggregation-prone regions. These sequences contain residues found missing in the most abundant deletion side products (see mass spectra of Figures 4A and 5A and Table S1) that are then mostly suppressed in the synthesis performed under the conditions of protocol II (see mass spectra of Figures 4B and 5B and Table S1).…”

“…As a fragment of the prototypical aggregating polypeptide Aβ , Aβ [26][27][28][29][30][31][32][33][34][35] has also strongly aggregating properties [18,31,32]. The AGGRESCAN prediction for this peptide (Figure S2, dotted line) suggested that region 29 AIIGL 33 exhibits a very high aggregation tendency.…”

“…Relevant impurities were seen at 10.32 and 10.93 min. The product at 10.32 min was identified as des(K;I/L)-Aβ [26][27][28][29][30][31][32][33][34][35] deleted peptide with a molecular mass at m/z: 803.45. Moreover, a des(I/L)-Aβ [26][27][28][29][30][31][32][33][34][35] by-product was coeluted with the correct peptide, but this was generated under both the conditions of protocol I and protocol II, as shown in Figure 3, right panels.…”

“…The product at t R : 10.93 min remained unidentified. The global purity of Aβ [26][27][28][29][30][31][32][33][34][35] obtained with protocol I was estimated to be 47% while the one obtained with protocol II was 90%.…”

“…We have observed that double sequential couplings with Oxyma/DIC and HATU/Sym-collidine lead to largely improved yields in some difficult synthetic steps, thus affording final products that can be more easily isolated and characterized. On this basis, we report here a comparative study of the use of sequential double couplings with HATU/Sym-collidine or HATU/Symcollidine combined to Oxyma/DIC to improve the efficiency in the synthesis of some bioactive polypeptides chosen from those used in our laboratories in ongoing projects and for which we have experienced synthesis problems (Aβ [26][27][28][29][30][31][32][33][34][35] [18][19][20][21][22] or selected as models of difficult sequences (α-Syn[68-78], Aib-Enkephalin) [23,15].…”