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.

Introduction

Although apples can significantly contribute to a healthy diet due to their bioactive ingredients,^1,2 50–70% of all birch pollen allergy sufferers in Central and Northern Europe develop a cross-allergy to apples (Malus domestica Borkh.) in the course of their lives.^3,4 Due to the structural homology between the allergens Bet v 1 in birch and Mal d 1 in apples, immunological crossreactivity is regularly observed between the two proteins.⁵⁻⁷ The 17.5 kDa allergen Mal d 1 is heat-labile and is degraded by proteases during gastric digestion.⁸ However, symptoms such as itching and burning of oral mucous membranes or tongue (oral allergy syndrome) are responsible that people suffering from apple allergy avoid the consumption of fresh apples.⁹

Variations in the amino acid sequence of allergens are well documented. Allergens with a sequence coverage of 67% and higher are called isoallergens. According to the WHO/IUIS allergen nomenclature, allergens are named after the abbreviation of their source name and an Arabic number.¹⁰ Isoallergens are designated by the first two digits after the decimal point. For Mal d 1, twelve different isoallergens are listed in Uniprot, with Mal d 1.01 to 1.06 being the most relevant. Allergens that match 90% in their amino acid sequences are called variants or isoforms and they are indicated by the last two digits in their names (e.g., Mal d 1.0101).

The allergenic potential, which has been investigated diagnostically at the effect level after oral provocation,¹¹⁻¹³ skin prick test/prick-to-prick test^3,11,14,15 or immunochemically with allergy sera,^14,16 differs significantly among various apple varieties.^11,13,17,18 However, these differences cannot be sufficiently correlated with variety- or cultivation-related differences in the Mal d 1 content quantified by gene expression,^{3,14,16,17,19,20} enzyme allergosorbent test (EAST),²¹ and enzyme-linked immunosorbent assay (ELISA).^{12,16,17,20−24} Furthermore, Zuidmeer et al. showed a marked impact of the immunoassay used and variations in sample preparation on the Mal d 1 content quantified.²¹ In an oral provocation study, published by Romer et al., a medium Mal d 1 content but a high allergenic potential was observed for Golden Delicious.¹² In contrast, varieties, containing much higher Mal d 1 contents, showed no allergenic potential. A further discrepancy was that the Mal d 1 content in Golden Delicious was two times higher in the following harvest year but the allergic potential was reduced.

Only limited information is available about differences in protein extraction among quantification methods used so far. Besides the main isoallergens 1.01 and 1.02, other isoallergens are known for which an altered allergenic potential is postulated.^3,16 For currently applied methods, it is unknown to what extent these individual isoallergens are covered.^12,24 Furthermore, to the best of our knowledge, no data are available about the variety-specific isoallergen profiles (1.01–1.09) in apples and how these differ among noncommercial and commercial cultivars.

Mass spectrometry-based bottom-up proteomics is a well-established approach to detect and quantify allergens in different food matrices.²⁵ Absolute quantification is usually performed by adding a known amount of an isotopically labeled standard peptide to the sample. This internal standard is similar in structure and physical–chemical properties to the peptide of interest (further information is provided in Supporting Information). For allergen research the advantage of this approach is that individual isoallergens, combinations thereof, and total allergen contents can be quantified in parallel, depending on the marker peptides identified. To correlate allergenicity with the Mal d 1 content or the isoallergen profile, a reliable Mal d 1 quantification method is required. Apples are a highly complex and polysaccharide-rich matrix, which is additionally characterized by changes during fruit ripening and storage.^2,26,27 Mass spectrometry allows the use of a recombinant protein (r-Mal d 1_mut), comparable in its extraction characteristics to the native Mal d 1, as an internal standard. This helps to monitor extraction efficiency and to correct variety-specific differences.

Therefore, we developed an isoallergen-specific quantification method for Mal d 1 based on mass spectrometry using a recombinantly expressed Mal d 1 mutant as an extraction standard. This method was applied to the flesh and peel of apple varieties containing high amounts of polyphenols (Ontario, Gewürzluiken, Brettacher, and Bohnapfel) grown primarily in orchard meadows. Furthermore, Boskoop, a variety commercially available in Southern Germany containing significant amounts of phenolics, Santana, a specific hypoallergenic cultivar, and new breeds like Sonnenglanz and Magic Star/Natyra were included in this study.

Materials and Methods

Chemicals and Materials

Based on the amino acid sequence of the Mal d 1.0101 isoform (UniProt P43211; https://www.uniprot.org/), a mutant (r-Mal d 1_mut, ESTD) with a terminal Histag and substitutions of valine at positions 24 and 68 into alanine and at position 38 and 42 vice versa (Figure 1) was recombinantly expressed and purified according to the procedure published previously.²⁸ A purity of >90% was estimated by SDS-PAGE (Figure S1) and the stock in 1 M urea buffer was stored at −20 °C until further use. Quantification differed markedly between UV spectroscopy and mass spectrometry. Information about ESTD quantification is available in the Supporting Information (Table S1).

