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Probes for ATP binding proteins

Acyl-ATP probes for identification of ATP-binding proteins in native proteomes

The comprehensive identification of ATP-binding proteins, such as the important class of kinases, and the dynamic analysis of nucleotide-protein interactions at the proteomic scale are fundamental for better understanding of the regulatory mechanisms of nucleotide-binding proteins[1].

ATP binding activities as well as specific binding sites of proteins can be detected globally in any biological sample or tissue from any species by using Biotin/Desthiobiotin-conjugated acyl-ATP probes (AcATP)[2-6].

The method is based on the following steps:

  • AcATP binds to the ATP pocket of ATP binding proteins and places the acyl group in close proximity to conserved lysine residues.
  • Nucleophilic attack by the lysine amino group, resulting in a covalent attachment of the acyl reporter and concomitant release of ATP.
  • Enrichment of Biotin/Desthiobiotin-tagged-proteins on streptavidin-coated solid support.
  • Digestion with proteases, isolation/purification of labeled peptides and identification / quantification using LC-MS/MS.

Selected references:

[1] Manning et al. (2002) The protein kinase complement of the human genome. Science 298:1912.
[2] Villamor et al. (2013) Profiling protein kinases and other ATP binding proteins in Arabidopsis using Acyl-ATP probes. Molecular & Cellular Proteomics 12 (9):2481.
[3] Xiao et al. (2013) Proteome-wide discovery and characterizations of nucleotide-binding proteins with affinity-labeled chemical probes. Anal. Chem. 85 (6):3198.
[4] Okerberg et al. (2019) Chemoproteomics using nucleotide acyl phosphates reveals an ATP binding site at the dimer interface of procaspase-6. Biochemistry 58 (52):5320.
[5] Nordin et al. (2015) ATP acyl phosphate reactivity reveals native conformations of Hsp90 paralogs and inhibitor target engagement. Biochemistry 54 (19):3024.
[6] Adachi et al. (2014) Proteome-wide discovery of unknown ATP-binding proteins and kinase inhibitor target proteins using an ATP probe. J. Proteome Res. 13 (12):5461.