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Keywords:

  • Affinity chromatography;
  • Chemical proteomics;
  • Label-free quantification;
  • Nucleotides;
  • Platelets

Abstract

Chemical proteomics or activity based proteomics is a functional proteomics technology where molecular probes are used to target a selective group of functionally related proteins. Its emergence has enabled specific targeting of subproteomes, overcoming the limitations in dynamic range of traditional large-scale proteomics experiments. Using a chemical proteomics strategy, we attempt to differentially profile the nucleotide-binding proteome of active and resting platelets. We apply an affinity chromatography protocol using immobilized adenosine triphosphate, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. The specificity of the immobilized nucleotides was demonstrated by competitive assays and by immunoblotting. LC coupled MS/MS was applied to identify the proteins recovered by our chemical proteomics strategy. When compared to a standard set of platelet lysate proteins, we confirmed that enrichment for nucleotide-binding proteins was indeed taking place. Finally, by employing label-free MS-based comparative quantification, we found a small number of platelet proteins that show statistically significant difference between the active and resting nucleotide-binding proteome.