Allergen content in mouse allergenic samples and products is more complex than a single Mus m 1 value

Abstract

Mus m 1 is a group of >21 polymorphic major urinary proteins (MUPs) and the specificity of available Mus m 1 immunoassays to any of these MUPs is unknown. In order to improve the diagnostic and therapeutic potency of commercial mouse allergen extracts, we need improved methods to characterize mouse allergens and the immune responses to them. We applied the following analytical methods to commercial extracts, urine from laboratory and wild mice, and environmental samples from homes of Baltimore asthma patients: isoelectric focusing, two-dimensional electrophoresis (2D-PAGE), liquid chromatography, immunostaining, mass spectrometry, and multiple reactions monitoring (MRM). By 2D-PAGE with immunostaining, MUPs are the major IgE-binding proteins (90-95%) in urine, but albumin is more abundant in commercial extracts. MUPs have complex migration patterns on 2D-PAGE, attributable to sequence polymorphisms and posttranslational modifications. For example, we identified at least 14 distinct MUP3-variants with variable glycosylation states. Using MRM, we determined that MUPs with >97% sequence similarity constitute 85-90% of the total MUPs in both commercial extracts and urine. Environmental dust samples, from bed, bedroom, and kitchen, contained MUP profiles consistent with those found in commercial extracts. Commercial extracts are highly variable, with up to 30-fold differences in specific MUP content. Using a full array of proteomic and mass spectrometric tools, we can assess the allergen content of commercial mouse allergen extracts and compare it to allergens in mouse urine and environmental dust samples. These tools will help guide further research, characterization, and standardization of mouse allergenic products.