Figure 1.

Overview of individual, combination, and global marker peptides used for quantification and their position in the Mal d 1 sequence. Substituted amino acids compared to the isoallergen 1.01 are colored in red. Marker peptides are highlighted with color, and for better readability, the red amino acids are bold. Cleavage sites of trypsin are marked by vertical lines.

Twelve isotopically labeled and absolute quantified peptides (ISTD*, SpikeTides TQL) were obtained from JPT Peptide Technologies GmbH (Berlin, Germany).²⁹ The carbons and nitrogen of the terminal lysine were substituted by ¹³C and ¹⁵N. Isotopic purity was specified as 97–99% and the purity of peptides was determined by HPLC-UV at 220 nm to 91–97%. The lyophilized peptides were mixed and aliquoted to 0.5 nmol of each peptide (absolute amount) per vial by JPT.

For extraction, urea buffer (6 M urea, 1 M thiourea, and 50 mM Tris-HCl, adjusted to pH 8.0 with NaOH) was used. A stock of 400 mM Tris buffer was adjusted with HCl to pH 7.8 and used to prepare digestion buffer (6 M urea in 100 mM Tris buffer), reduction reagent (200 mM 1,4-dithiothreitol in 100 mM Tris buffer), alkylation reagent (200 mM iodoacetamide in 100 mM Tris buffer) and tryptic solution (200 μg/mL in 100 mM Tris buffer). Strata-X 33 polymer reversed-phase solid-phase extraction cartridges (30 mg/1 mL) for sample clean-up were acquired from Phenomenex (Aschaffenburg, Germany).

All chemicals were of analytical grade. Except for mass spectrometry, here, MS-grade solvents and formic acid were used. Acetonitrile, acetone, and methanol were from Fisher Scientific (Loughborough, U.K.). Hydrochloric acid (HCl, 37%) and acetic acid were obtained from Grüssing (Filsum, Germany) and formic acid from Merck (Darmstadt, Germany). Urea, thiourea, Tris-HCl, 1,4-dithiothreitol, potassium chloride, potassium dihydrogen phosphate, calcium chloride, sodium azide (NaN₃, purity ≥99%), and sodium ethylenediaminetetraacetic acid dihydrate (EDTA, purity ≥99%) were bought from Carl Roth (Karlsruhe, Germany). Iodacetamide (purity ≥99%), ammonium bicarbonate, polyvinylpolypyrrolidone (PVPP), and sodium diethyldithiocarbamate trihydrate (DIECA) were from Sigma-Aldrich (Taufkirchen, Germany). Trypsin, sequencing grade, modified from porcine pancreas, was purchased from Serva (Heidelberg, Germany). Ultrapure water (ELGA PurLab flex, Veolia Waters, Celle, Germany) was used throughout the experiments.

Samples

Nine different apple varieties were either donated by A. Siegle from the Obstbauberatung Stuttgart (Liegenschaftsamt, Stuttgart, Germany; year 2020, Boskoop, Gewürzluiken, Ontario, Brettacher, Bohnapfel) or by D. Neuwald from the Competence Centre for Fruit-Growing Bodensee (Bavendorf, Germany; year 2021, Santana, Sonnenglanz, Natyra, Magic Star). Water contents of the flesh and peel as well as the proportions of the flesh and peel to the whole apple are listed in Table S2.

For each variety, eight (2020) and five (2021) apples were washed, quartered, and, after the removal of the stem and carpel, peeled with a peeler by hand. Before freezing in liquid nitrogen, the weights of peel and flesh were determined individually. The frozen samples were freeze-dried for 2–4 days using a Labconco Freezone 12 Liter Console Freeze Dry System (2020; Kansas City, MO) or a PIO-K-W (2021; Piatkowski Forschungsgeräte, Peterhausen, Germany). The dried peel was milled (30 s, 10 000 rpm) with an IKA Tube Mill Drive (Staufen, Germany) and the flesh (10 s, 10 000 rpm) using a Retsch Grindomix GM 200 (Haan, Germany). Homogenized samples were stored under argon and kept in the dark at room temperature (2020) and −20 °C (2021) until further use. The freeze-dried flesh and peel of Golden Delicious and Braeburn (local store) were used for preliminary experiments and method optimization.

Sample Extraction Procedure

Proteins were extracted from the flesh (1 g) and peel (0.5 g) using 10 mL of urea buffer containing r-Mal d 1_mut (10 mg/L, equal to 5 mg/L in final extraction volume) as an extraction standard (ESTD). Samples were extracted by an overhead shaker at room temperature for 1.5 h. After centrifugation for 1 h (10 410 rcf, 4 °C), 9 or 7 mL of the supernatant were removed for the flesh and peel samples, respectively. The second extraction was performed in a similar manner using 10 mL of urea buffer without ESTD. The combined supernatants were centrifuged (1 h, 10 410 rcf, 4 °C) once more and 250 μL of the extracts were precipitated with 1.5 mL of ice-cold acetone. The samples were stored at −20 °C overnight and centrifuged at 6000 rpm (VWR MiniStar Silverline), and the supernatant was discarded.

