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

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.

Optimization of a sample preparation workflow based on UHPLC-MS/MS method for multi-allergen detection in chocolate: An outcome of the ThRAll project

“…These buffers can be used in combination with denaturing, reducing agents, and/or surfactants such as urea, thiourea, dithiotreitol, sodium dodecyl sulphate (SDS), tween, octyl β-D-glucopyranosid and RapiGest SF, in order to improve the protein extraction rate [Martinez-esteso 2020, Monaci 2014, New 2018, 2020, Sayers 2018, Sagu 2021, Xiong 2021]. However, some of these additives can interfere with the enzymatic digestion step (e.g., proteases like trypsin are inhibited by urea concentrations higher than 1 M) or may adversely affect the MS analysis (e.g., SDS is not MS compatible); therefore whenever added to improve the protein extraction yield, such additives require proper removal steps in the preparation workflow such as solid phase extraction, cut-off filtration or dilution down to compatible concentrations, to avoid any interference with the final detection [Boo 2018, Croote 2019, Monaci 2020, Planque 2016, Xiong 2021].…”

Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.)

“…The digestion buffer (100 mM of ammonium bicarbonate), and the proteomics grade trypsin solution were then added to the mixture (ratio of approximately 20:1, protein/enzyme) and the digestion was performed for 20 h at 37 • C. A solution of 40% formic acid was used to stop the digestion. The samples were filled into the vials for the analysis while the solid phase extraction (SPE) was applied as previously described [49] to the digested crude extract prior to the analysis.…”

Targeted Bottom–Up Mass Spectrometry Approach for the Relative Quantification of Post-Translational Modification of Bovine κ-Casein during Milk Fermentation

“…A targeted LC-MS/MS method was developed to evaluate the PTM behavior of milk κ-CN during the fermentation process. To this end, the approach used was similar to the method previously described, based on the Skyline software for the in-silico digestion as well as the development and optimization of mass spectrometry parameters for the analysis of the targeted modified peptides [35][36][37].…”