New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1

Abstract

Patients who suffer from birch pollinosis often develop adverse reactions to the consumption of fresh apples due to the structural similarity of the allergens Bet v 1 and Mal d 1 from birch and apples, respectively. A different allergenic potential for Mal d 1 isoallergens is postulated, but approaches to quantify the Mal d 1 isoallergen-specific are missing. Therefore, a bottom-up proteomics approach was developed to quantify Mal d 1 by stable isotope dilution and microHPLC-QTOF analyses. Marker peptides for individual isoallergens (Mal d 1.01–Mal d 1.03 and Mal d 1.06), combinations thereof (Mal d 1.01 + 1.02, Mal d 1.02 + 1.06, and Mal d 1.04 + 1.05), and two global marker peptides, comprising Mal d 1.01 + 1.02 + 1.04 + 1.05 and Mal d 1.03 + 1.06 + 1.07 + 1.08 + 1.09, were identified. By the use of an extraction standard (r-Mal d 1_mut), an optimized protocol for extraction and tryptic digestion of apple proteins was developed, and the variety-specific extraction efficiency was monitored for the flesh and peel of apples. The Mal d 1 contents in flesh and peel of five commercial apple breeds and four apple varieties from orchard meadows were quantified isoallergen-specific.

Characterization and Quantification of Mal d 1 Isoallergen Profiles and Contents in Traditional and Commercial Apple Varieties by Mass Spectrometry

“…For the latter three, less information about the allergenic potential is available, except for Sonnenglanz, for which Nothegger et al determined a high allergenic potential by skin prick tests. 21 However, our data for Mal d 1 contents as well as the isoallergen profiles (Mal d 1.01−1.09) of the varieties investigated 19 did not allow us to differentiate between traditional and commercial varieties or to facilitate the prediction of the allergenic potential. Therefore, we extended the study, investigating 10 traditional and 10 commercial apple varieties harvested in 2019. respective ISTD* and fragmentation pattern (Table S2).…”

“…Data for 2020 were published previously. 19 in Baya Marisa and Jonagold, with 123 mg/kg DW and 109 mg/kg DW, respectively. Except for Bohnapfel and Goldparmane, where both isoallergens were detected in the peel only in traces, 1.03 and 1.06 comprised up to 10−25% of total Mal d 1 in the apples studied.…”

“…Our data for 20 different apple varieties showed noticeable differences from the data published in a previous study. 19 Significant differences between different years of harvest probably due to fruit maturity, climate, and storage until sample preparation have been reported 11,32 and were also obvious for the five traditional varieties from orchard meadows (Bohnapfel, Boskoop, Brettacher, Gewurzluiken, and Ontario) harvested in 2019 and 2020 (Figure 4). The quantified allergen content was higher in the peel than in the flesh, which agreed with other studies and supported the recommendation for patients to remove the peel before apple consumption.…”

“…In a previous study, we quantified traditional apple varieties from 2020. 19 Therefore, it is possible to compare the Mal d 1 contents and the isoallergen profiles between the years 2019 and 2020 for Boskoop, Brettacher, Gewurzluiken, Bohnapfel, and Ontario. Figure 4 provides information about the changes in total Mal d 1 contents and isoallergen profiles.…”

“…To obtain profounder knowledge on the isoallergen profile of Mal d 1 in apples, a method for an isoallergen-specific absolute quantification, based on isotope dilution analysis, was developed for freeze-dried apple samples using a mass spectrometric bottom-up approach. 19 In this preliminary study, we investigated five traditional apple varieties from orchard meadows (Bohnapfel, Brettacher, Boskoop, Gewurzluiken, Ontario; harvest year 2020), characterized by a high phenolic content and a proposed low allergenic potential; 13,20 Santana, a proposed hypoallergenic commercial breed, but often evaluated only as a medium allergenic; 11,21 and three new breeds (Natyra, Magic Star, Sonnenglanz; harvest year 2021). For the latter three, less information about the allergenic potential is available, except for Sonnenglanz, for which Nothegger et al determined a high allergenic potential by skin prick tests.…”