Sample Digestion Procedure

After removal of acetone, the protein pellets were dissolved in 70 μL digestion buffer. Then, 900 μL of acetonitrile/carbonate buffer (0.1 M, 1:3, v/v) was added to one vial of ISTD*s to dissolve the peptides and 30 μL was added to each sample, followed by 5 μL of the reduction reagent. After 1 h, the samples were incubated for another hour with 20 μL of the alkylation reagent in the absence of light. Then, 20 μL of the reducing reagent was added once more and after 1 h, the solutions were diluted with 365 μL of water. Digestion was started by the addition of 50 μL of the trypsin solution, and the samples were incubated overnight for at least 17 h in a thermoshaker (MB-102, BIOER, Hangzhou, China, 37 °C, 300 rpm). Digestion was stopped by adding 2.5 μL of glacial acetic acid, and the samples were purified by solid-phase extraction. After conditioning the cartridge with methanol and washing it with 1% formic acid, the samples were added and washed with 1% formic acid. Peptides were eluted with 90% methanol/1% formic acid. The solvent was removed via a vacuum concentrator (RVC 2-18 CD plus, Christ, Osterode am Harz, Germany) and the residue was dissolved in 50 μL of HPLC eluent and stored at −20 °C. Prior to LC–MS analyses, the samples were diluted 1:50 for flesh and 1:75 for peel with the eluent.

Mass Spectrometry

The digested samples were separated by reversed-phase liquid chromatography on a YMC-Triart C18 column (150 × 0.3 mm², 3 μm; YMC Europe GmbH, Dinslaken; Cat. No. TA12S03-15) at 40 °C, a flow rate of 7 μL/min, and an injection volume of 5 μL using an M3 MicroLC System coupled via a duo spray ion source to a Sciex Triple TOF 6600 (Sciex, Darmstadt, Germany). Marker identification was performed by data-dependent acquisition (DDA) according to Kaeswurm et al.,²⁸ and for quantification, the peptides were eluted with aqueous 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B) within 15 min. Starting with 3%, B was raised to 40% in 9.5 min, increased to 100% in 30 s, and kept for 2.5 min before reconditioning. The ESI source was operated at 100 °C and 5500 V, with nitrogen at 30 psi as the curtain gas. Multiple reaction monitoring (pseudo-MRM) product ion scans were performed in the high-resolution mode (m/z 100–1500, 950 cycles, cycle time 876.6 ms). Analyst TF 1.7 software was used for data acquisition, and with PEAKS (Peaks X+), untargeted data were processed. Data analyses and quantification were done using SKYLINE (Version 21.1.0.146, MacCoss Lab, Department of Genome Science, UW). Statistical analysis was carried out with the add-in Real Statistics release 7.6 in Excel 2016.³⁰ Data were checked for normality and homogeneity of variance by Shapiro–Wilk and Levene’s test, respectively. Either one-way analysis of variance (ANOVA, α = 0.05) or Kruskal–Wallis test (α = 0.05) was used to test for statistical significances between groups.

Identification of Marker Peptides

Based on 46 UniProt entries for Mal d 1, 34 potential marker peptides for isoallergens 1.01–1.06 were identified by in silico digestion. Selection criteria were that the marker (I) represented all isoforms of one isoallergen, (II) did not contain cysteine or post-translational modifications, and (III) had a length between 6 and 20 amino acids. Nontargeted experiments (data-dependent acquisition scans) on r-Mal d 1_mut and apple protein extracts revealed difficulties to find markers, in particular at the end of the Mal d 1 sequence, resulting in 21 prospective marker peptides. Optimization of collision energy (CE) and declustering potential (DP) was carried out with commercial peptides based on the intensity of the five most intense transitions.

The optimized targeted approach was used to confirm the potential marker peptides in different apple samples. Except for isoallergens 1.04 and 1.05, at least one marker peptide for each individual isoallergen was identified (Figure 1 and Table S3). In addition, combination markers, representing at least two isoallergens, were used to verify the quantification of the isoallergens. DNFTYSYSMIEGDTLSDK (1.04) and HNFTYSYSMIEGDALSDK (1.05) showed only poor signal intensities. Therefore, only the combination marker for both isoallergens (AFILDADNLIPK) was used. However, this combination marker was isobar to AFVLDADNLIPK (1.01 + 1.02) and in addition to different retention times, DP and CE were optimized to obtain different transitions (Table S3). Since LAPQAVK (1.08 + 1.09) and IAPQAVK (1.03 + 1.06 + 1.07) are not distinguishable by HPLC and mass spectrometry, the marker IAPQAVK reflects the isoallergens 1.03 + 1.06 + 1.07 + 1.08 + 1.09. Therefore, using IAPQAIK and IAPQAVK as global markers, the total Mal d 1 content (1.01–1.09) was expressed. The specificity of the selected peptide markers for Mal d 1 in the apple matrix was checked in the NCBI nonredundant protein sequence (nr) database using the protein–protein algorithm of the “Basic Local Alignment Search Tool” (BLASTP, search conducted in March 2021).^31,32

