Cell-Wide Survey of Amide-Bonded Lysine Modifications by Using Deacetylase CobB

Significance of the Topic

Lysine post-translational modifications such as acetylation, succinylation and crotonylation modulate protein function across chromatin remodeling, metabolism and transcriptional control. Comprehensive profiling of these PTMs in human cells is needed to understand their dynamic roles in health and disease.

Objectives and Study Overview

This study aimed to develop a general cell-wide survey method for amide-bonded lysine modifications in human tissue and cell proteomes using the Salmonella enterica deacetylase CobB. By exploiting CobB’s newly discovered nonspecific amidase activity, the protocol seeks to release diverse amide-linked lysine modifiers for subsequent chromatographic and mass spectrometric analysis.

Methodology and Instrumentation

The workflow entails proteome extraction from HCC tissues or HEK293T cells followed by subcellular fractionation. Denatured proteins undergo extensive acetone washes to remove non-covalent metabolites. Immobilized recombinant CobB cleaves ε-amide bonds at 37 °C, liberating modifiers into solution, which is then separated by centrifugation.
Released amide-linked metabolites are derivatized for LC/MS (butylation with HCl in butanol) and GC/MS (methoximation and silylation with MTBSTFA) and analyzed using an Agilent UPLC–MS/MS system with an ion-pairing HFBA gradient and an Agilent 7890B/5977B GC–MS. Data are processed by peak area comparison between CobB-treated and control samples followed by MS/MS library matching and validation against authentic standards.

Key Results and Discussion

The CobB-based protocol identified over 40 known and novel amide-bonded lysine modifications in the human proteome, including amino acid acylations. CobB exhibited robust cleavage of synthetic propionyl-, succinyl-, crotonyl- and acetyl-lysine peptides but did not hydrolyze peptide backbone bonds.
Quantitative comparison showed significant (>2-fold) increases in targeted modifier peaks upon CobB treatment. MS/MS spectra confirmed metabolite identities via NIST library matching and standard co-elution studies.

Benefits and Practical Applications

This method enables simultaneous discovery and quantification of multiple amide-based lysine modifications across cellular compartments without requiring PTM-specific antibodies or enrichment strategies. It provides a versatile tool for PTM profiling in basic research and QA/QC workflows in industrial proteomics.

Future Trends and Opportunities

The protocol could be extended to other covalent PTMs by identifying cognate cleavage enzymes, potentially enabling surveys of cysteine acylations, glycosylations or ubiquitin-like modifications. Integration with quantitative proteomics and targeted MS assays may further elucidate PTM dynamics in disease models.

Conclusion

A CobB-based amidase approach offers an accessible, generalizable platform for global analysis of amide-bonded lysine modifications in human cells, expanding the toolbox for PTM discovery and quantification.

References

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