Utilizing AAC04-Beads to enrich acetylated peptides for detection via mass spectrometry analysis

Is dysfunctional acetylation present in metabolic disease?

Lysine acetylation of histone and non-histone proteins is a critical post-translational modification (PTM) that can regulate many cellular functions in health and disease¹. Dysfunctional acetylation contributes to many diseases, including neurological, metabolic, cancer, obesity, mitochondrial, and cardiovascular diseases¹.

Many of these diseases are related to altered metabolic states, and emerging evidence shows a strong relationship between acetylation and metabolic sensing². This may not be surprising as there is a strong interdependence between lysine acetylation and metabolites/co-factors like Acetyl-CoA, nicotinamide, zinc, and others³. For example, deciphering how dysfunction of acetylation can impact Acetyl-CoA, which is important in energetics and biosynthesis processes, may be important to gain a comprehensive understanding of how acetylation affects metabolic processing⁴.

Importantly, Acetyl-CoA can produce at least eight other acylation PTMs that also bind lysine residues to modify proteins, such as lactylation, succinylation, and glutarylation⁵. Because of the overlap of these acylation modifications in substrate, targets, and function⁶, it is important to understand the interplay between these PTMs and how dysregulation of one acylation may affect acetylation both physiologically and pathologically.

What approaches can we use to effectively study both target protein acetylation and global acetylome changes in response to metabolic stressors?

Our understanding of the total number of proteins that are acetylated has grown tremendously due to global proteomics analyses. Complementarily, in-depth functional studies examining target protein acetylation in specific scenarios has helped us pinpoint critical acetylation-dependent mechanisms in an array of diseases.

Early acetylation studies relied on tagged-overexpression or mutagenesis approaches to characterize target-protein acetylation. Enhanced tools such as the Signal-Seeker Acetyl-Lysine Detection Kit (Cat. # BK163) support these types of studies while adding a critical piece of information by capturing the global endogenous acetylated proteins in an experimental model and allowing investigators to determine if their target protein is acetylated under physiological or treatment conditions.

In fact, recent metabolic studies looking at target protein acetylation utilized these Acetyl-Lysine Enrichment Beads (AAC04-Beads) to effectively capture changes in the acetylation state of their target proteins. These include detecting target protein acetylation in the following examples: effects on lipogenesis under nutrient stress⁷, adipose plasticity and metabolic rhythms⁸, and tumor progression via metabolic and epigenetic modulation⁹.

Two recent studies, however, utilized the AAC04-Beads in a different, but useful way, which was to combine the bead’s ability to capture a global acetylome profile with the detection ability of mass spectrometry. In the first example, the group identified a sphingosine kinase-2/(sphK2)/acetyl-CoA carboxylase-1/phospholipid axis that functions as a metabolic checkpoint to regulate MDSC immunogenicity in cancer¹⁰. As part of their study, they sought to determine how the knockout of sphK2 altered global acetylation, which they determined with AAC04-beads-enriched acetylome mass spectrometry studies.

In the second study, another group sought to understand whether the deacetylase, SIRT2, regulated chaperone-mediated autophagy (CMA)¹¹. HSC70, whose acetylation profile was altered by SIRT2, was identified as a key regulator of CMA. The group expressed HA-tagged HSC70 in SIRT2 knockdown cells then isolated the HA-HSC70 protein, digested it into peptides, and captured the acetylated peptides with AAC04-Beads to determine which lysine residue was deacetylated by SIRT2 (see figure 1).

These peptides were then subjected to mass spectrometry and K557 was identified as the key lysine residue¹¹. Importantly, this is the first report highlighting the ability of this acetyl-lysine affinity bead to capture acetylated peptides.

References

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2. Eftekhari, A., U. Sabir, and T. Kasumov, The role of lysine acetylation in metabolic sensing and proteostasis. Pharmacol Ther, 2025. 274: p. 108908.

3. Menzies, K.J., et al., Protein acetylation in metabolism - metabolites and cofactors. Nat Rev Endocrinol, 2016. 12(1): p. 43-60.

4. Wang, Y., et al., The multiple facets of acetyl-CoA metabolism: Energetics, biosynthesis, regulation, acylation and inborn errors. Mol Genet Metab, 2023. 138(1): p. 106966.

5. Shang, S., J. Liu, and F. Hua, Protein acylation: mechanisms, biological functions and therapeutic targets. Signal Transduct Target Ther, 2022. 7(1): p. 396.

6. Jiang, W., et al., Lactylation and acetylation: parallel paths, divergent deeds, and research dilemmas. J Transl Med, 2026. 24(1).

7. Karim, R., et al., SIRT2-mediated ACSS2 K271 deacetylation suppresses lipogenesis under nutrient stress. Elife, 2025. 13.

8. He, Y., et al., PPARgamma Acetylation Orchestrates Adipose Plasticity and Metabolic Rhythms. Adv Sci (Weinh), 2023. 10(2): p. e2204190.

9. Williams, D., et al., Type II Interleukin-4 Receptor Activation in Basal Breast Cancer Cells Promotes Tumor Progression via Metabolic and Epigenetic Modulation. Int J Mol Sci, 2024. 25(9).

10. Chakraborty, P., et al., Sphingosine kinase-2 inhibition promotes immunogenic differentiation of myeloid-derived suppressor cells through an Acetyl-CoA carboxylase-phosphatidylcholine axis. Nat Commun, 2026. 17(1).

11. Ahn, B., et al., Deacetylation of HSC70 by SIRT2 promotes chaperone mediated autophagy. Autophagy Rep, 2025. 4(1): p. 2580781.