Diversity and complexity of mouse allergens in urine, house dust, and allergen extracts assessed with an immuno‐allergomic approach

Abstract

Background: Mouse allergy is an important cause of indoor asthma and allergic rhinoconjunctivitis. The major mouse allergen, Mus m 1, is a complex of homologous pheromone-binding lipocalins called major urinary proteins (MUPs). Methods:We analyzed the proteome of MUPs in mouse urine, commercial mouse epithelial extracts, and environmental samples using several approaches. These include as follows: two-dimensional electrophoresis and immunoblotting; liquid chromatography-high-resolution mass spectrometry (LC/HRMS); multiple reaction monitoring (MRM) mass spectrometry; and LC/HRMS analysis of glycans at the N-66 residue of MUP3.Results: Albumin is predominant in the extracts, while MUPs are predominant in urine. LC/HRMS of 4 mouse allergen extracts revealed surprising heterogeneity. Of 22 known mouse MUPs, only 6 (MUP3, MUP4, MUP5, MUP13, MUP20, and MUP21) could be identified with MRM using unique peptides. Assessment of MUP content in urine, extracts, and dust samples showed good correlation between MRM and other methods working with different detection principles. All 6 identifiable MUPs were found in electrophoretically separated urine bands, but only MUP3 and MUP20 were above LOQ in unseparated mouse urine, and only MUP3, MUP4, and MUP20 were found in mouse epithelial extracts. Glycan heterogeneity was noted among 4 individual inbred mice: of 13 glycan structures detected, 8 were unique to one mouse, and only 2 glycan modifications were present in all 4 mice. Conclusions:Using mass spectrometry and MRM, mouse allergen extracts and urine samples are shown to be complex and heterogeneous. The efficacy and safety of commercial mouse allergen extracts will be improved with better controls of allergen content.

The History, Present and Future of Allergen Standardization in the United States and Europe

“…The MRM approach has been successfully used for identification and quantification several allergens in extracts prepared from timothy grass pollen (94), cockroach (43,93), house dust mites (44), mouse urine (95), and to quantify milk, soy, peanut, fish, and egg allergens in several food products (96)(97)(98)(99)(100)(101) reviewed in (91,92). The broad applicability of MS-based MRM was further demonstrated by Mindaye et al in proof-of-concept studies (43,93), demonstrating the accurate quantification of German cockroach allergens in complex extracts.…”

Ex vivo assays show human gamma-delta T cells specific for common allergens are Th1-polarized in allergic donors

“…Different allergen extracts, owing to their complexity, 44 might contain different immunogenic compounds recognized by γδ T cells. Indeed, in the case of MO allergen extract, the γδ T cell responses are elicited by the low molecular weight fraction of the extract (i.e., <3 kDa), while the antigenic fraction in CR allergen extract is of high molecular weight (i.e., >3 kDa) ( Figures S4 A–S4C).…”

Component-Resolved Diagnosis Based on a Recombinant Variant of Mus m 1 Lipocalin Allergen

“…Biologic potency and standardization of allergen content are well-known critical issues [ 39 ]. In different mouse epithelial allergen extracts, total and isoform specific MUP-quantification turned out to be variable and low, while urine samples revealed a different composition in terms of isoforms and concentrations when compared with commercial epithelial extracts [ 40 , 41 ]. Because of all these discrepancies, the use of epithelial extracts in the diagnosis of mouse allergy has been a matter of debate.…”