A Robust Proteomics-Based Method for Identifying Preferred Protein Targets of Synthetic Glycosaminoglycan Mimetics

ABSTRACT A robust technology is critically needed for identifying preferred protein targets of glycosaminoglycans (GAGs), and synthetic mimetics thereof, in biological milieu. We present a robust 10-step strategy for identification and validation of preferred protein targets of highly sulfated, synthetic, small, GAG-like molecules using diazirine-based photoaffinity labeling– proteomics approach. Our work reveals that optimally designed, homogeneous probes based on minimalistic photoactivation and affinity pulldown groups coupled with rigorous proteomics, biochemical and orthogonal validation steps offer excellent potential to identify preferred targets of GAG mimetics from the potentially numerous possible targets that cloud GAG interaction studies. Application of this 10-step strategy for a promising highly sulfated, small GAG mimetic led to identification of only a handful of preferred targets in human plasma. This new robust strategy will greatly aid drug discovery and development efforts involving GAG sequences, or sulfated small mimetics thereof, as leads. Competing Interest Statement The authors have declared no competing interest. Abbreviations - Bf - bound fraction - CW - Cardin-Weintraub motifs - GAGs - glycosaminoglycans - Hp - heparin - HS - heparan sulfate - HS06 - heparan sulfate hexasaccharide - NSGMs - non-saccharide glycosaminoglycan mimetics - PAL - photoaffinity labeling