Quantification by Stable Isotope Dilution and Internal Extraction Standard

Marker identification was based on transitions and respective ISTD*s and quantification of the intensity, calculated from the sum of the three most intense transitions, with a minimum signal-to-noise ratio higher than 3 according to Table S3. For 1.01 and 1.02 and the combination 1.01 + 1.02 + 1.05, two marker peptides were required. The respective ions containing the additional lysine (1.01K, 1.02K, K(1.01 + 1.02 + 1.05)) were quantified separately. The final content of each isoallergen and combinations thereof in apples was corrected by the recovery of the ESTD. Mass concentrations were calculated with respective molecular weights (Table S3).

Results

Optimization of Extraction and Evaluation of Sample Preparation

According to the literature³³ protein extraction was performed with 10 mM phosphate buffer pH 7, containing 2% PVPP, 2 mmol EDTA, 10 mmol of DIECA, and 3 mmol of NaN₃; furthermore, urea buffer and urea buffer with PVPP were tested to find optimal extraction conditions. PVPP is described to adsorb phenolics and thus interactions between polyphenols and proteins during extraction might be inhibited.³⁴ The buffer used had a marked impact on the extracted total protein content (Figure 2A); however, variety-specific differences were obvious. Sample purification after tryptic digestion by SPE was problematic for the phosphate buffer extracts and the recoveries of the ESTD in Brettacher and Golden Delicious were very low. Extraction with urea buffer was much more efficient. Since PVPP did not significantly improve ESTD recovery and to avoid unfavorable adsorption of proteins,³⁴ it was not added routinely. The recovery of ESTD was significantly higher for the variety Golden Delicious than for Brettacher, pointing out the importance of using an extraction standard. However, differences between both ESTD marker peptides were obvious.

Figure 2.

(A) Recovery of ESTD (n = 2) in different urea and phosphate (Pi) buffers. Preliminary experiments with Golden Delicious and Brettacher based on the two marker peptides using nonoptimized extraction conditions. (B) Evaluation of extraction conditions (n = 2) using urea buffer for Brettacher (B) and Golden Delicious (GD) flesh. (a) 2 g of the sample extracted once, 250 μL precipitated, (b) 2 g of the sample extracted once, 125 μL precipitated, (c) 1 g of the sample extracted once, 125 μL precipitated, (d) 0.5 g of the sample extracted once, 250 μL precipitated, (e) 1 g of the sample extracted twice, 250 μL precipitated and (f) 2 g of the sample extracted twice, 125 μL precipitated.

To find optimal extraction conditions for the flesh and peel, the sample amount, the volume of extraction buffer and the amount of the protein extract precipitated with acetone for tryptic digestion were varied (Figure 2B and SDS-PAGE Figure S2). Optimal extraction was performed with 1 g for the lyophilized flesh and 0.5 g for the peel. Extraction was most efficient and reproducible with 2 × 10 mL of extraction buffer, and precipitation of 250 μL of the combined supernatants with acetone was sufficient for analyses (Figure 2B(e)). Sample stability was checked based on data for ESTD marker peptides and both global markers (IAPQAIK and IAPQAVK). confirming that samples were stable during storage at −20 °C up to 8 months, independent of the concentration (Figure S3).

The isotope-labeled peptides (ISTD*s) and the peptides to be quantified should be in a similar intensity range. Therefore, the amount of the isotope-labeled peptide mix added prior to digestion was evaluated. The addition of 30 μL (16.67 pmol/isoallergen) to each sample turned out to be optimal. Despite a marked variation in the isoallergen profile in the apples investigated, the ratios of ISTD*s to the marker peptides were in an adequate range (1:10–10:1, Figure S4). To investigate the impact of the matrix on the signal intensity of the ISTD*s, the protein matrix and ISTD*s were digested individually and mixed prior to mass spectrometry in different ratios. While no significant impact was observed for higher matrix proportions, the absence of the matrix provoked a reduction in signal intensity for some ISTD*s (Figure S5A). This might be due to reduced ionization and/or elevated in-source decay of the precursor peptide in the absence of the matrix.

