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Immunoenrichment microwave and magnetic proteomics for quantifying CD47 in the experimental autoimmune encephalomyelitis model of multiple sclerosis

Mahesula, S;Raphael, I;Raghunathan, R;Kalsaria, K;Kotagiri, V;Purkar, AB;Anjanappa, M;Shah, D;Pericherla, V;Jadhav, YL;Gelfond, JA;Forsthuber, TG;Haskins, WE;

We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating immunoenrichment prior to rapid microwave and magnetic (IM(2) ) sample preparation, might enable correlation of the relative expression of CD47 and other low abundance proteins to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, anti-CD47 antibodies were used to enrich for low abundance CD47 prior to microwave and magnetic proteomics in EAE. Decoding protein expression at each time point, with CD47-immunoenriched samples and targeted proteomic analysis, enabled peptides from the low abundance proteins to be precisely quantified throughout disease progression, including: CD47: 86-99, corresponding to the marker of self overexpressed by myelin that prevents phagocytosis, or cellular devouring, by microglia and macrophages; myelin basic protein: 223-228, corresponding to myelin basic protein; and migration inhibitory factor: 79-87, corresponding to a proinflammatory cytokine that inhibits macrophage migration. While validation in a larger cohort is underway, we conclude that IM(2) proteomics is a rapid method to precisely quantify peptides from CD47 and other low abundance proteins throughout disease progression in EAE. This is likely due to improvements in selectivity and sensitivity, necessary to partially overcome masking of low abundance proteins by high abundance proteins and improve dynamic range.