Figure 2. AutoSTOMP selectively enriches macrophage-associated proteins in the CD68⁺ regions of rat cardiac infarcts.

CD68⁺ regions of the cardiac infarcts are biotinylated by AutoSTOMP as described in Figure 1. A, After AutoSTOMP crosslinking, coverslips were washed of unconjugated Biotin-BP and lysed (1). Biotinylated proteins (‘CD68’ fraction) were bound to streptavidin beads (2) and pelleted (3). Unbound proteins were reserved as a ‘flow through’ control (3). Both fractions are trypsin/LysC digested and analyzed by LC-MS(4). B-D Protein abundance was determined by MaxQuant label free quantification (MaxLFQ). B, Of the 1,671 rat proteins identified, 94.2% were observed with one or more valid readouts in each of the three ‘CD68’ and/or ‘flow through’ fraction replicates. Heat map represents z-score for each protein across all the samples. Hierarchical clustering (left) indicates three enrichment patterns. C, T-distributed stochastic neighborhood embedding (t-SNE) analysis of variation among the ‘CD68’ fractions (cd1, cd2, cd3) and ‘flow through’ fractions (ft1, ft2, ft3) belonging to 3 paired replicates. D, Of the 1,671 proteins identified, 28.2% of were significantly enriched and 33.7% were significantly lower in the ‘CD68’ fractions relative to the ‘flow through’ (red circles p < 0.05). Plotted as −log₁₀ p-value (y axis) versus the log₂ fold-change (x axis) of the protein abundance averaged between replicates with a false discovery of < 0.1. Some significantly expressed proteins annotated are in blue dots.