Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS.

Sorel Tchewonpi Sagu,Gerd Huschek,Thomas Homann,Harshadrai M Rawel

doi:10.3390/molecules26154698

Sorel Tchewonpi Sagu, Gerd Huschek + Show 2 more

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https://doi.org/10.3390/molecules26154698

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Abstract

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Highlights

Sensitive and selective analytical multi-parameter methods are necessary to evaluate whether food labelling complies with the legal regulations to protect consumers [1,2]
The almond extracts obtained from the PVPP, sodium dodecyl sulfate (SDS) and TCA/acetone methods contained similar protein concentrations of about 15 mg/mL while the CM/NaCl extract showed a concentration of only 03.82 ± 0.13 mg/mL
The analysis of the protein composition was performed comparing the protein profiles in different ranges (0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa) It should be noted that the main allergenic proteins of the different nut samples selected for this study show molecular weights ranging from 5 to 70 kDa (See Table S1, Supplementary Materials)

Summary

Introduction

Sensitive and selective analytical multi-parameter methods are necessary to evaluate whether food labelling complies with the legal regulations to protect consumers [1,2]. A particular analytical challenge concerns the labelling of allergens, as minimal amounts of allergenic foods are sufficient to cause severe or even fatal allergic reactions. Only recommendations for specific allergenic foods are made ranging between 10 to 100 mg allergenic food/kilogram [4]. Nuts represent a special group causing often severe allergenic reactions and affecting especially children. The most commonly used nuts include almond, walnut and peanut. Other important nuts such as pistachio, hazelnut, macadamia, pecan or even cashew are among the most popular varieties [6,7].

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