Measurement of German cockroach allergens and their isoforms in allergen extracts with mass spectrometry

Abstract

Background: Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non-standardized.More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency.Objective: To expand the multiple reaction monitoring (MRM) assay for an in-depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts. Methods:We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM-MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform-specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables.

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

“…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. As a first step, the authors used an in silico prediction together with high-resolution MS for peptide mapping and for the selection of the best representative peptides to serve as standards in the quantification analysis.…”

“…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. As a first step, the authors used an in silico prediction together with high-resolution MS for peptide mapping and for the selection of the best representative peptides to serve as standards in the quantification analysis.…”

“…For the selection of peptide standards, untargeted analysis with high-resolution MS instruments is carried out to identify peptides fulfilling a number a criteria, including sequence-based features (e.g. not prone to missed proteolytic cleavages and modifications; precursor ion’s charge, preferably doubly charged), and specificity, including the issue of multiple protein isoforms ( 93 ).…”

“…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 – 101 ) reviewed in ( 91 , 92 ). The broad applicability of MS-based MRM was further demonstrated by Mindaye et al.…”

Developments in the field of allergy in 2020 through the eyes of Clinical and Experimental Allergy

“…Clinical and Experimental Allergy continues to report important reviews or new findings related to less common forms of allergy. Allergy to horsefly and deerfly was reviewed, 111 and the use of mass spectrometry for measuring cockroach allergens was investigated 112 . Important advances in the investigation of tropomyosin allergenicity were reported 113 and the development of immunotherapy for Blomia tropicalis 114 …”