The amount of ESTD added to the extraction buffer had no impact on its quantification and recovery rate in the flesh and peel (Table S4). Buffer containing in total 5 mg of ESTD/L turned out to be a good compromise for the flesh and peel samples. The ratios in signal intensity between the ESTD marker peptides (AFALDADNLIPK and IAPQVIK) and respective ISTD*s were around 2 for this concentration (Figure S5B). It is obvious, that recovery was lower for flesh samples than for peel samples. This might be due to lower Mal d 1 contents in the flesh than in the peel but can also be attributed to higher contents of soluble fibers in flesh than those in peel or different polysaccharide compositions in the flesh and the peel. Recoveries between both ESTD marker peptides differed significantly between extractions (Table S4). For repeated measurements (measurement stability), recovery rates varied only marginally; however, the deviation was generally higher for the peptide IAPQVIK than for AFALDADNLIPK (Figure S6A). Since IAPQVIK turned out to be problematic when determining the initial ESTD concentration (Table S4) and showed higher deviations in reproducibility (Figures S5B and S6A), the marker AFALDADNLIPK was used to correct the quantified isoallergen contents for the extraction efficiency in apple varieties.

The samples required dilution prior to analyses to avoid overloading of the detector. Therefore, they were diluted with the eluent (3% acetonitrile and 0.1% formic acid in water) in the ranges of 1:50–1:300 and 1:75–1:300 for the flesh and the peel, respectively. Quantification based on three transitions (S/N > 3) was possible up to a dilution factor of 1:300 for all marker peptides despite the individual markers for isoallergens 1.03 and 1.06 and the combination markers of 1.04 + 1.05 and 1.03 + 1.06 (data not shown). The recovery of the ESTD from Santana samples, based on its marker peptides, was independent from dilution(Table S5).

Evaluation of Individual and Global Markers

The isoallergens 1.01, 1.02, 1.03, and 1.06 have been identified and quantified in the apples investigated. Despite the fact that the combination marker for 1.04 + 1.05 (AFILDGDNLIPK) was found to give intense signals with synthesized peptides (Figure 3A), it was not detected in the apple samples at the dilution used.

Figure 3.

(A) HPLC-MS chromatogram for the commercial marker peptides (not isotopically labeled). The concentrations among the peptides are similar, and differences in signal intensity of the precursors are due to ionization. (B) Percentage of 1.02K to the sum of 1.02 + 1.02K in the flesh and peel (n = 2).

The markers for 1.01 and 1.02, the major isoallergens, suffer from a missed cleavage site with two successive lysines in the protein (Figure 1). Due to the absence of alternative marker peptides and no positive impact of increased trypsin concentrations and prolonged digestion time, both peptides 1.01 + 1.01K (QAEILEGNGGPGTIK(K)) and 1.02 + 1.02K (HAEILEGDGGPGTIK(K)) needed to be quantified according to their respective ISTD*s. While the precursor and fragmentation pattern for 1.02K is comparable to that of 1.02, the precursor of 1.01K is not detectable (Table S3). Therefore, the variety-specific proportion of 1.02K (20–53%, Figure 3B) was used to approximate the amount of 1.01K. Due to the fact that specific ISTD*s were unavailable for 1.01K and 1.02K, these values are provided separately in all tables and figures.

Ideally, the sum of individual isoallergens matches the corresponding combination markers. In the range of statistical deviations, this was proven for all combination markers, except for (K)ITFGEGSQYGYVK (1.01 + 1.02 + 1.05; Table 1). This peptide also suffered from incomplete cleavage. For the other markers, a trend of underestimation by 10 to 20% was observed for individual markers compared to the combination markers, even though the estimated amounts for 1.01K and 1.02K were included in the calculation. This effect was more pronounced for the peel than for the flesh and for varieties with higher amounts of the isoallergen 1.01. This leads to the conclusion that 1.01K might be generally underestimated and respective values should be interpreted with caution. Based on the comparable results for the sum of the individual markers for 1.03 and 1.06 and the global marker IAPQAVK (1.03 + 1.06 + 1.07 + 1.08 + 1.09), it is concluded that the isoallergens 1.07, 1.08, and 1.09. are not relevant in the apple varieties investigated.

Table 1. Proportion of the Sum of Isoallergens Quantified Individually to the Quantification Based on the Combination Markers.

isoallergen(s)	peptide	flesh [%]a	peel [%]a
1.04 + 1.05	AFILDGDNLIPK	not found	not found
1.01 + 1.02	AFVLDADNLIPK	79.8 ± 9.4	71.5 ± 6.4
1.02 + 1.06	LVASGSGSIIK	108.7 ± 14.3	92.7 ± 16.4
1.01 + 1.02 + 1.05	(K)ITFGEGSQYGYVK	123.8 ± 37.8b	111.3 ± 35.9b
1.01 + 1.02 + 1.04 + 1.05	IAPQAIK	88.7 ± 9.3	79.6 ± 7.5
1.03 + 1.06 + 1.07 + 1.08 + 1.09	IAPQAVK	90.0 ± 9.9	88.2 ± 15.2

^an = 8, values calculated as an average of all apple varieties investigated, except Santana due to low 1.02 contents, prohibiting calculation of the factor for the major isoallergen 1.01.

