Effect of sample preparation on the detection and quantification of selected nuts allergenic proteins by LC-MS/MS
- 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 ofThe 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.…
Author details: | Sorel Sagu TchewonpiORCiD, Gerd HuschekORCiDGND, Thomas HomannORCiD, Harshadrai Manilal RawelORCiDGND |
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DOI: | https://doi.org/10.3390/molecules26154698 |
ISSN: | 1420-3049 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/34361856 |
Title of parent work (English): | Molecules : a journal of synthetic chemistry and natural product chemistry / Molecular Diversity Preservation International |
Publisher: | MDPI |
Place of publishing: | Basel |
Publication type: | Article |
Language: | English |
Date of first publication: | 2021/07/09 |
Publication year: | 2021 |
Release date: | 2021/10/18 |
Tag: | LC-MS/MS; SDS PAGE; microwave assisted digestion; nut allergenic proteins; protein extraction; sample preparation; tryptic digestion |
Volume: | 26 |
Issue: | 15 |
Article number: | 4698 |
Number of pages: | 21 |
Funding institution: | Universität Potsdam |
Funding institution: | Alexander von Humboldt Foundation under the Georg Forster Research Fellowship Alexander von Humboldt Foundation |
Funding number: | PA 2021_080 |
Funding number: | 3.4-CMR-1164093-GF-P |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft |
DDC classification: | 5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften |
Peer review: | Referiert |
Grantor: | Publikationsfonds der Universität Potsdam |
Publishing method: | Open Access / Gold Open-Access |
License (German): | CC-BY - Namensnennung 4.0 International |
External remark: | Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 1157 |