Related Experiment Video
Updated: May 16, 2026
Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis
Published on: December 1, 2023
Immunoenrichment microwave and magnetic proteomics for quantifying CD47 in the experimental autoimmune
Swetha Mahesula1, Itay Raphael, Rekha Raghunathan
1Pediatric Biochemistry Laboratory, University of Texas at San Antonio, San Antonio, TX, USA.
Abstract:
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.
Insights
Quantitative proteomics using IM(2) sample preparation precisely quantified CD47 and other low-abundance proteins during multiple sclerosis progression in EAE. This method aids in understanding disease mechanisms by tracking key protein changes.
Area of Science:
- Proteomics
- Immunology
- Neuroscience
Background:
- Multiple sclerosis (MS) is a neuroinflammatory disease.
- Understanding protein expression changes during disease progression is crucial.
- Low-abundance proteins are challenging to quantify in complex biological samples.
Purpose of the Study:
- To correlate the relative expression of CD47 and other low-abundance proteins with disease progression in the experimental autoimmune encephalomyelitis (EAE) model of MS.
- To evaluate the utility of quantitative MS/MS-based proteomics with immunoenrichment and rapid microwave and magnetic (IM(2)) sample preparation for this purpose.
Main Methods:
- Utilized anti-CD47 antibodies for immunoenrichment of CD47.
- Employed rapid microwave and magnetic (IM(2)) sample preparation for proteomics.
- Performed quantitative MS/MS-based proteomics at multiple time points in the EAE model.
- Targeted proteomic analysis to quantify specific peptides from low-abundance proteins.
Main Results:
- Precisely quantified peptides from CD47 (marker of self), myelin basic protein, and migration inhibitory factor throughout EAE disease progression.
- Demonstrated overexpression of CD47 on myelin, potentially preventing phagocytosis by microglia and macrophages.
- Identified migration inhibitory factor as a proinflammatory cytokine inhibiting macrophage migration.
Conclusions:
- IM(2) proteomics is a rapid and effective method for precisely quantifying low-abundance proteins, including CD47, during EAE progression.
- The method's sensitivity and selectivity help overcome the masking effect of high-abundance proteins.
- This approach holds promise for advancing the understanding of MS pathogenesis and identifying potential therapeutic targets.
