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.

Keywords: glycosaminoglycan-binding proteins; glycosaminoglycans; interactome; photoaffinity; proteomics.

FIGURE 1.

Structures of synthetic GAG mimetic G2.2 (A) and its photoaffinity probe G2P (B) containing diazirine and alkyne moieties (shown in red). (C) Molecular models of G2.2 (left) and G2.2 (blue) overlaid on HS06 (green) with sulfates shown in atom-type color. (D) Structure of HS06, which is functionally and structurally mimicked by G2.2.

FIGURE 2.

(A) Synthesis of G2P from quercetin (1) in eight steps. (B) Binding and photolabeling property of G2P against two test proteases, thrombin (FIIa) and factor IXa (FIXa), of the coagulation cascade. Increasing levels of G2P levels resulted in higher photoaffinity labeling (fluorescence gels) as a proportion of the total protein (silver stained gels), which can be analyzed as hyperbolas for affinity calculations (graphs).

FIGURE 3.

The 10-step strategy based on PAL-proteomics to identify the preferred targets of G2P, and thereby G2.2, in human plasma. Steps 1 – 4 involve PAL methods, steps 5 – 8 correspond to proteomics, and steps 9 & 10 involve validation of targets. This strategy rests on rigorous application of steps 7, 8 and 10, which focus on identifying the ‘preferred’ targets. The human plasma G2P targetome was reliably identified to contain 12 proteins (step 8) from >80 identified as plausible targets. See text for details.

FIGURE 4.

(A) Distribution of proteins identified as preferred targets of G2P using literature reports and Cardin-Weintraub (CW) consensus sequence analysis. See also Table S5 for details. (B) Binding affinities of G2.2 for two of the representative six protein targets identified as preferred ones through PAL-proteomics. His-tagged proteins, labeled with RED-tris-NTA dye, were used in microscale thermophoresis (MST)-based titrations to measure K_D of interaction. (C) Comparison MST-measured K_Ds for G2.2 (red) and G2P (cyan). Error bars show ±1 SE. See also Figures S5 and S6 for all profiles.