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Accurate quantification of 5 German cockroach (GCr) allergens in complex extracts using multiple reaction monitoring mass spectrometry (MRM MS)
Author(s) -
Mindaye S. T.,
Spiric J.,
David N. A.,
Rabin R. L.,
Slater J. E.
Publication year - 2017
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/cea.12986
Subject(s) - chromatography , chemistry , allergen , selected reaction monitoring , mass spectrometry , peptide , multiplex , tandem mass spectrometry , allergy , medicine , biology , immunology , biochemistry , bioinformatics
Summary Background German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy. Objective and Methods The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC‐MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts. Results We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high‐resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD < 10%), linear over a wide range ( r > 0.99, 0.01‐1384 fmol/μL), and sensitive (LLOD and LLOQ <1 fmol/μL). Having established the parameters for LC‐MRM MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy. Conclusions and Clinical Relevance Our data demonstrate that the LC‐MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies.