Fig 1: Red blood cell (RBC) membrane proteomics is integrated into an in-silico model to predict surface crowding heterogeneity validated by theory and experiments.A Relevant data from RBC proteomics for predicting surface crowding variation with extracellular height. Four of the most abundant RBC proteins with extracellular domains35 are characterized according to copy number and extracellular domain size: Band 3, glycophorin A (GYPA), aquaporin 1 (AQP1), and CD4735–37, 101–104. Simplified schematics of transmembrane and extracellular domains are sketched in a lipid bilayer, with black peptide sequences, blue N-glycans, and magenta O-glycans. GYPA and Band 3 are the only proteins of sufficient extracellular size and density to be considered in our model. B MD simulations and analytical theory describe steric repulsion between an IgG antibody and a model RBC glycocalyx. (Left) Repulsive energy penalty ΔU from both analytical theory and MD simulation is plotted as a function of height. (Right) Snapshot of MD simulations with spherical IgG adsorbing amongst course-grained Band 3 and GYPA. C Molecular probes verify in-silico crowding heterogeneity predictions. (Upper left) Fluorescence micrographs of cholesterol-PEG-FITC and anti-FITC IgG binding to RBCs (scale bar 5 μm). (Lower left) Schematic of IgG binding to synthetic antigen sensors on a RBC surface, with GYPA and Band 3 dominating crowding. (Right) Mean crowding energy ΔU, normalized by the surface value on wild-type (WT) human RBCs \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\Delta }}{U}_{{{{{{{{\rm{0.5k}}}}}}}}}^{{{{{{{{\rm{WT}}}}}}}}}$$\end{document}ΔU0.5kWT is plotted against mean cholesterol-PEG-FITC sensor height 〈h〉 for WT RBCs (blue circles) and RBCs treated with neuraminidase (NA, green triangles). Analytical theory (black curve) and MD simulation (black crosses) predict weaker free energy penalties of crowding with increasing antigen sensor height on WT cells. Dashed lines connecting experimental data are guides to the eye. Height data (x-axis) is CSOP data presented in Fig. 1. Experimental data points are derived from binding isotherm curve fits, and vertical error bars are derived as described in Fig. 2C. Curve fits are generated from populations of n = 912, 905, 1062, and 635 beads, for KD,0 and populations of n = 6113, 14476, 5680, and 3335 RBCs for KD.