^bThe combination marker is probably underestimated due to missed cleavage.

Variety-Specific Recovery of ESTD from the Flesh and Peel and Quantification of the Total Mal d 1 Content

Except for Bohnapfel, the recovery rates were higher in the peel than those in the flesh (Table 2). Recovery rates of above 100% were determined for the ESTD using the peptide IAPQVIK pointing out the difficulties with this marker and supporting the decision to correct the Mal d 1 content based on the recovery rate calculated for the marker peptide AFALDADNLIPK. No trend in the recovery rates was observed among the varieties from orchard meadows and commercial breeds harvested in 2020 and 2021, respectively. Unexpectedly, significantly higher Mal d 1 contents were quantified in the flesh of noncommercial varieties (53.3–249.2 mg/kg dry weight (DW)) than those in commercial breeds (16.7–64.9 mg/kg DW; Table 2 and Figure 4A). In contrast, higher Mal d 1 contents were observed in the peel of the commercial breeds (426.5–726.5 mg/kg DW) compared to varieties from orchard meadows (235.3–376.0 mg/kg DW). Santana, a hypoallergenic breed, showed the lowest Mal d 1 contents in the flesh and medium contents in the peel compared to the other varieties investigated. In the peel, higher Mal d 1 contents were quantified, which is in accordance with the literature.^16,35 It is obvious that the ratio between the Mal d 1 contents in the peel and flesh is significantly lower (1.1–5.1) for the noncommercial than that for commercial varieties (∼12, Santana 25.3).

Table 2. Variety-Specific Recovery of ESTD for Both Marker Peptides and the Total Mal d 1 Content in the Flesh, Peel, and Whole Apples Quantified by Global Markers.

	recovery of AFALDADNLIPK [%]		recovery of IAPQVIK [%]
variety	flesh	peel	flesh	peel	flesh DWa [mg/kg]	peel DWa [mg/kg]	ratio peel/flesh	whole applesb [mg/FW kg]
Bohnapfel	99.8 ± 1.7	65.1 ± 1.1	126.6 ± 3.2	75.6 ± 0.4	53.3 ± 3.9	235.3 ± 5.8	4.4	17.2 ± 0.3
Boskoop	46.5 ± 1.7	81.8 ± 2.6	57.2 ± 0.1	101.2 ± 8.7	195.5 ± 14.0	322.6 ± 2.5	1.6	37.7 ± 1.9
Brettacher	79.5 ± 15.2	85.7 ± 0.7	100.2 ± 17.5	98.9 ± 1.8	63.4 ± 0.0	321.0 ± 3.6	5.1	14.8 ± 0.1
Gewürzluiken	48.1 ± 4.7	84.6 ± 4.1	63.5 ± 0.3	104.8 ± 4.6	182.2 ± 14.9	376.0 ± 7.6	2.1	39.7 ± 1.9
Ontario	39.3 ± 0.0	95.9 ± 4.3	46.0 ± 1.9	113.3 ± 4.8	249.2 ± 10.4	287.0 ± 28.7	1.1	42.6 ± 2.2
mean							2.9 ± 1.6
Magic Star	37.6 ± 1.9	52.7 ± 1.5	44.9 ± 0.2	60.5 ± 0.4	64.9 ± 2.9	726.4 ± 0.6	11.2	25.7 ± 0.0
Natyra	39.3 ± 3.7	57.9 ± 3.6	51 ± 4.7	67.2 ± 3.2	58.8 ± 1.4	725.7 ± 12.0	12.3	27.4 ± 0.1
Sonnenglanz	54.6 ± 0.5	86.8 ± 12.9	62.5 ± 4.0	102.8 ± 14.1	54.3 ± 2.8	660.7 ± 9.9	12.2	27.0 ± 0.0
Santana	55.5 ± 2.2	39.7 ± 2.2	56.1 ± 0.6	46.5 ± 1.0	16.7 ± 2.0	426.5 ± 33.5	25.5	14.1 ± 1.4
mean							15.2 ± 5.8
meanc							11.9 ± 0.6

^aValues calculated by the use of global markers and corrected according to the recovery of the ESTD marker AFALDADNLIPK (n = 2).

^bFresh weight, calculated according to the proportion of the flesh and peel (Table S2).

^cWithout Santana.

Figure 4.

(A) Sum (n = 2) of individual isoallergens 1.01–1.06 (bar 1), sum of 1.01K + 1.02K (bar 2), sum of all individual markers (bar 3), and the two combination markers representing the isoallergens 1.01–1.09 (bar 4) for the flesh and peel of each apple variety. (B) Isoallergen profile (n = 2) for commercial breeds and varieties from orchard meadows in the flesh (f) and peel (p) for the different apple varieties.

