Design and Synthesis of Photoaffinity Probe for Identification of Protein Targets of Glycosaminoglycan Mimetics

Abstract

Glycosaminoglycans (GAGs) are complex biomacromolecules ubiquitously distributed in the animal kingdom where they play major roles in the large number of physiological and pathological processes. Identification of protein targets of GAGs and GAG mimetics has been a major challenge because of the size of the library of GAG‐binding proteins. We reasoned that photoaffinity probes of bioactive GAG mimetics would serve as a useful method for identification of protein targets. This work presents synthesis and mass spectrometric characterization of novel photoactivatable analogs of a lead anti‐cancer agent for identification of its plausible protein targets. Photoactivatable analog of the lead GAG mimetic G2.2 containing diazirine and alkyne groups G2PA was synthesized through a series of 10 steps. Structural characterization was performed using 1H/13C NMR and HRMS analysis. A test case of photoactivation was performed by first incubating insulin‐like growth factor‐1 with mimetic G2PA at 4 °C and then photo‐irradiated for 5 min at 368 nm. The sample was reduced using DTT, alkylated with iodoacetamide in dark, digested with trypsin and cleaned up using C18 StageTip. Proteomics analysis of the photoactivated IGF‐1–G2PA complex was performed on nLC‐Q Exactive (Thermo) in a top 12 data dependent acquisition. Proteomic analysis of the IGF‐1–G2PA complex gave a mass peak at 2688 m/z corresponding to the peptide APQTGIVDEccFR covalently linked to the photoactivated G2PA. MS/MS analysis of this fragment showed peaks 1) at 2391 m/z corresponding to TGIVDEccFR+G2PA; 2) 1098 m/z corresponding IVDEccFR; and 3) 297 m/z corresponding APQ. Proteomic analysis indicated G2PA covalently modified the thyronine residue present in APQTGIVDEccFR sequence of IGF‐1. Thus, the diazirine‐based photo‐activation approach for the test case of IGF‐1 identified the site of covalent conjugation and validated the approach. This approach, in principle, could be more generally used to identify protein targets of GAGs and GAG mimetics.Support or Funding InformationThis work is supported by HL107152, HL090586 from National Heart, Lung and Blood Institute to URDThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.