Radical Footprinting in Mammalian Whole Blood

Abstract

Hydroxyl Radical Protein Footprinting (HRPF) is a powerful tool to probe protein higher-order structure, as well as protein-protein and protein-carbohydrate interactions. It is mostly performed in vitro, but recent advances have extended its use to live cells, nematodes, and 3D cultures. However, application in living mammalian tissues has not been accomplished. Here, we present the first successful use of radical protein footprinting (RPF) in mammalian whole blood from wild-type (WT) and type 2 diabetes mellitus (T2DM) BKS. Cg Dock7 ^m +/+ Lepr ^db /J mice. Using persulfate photoactivated with the FOX Photolysis System, we achieved effective protein labeling without significant disruption to blood cell morphology. An optimized quenching protocol eliminated background labeling. We report oxidative modifications in 11 selected proteins, revealing disease-associated conformational changes in multiple proteins. These findings demonstrate the feasibility of RPF in mammalian blood and open new opportunities for structural proteomics in preclinical models and clinical samples.

Keywords: Fast photochemical oxidation of proteins; Protein Structural Biology; Radical Protein Footprinting; Type 2 Diabetes Mellitus.

Figure 1.

Experimental workflow of in-blood RPF. The figure was created with BioRender.com.

Figure 2.. Test of inhibitors of catalase.

(A) Foam height after 30 minute incubation (5:1 v/v blood to inhibitor) at room temperature. TSC is insoluble at 200 mM. Error bars represent one standard deviation of triplicate measurement. (B) Representative samples of blood spiked with varying final concentrations of hydroxylamine: 0 mM (green A-B), 5 mM (orange C-D), 25 mM (blue E-F), 100 mM (orange G-H), and 142 mM (blue J-K). (C) Blood spiked with equal volumes of 30% H₂O₂ (blue) or 2.5 M sodium persulfate (red).

Figure 3.. Adenine dosimetry response to SO₄⁻•.

(A) Adenine absorbance after illumination using different lamp voltages, (B) change in absorbance for different persulfate concentrations at 900 V versus 0 V, and (C) adenine absorbance at 900V in the presence of different concentrations of sucrose radical scavenger. Error bars represent one standard deviation of a triplicate measure.

Figure 4.. Selection of effective quench solutions for background oxidation.

(A) Evaluation of Quench I (without imidazole); (B) Evaluation of Quench II (with imidazole). Statistical significance was calculated by a two-tailed unpaired t-test (α = 0.05) versus the 0 mM persulfate control under the same conditions; peptides showing a significant difference in oxidation are marked with an asterisk. Myoglobin peptides are denoted by their position. The myoglobin peptide corresponding to the 2–17 residues displays significant in-source oxidation. Error bars represent one standard deviation.

Figure 5.. Effect of sodium persulfate on blood cell morphology.

(A) Blood cells from canine blood. (B) Same blood with 200 mM sodium persulfate. Cells show a moderate increase in hypertonicity, evidenced by an increase in wrinkled cells. (C) An equal concentration of sodium chloride results in cells with an identical morphology to the persulfate sample

Figure 6.. Comparison of in-blood RPF of top 6 intracellular proteins detected in WT and T2DM whole blood.

Significantly higher modification was observed in extracellular proteins of WT (A) and T2DM (B) compared to intra-cellular proteins, Each data point represents a peptide.The modification extent at 0V was subtracted for each peptide; (C-H) Peptide-level oxidation of peptides from six intracellular proteins, after subtraction of 0V background oxidation values: (C) alpha-globin; (D) beta-globin; (E) peroxiredoxin-2; (F) Flavin reductase; (G) 60S ribosomal protein-L40; (H) Serine protease inhibitor. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired T-test after Šídák correction.

Figure 7.. Comparison of the in-blood RPF of top extracellular proteins detected in WT and T2DM whole blood.

(A) Transthyretin; (B) Apolipoprotein A-I; (C) Albumin; (D) Serotransferrin; (E) Complement C3; (F) Volcano plot of disease-associated conformational changes in T2DM, pink dots: peptides from complement c3, green dots: peptides from serotransferrin, (G) Topographical changes of protein serotransferrin in T2DM: peptide color represents fold change in oxidation, colors coded to match with (F), gray peptides showed no measurable oxidation; (H) Topographical changes of protein complement c3 in T2DM: peptide color represents fold change in oxidation, colors coded to match with (F), gray peptides showed no measurable oxidation. The modification extent at 0V was subtracted for each peptide. All statistics performed were unpaired two-tailed t-test with Šídák correction; * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired t-test.

Figure 8.. Comparison of in vitro and in-blood SO₄⁻• RPF of protein complement c3.

Error bars represent one standard deviation.