When estimating the Mal d 1 content for a whole (flesh and peel) cored apple (according to Table S2), Bohnapfel, Brettacher, and Santana showed lower Mal d 1 contents (below 17 mg/kg fresh weight (FW)) than Sonnenglanz, Natyra, and Magic Star (25–28 mg/kg FW), whereas the contents in Boskoop, Gewürzluiken, and Ontario were significantly higher (>37 mg/kg FW, Table 2).

Isoallergen Profile in the Flesh and Peel of Apple Varieties

Except for the flesh of Santana, Mal d 1.02 was the major isoallergen in all apple varieties, followed by 1.01. There might be a trend, that the proportion of 1.01 is elevated in the flesh of apples having low total Mal d 1 contents (Figure 4A,B). The isoallergens 1.03 and 1.06 were only present in a minor amount with a higher proportion in the peel than that in the flesh (Figure 4B). It is conspicuous that in commercial breeds, these isoallergens were absent in the flesh. The isoallergens 1.04 and 1.05 were not found in the apples studied, and our data indicate that the isoallergens 1.07, 1.08, and 1.09 were negligible or even absent.

In apple varieties from orchard meadows in sum, 4–9 mg/kg DW and 16–66 mg/kg DW of Mal d 1.03 + 1.06 in the flesh and peel were quantified using the global marker, respectively. In contrast, 27–84 mg/kg DW of both minor allergens were quantified in the peel of commercial breeds. The lowest contents of 1.03 + 1.06 in the peel were found for Santana (27 mg/kg DW) and Brettacher (16 mg/kg DW). The exact values for each isoallergen are provided in Tables S6 and S7, respectively.

Discussion

The Mal d 1 contents provided in the literature⁹ differed markedly for the same varieties, which might be attributed to the use of different immunoassays and differences in sample preparation.²¹ The use of urea buffer denaturizes the proteins including the polyphenol oxidase.³⁶ This inhibits oxidation of phenolic compounds to highly electrophilic quinones, which might react with Mal d 1 or further phenolic compounds, forming browning products that might also interact with Mal d 1.^24,37−39 Both mechanisms might reduce the quantifiable Mal d 1. Furthermore, the high polysaccharide content in apples accompanying the low protein content⁴⁰ complicates protein extraction. Our comprehensive evaluation of the protein extraction conditions and monitoring of the extraction efficiency points out the importance of an internal extraction standard. The extraction efficiency has a strong impact on the data and might explain some differences in data previously published. The marked variety-specific differences observed are probably related to alterations in the polysaccharide composition. Furthermore, the texture of apples changes during storage,^26,27 which is related to changes in the polysaccharide structures, and therefore, extraction efficiency might also vary with storage time. Similar (low) recoveries for Magic Star and Natyra support this interpretation, since they are identical varieties and are harvested and stored together, only differing in cultivation conditions that have no effect on polysaccharide composition. The (small) variations in Mal d 1 contents between both are probably related to inter-fruit variability and the integrated (Magic Star) and organic (Natyra) cultivation. However, the data do not support the findings of Takács et al., which proposed lower Mal d 1 contents in apples from organic farming.⁴¹ The data reported by Siekierzynska et al. also do not support a trend to lower Mal d 1 contents in the same variety when the fruits are grown under organic farming conditions.¹⁶

The quantified Mal d 1 contents of the apples investigated by mass spectrometry are in the range of contents reported in the literature determined using ELISA (1–50 mg/kg FW)⁹ but are higher when comparing individual varieties. In particular, for Boskoop (1 mg/kg FW)²² and Santana (0.5–5 mg/kg FW),¹² significantly lower values were reported in previous studies based on ELISA. Besides natural variations in the samples and differences in sample preparation, this might be related to the fact that antibodies are often not isoallergen-specific or at least not equally reactive to all isoallergens. This might lead to systematic underestimation of the total Mal d 1 content. However, our data do not reveal differences in the Mal d 1 content between varieties from orchard meadows and commercial breeds. Nonetheless, the two groups of apples investigated were harvested in different years and stored under dissimilar conditions, which might exclude comparison. The ratio of Mal d 1 contents between the flesh and the peel was extremely high for commercial breeds, supporting the recommendation to peel the fruits when reacting sensitively to fresh apples. The absence of the isoallergens 1.03 and 1.06 in the flesh of commercial breeds is surprising because an increased allergenic potential is postulated at least for 1.06A.^3,16 Postulations about the allergenic potential of an individual isoallergen are based on the correlation of the allergenicity of an apple variety with the quantified isoallergen. Uncertainties in the quantification methods used so far might therefore conceal interrelationships between the allergenic potential and the isoallergen profile. However, for the new apple breeds we investigated (Sonnenglanz, Natyra/Magic Star), allergenicity is unknown, and further research on well-known highly allergenic apple varieties like Gala, Golden Delicious, and Granny Smith is a focus of future research.

