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 establi...
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| Format: | Article |
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MDPI AG
2021-08-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/15/4698 |
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| author | Sorel Tchewonpi Sagu Gerd Huschek Thomas Homann Harshadrai M. Rawel |
| author_facet | Sorel Tchewonpi Sagu Gerd Huschek Thomas Homann Harshadrai M. Rawel |
| author_sort | Sorel Tchewonpi Sagu |
| collection | DOAJ |
| description | 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. |
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| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T09:10:33Z |
| publishDate | 2021-08-01 |
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| series | Molecules |
| spelling | doaj.art-d52413a158074efd9bbce323e7194fbe2023-11-22T06:02:07ZengMDPI AGMolecules1420-30492021-08-012615469810.3390/molecules26154698Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MSSorel Tchewonpi Sagu0Gerd Huschek1Thomas Homann2Harshadrai M. Rawel3Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, GermanyGV-Institut für Getreideverarbeitung GmbH, Arthur-Scheunert-Allee 40/41, 14558 Nuthetal, GermanyInstitute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, GermanyInstitute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, GermanyThe 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.https://www.mdpi.com/1420-3049/26/15/4698nut allergenic proteinssample preparationprotein extractiontryptic digestionmicrowave assisted digestionSDS PAGE |
| spellingShingle | Sorel Tchewonpi Sagu Gerd Huschek Thomas Homann Harshadrai M. Rawel Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS Molecules nut allergenic proteins sample preparation protein extraction tryptic digestion microwave assisted digestion SDS PAGE |
| title | Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS |
| title_full | Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS |
| title_fullStr | Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS |
| title_full_unstemmed | Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS |
| title_short | Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS |
| title_sort | effect of sample preparation on the detection and quantification of selected nuts allergenic proteins by lc ms ms |
| topic | nut allergenic proteins sample preparation protein extraction tryptic digestion microwave assisted digestion SDS PAGE |
| url | https://www.mdpi.com/1420-3049/26/15/4698 |
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