We could show that the mass spectrometric method allows the parallel quantification of the isoallergens 1.01–1.06, even though 1.04 and 1.05 could not be detected in the apples studied. Our data demonstrate the importance of the experimental proof of the theoretically predicted individual marker peptides and validate the quantification based on individual marker peptides by the combination and global markers. In summary, the parallel use of different marker peptides allows the determination of the isoallergen profile (individual markers), their verification (combination markers), and the determination of the total content of Mal d 1 based on the isoallergens 1.01–1.09 (global markers). In addition, the data emphasize that an extraction standard is required to compare Mal d 1 contents in different apple varieties.

The apples investigated showed differences in the total Mal d 1 content and profile between the flesh and the peel as well as among new breeds, harvested in 2021, and varieties from orchard meadows (2020). Although our data cannot explain differences in the allergic potential, this mass spectrometric approach is helpful for further investigations into variety-specific differences and other impact factors such as climate, cultivation, harvesting and storage on the allergen content and the isoallergen profile. The mass spectrometric approach supports further research to provide reliable information about the Mal d 1 content and the isoallergen profile in apples.

Glossary

Abbreviations

CE

collision energy

DIECA

sodium diethyldithiocarbamate trihydrate

DP

declustering potential

DW

dry weight

EDTA

sodium ethylenediaminetetraacetic acid dehydrate

ESTD

extraction standard

FW

fresh weight

ISTD*

isotopically labeled internal standard peptides

PVPP

polyvinylpolypyrrolidone

SPE

solid-phase extraction

Tris

2-amino-2-(hydroxymethyl)propane-1,3-diol

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.2c03745.

• Table S1: Quantification of the extraction standard (ESTD, r-Mal d 1_mut) after digestion with and without the protein matrix based on the two marker peptides; Table S2: Water content and proportion of the flesh and peel to the whole apple; Table S3: Marker peptides and their mass spectrometric properties for individual isoallergens and combinations thereof; Table S4: Quantification and recovery of extraction standard (ESTD) at different mass concentrations; Table S5: Recovery of the ESTD depending on final sample dilution in Santana; Table S6: Individual quantification of Mal d 1 isoallergens in mg/kg DW based on different marker peptides for the flesh (F) and the peel (P) in traditional apples harvested in 2020; Table S7: Individual quantification of Mal d 1 isoallergens mg/kg DW based on different marker peptides for the flesh (F) and the peel (P) in commercial apple breeds harvested in 2021; Figure S1: SDS-PAGE of r-Mal d 1_mut (ESTD) under denaturizing conditions (DDT); as a comparison, a protein marker (STD) from the same gel is shown. Purity was determined with Fiji to 92%; Figure S2: SDS-PAGE of protein extracts using urea buffer and different extraction procedures for Golden Delicious and Brettacher flesh (n = 2), according to Figure 2 in the article. M, marker (Serva dual color protein standard III); samples were diluted with buffer 1 + 1 prior to SDS-PAGE; Figure S3: Sample stability during storage at −20 °C for 3 and 8 months based on ESTD marker peptide recovery and global markers (uncorrected). The samples from the flesh were stored concentrated and at a final 1:50 dilution; Figure S4: Intensity ratio of marker peptides to respective ISTD*s in Brettacher (pink) and Golden Delicious (green) flesh samples. Data for AFILDGDNLIPK (1.04 + 1.05) in both varieties and YSVIEGDAISETIEK (1.03) in Brettacher are not shown due to the absence of these isoallergens in the apple samples; Figure S5: Impact of the matrix (Golden Delicious, flesh) on the response of ISTD*s. An ISTD* value of 1 corresponds to 16.67 pmol added to the digested matrix (equivalent to 1–8 g of the freeze-dried sample). The area and ratio for marker peptides and respective ISTD*s depend on the amount of ESTD added to the extraction buffer for the flesh (1:50 final dilution) and peel (1:75 final dilution) samples; and Figure S6: Measurement stability. Deviation in the recovery of the ESTD marker peptides (2–8%) and the quantification of global markers (4–7.5%) during HPLC-QqQ-MS/MS analyses for 16 successive injections of the flesh (F) and peel (P) samples. Braeburn peel samples were injected 16 times at the beginning of the sequence and at the end after 5 days of measurement (PDF)

Author Contributions

Experiments were performed by J.A.H.K., A.K., and L.V.S. Data analyses and curation were performed by J.A.H.K. and L.V.S. and discussed with M.B. and J.B. The manuscript was written by J.A.H.K. and M.B. J.B. and L.V.S. supported visualization and manuscript review and editing. M.B., J.A.H.K., and J.B. conceptualized the study. M.B. supervised the study and was responsible for project administration and funding acquisition. All authors have given approval to the final version of the manuscript.

This research was funded by the German Research Foundation (DFG, Grant Number 3811/1-1), the Ministry of Science, Research and the Arts Baden-Württemberg (M.B., M. v. Wrangell program), the Dr. Leni Schöninger Foundation, and funds of the chemical industry, Germany (FCI).

The authors declare no competing financial interest.

Notes

The data generated during the study are included in this published article and its supplementary files.