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

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4698
Author(s):  
Sorel Tchewonpi Sagu ◽  
Gerd Huschek ◽  
Thomas Homann ◽  
Harshadrai M. Rawel
Keyword(s):  
Sample Preparation ◽  
Extraction Method ◽  
Trichloroacetic Acid ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Internal Standards ◽  
Allergenic Protein ◽  
Microwave Assisted ◽  
Allergenic Proteins ◽  
Detection And Quantification

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.

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Gram Negative ◽  
Protein Quantitation ◽  
System P ◽  
Automated Protocol ◽  
Chloroform Methanol ◽  
Extract Protein

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v1

2020 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Gram Negative ◽  
Protein Quantitation ◽  
System P ◽  
Automated Protocol ◽  
Chloroform Methanol ◽  
Extract Protein

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Protein Quantitation ◽  
System P ◽  
Chloroform Methanol

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system

scholarly journals Evaluation and refinement of sample preparation methods for extracellular matrix proteome coverage

2020 ◽  
Author(s):  
Maxwell C. McCabe ◽  
Lauren R. Schmitt ◽  
Ryan C. Hill ◽  
Monika Dzieciatkowska ◽  
Mark Maslanka ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Sample Preparation ◽  
Real World ◽  
Extraction Method ◽  
Protein Extraction ◽  
Preparation Methods ◽  
Proteome Coverage ◽  
Ecm Proteins ◽  
Sample Preparation Methods

ABSTRACTThe extracellular matrix is a key component of tissues, yet it is under-represented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared to a standard in-solution digest, the optimized method yielded a 4-fold improvement in unique ECM peptide identifications.Abstract Figure

Automated Protein Quantification with the Biomek-FX Liquid Handler System v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Tad Ogorzalek ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Protein Quantification ◽  
Tryptic Digestion ◽  
System P ◽  
Plate Reader ◽  
Separate Control ◽  
Control Plate ◽  
Chloroform Methanol

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Tryptic Digestion ◽  
Protein Quantitation ◽  
System P ◽  
Chloroform Methanol

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system

Automated Protein Quantification with the Biomek-FX Liquid Handler System v2

2022 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Tad Ogorzalek ◽  
Jennifer Gin ◽  
Christopher J Petzold
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Protein Quantification ◽  
Tryptic Digestion ◽  
System P ◽  
Plate Reader ◽  
Separate Control ◽  
Control Plate ◽  
Chloroform Methanol

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

Verification of Protein Extraction Method by Magneto-Ferrite Treatment with EDS Analysis

2020 ◽  
Vol 140 (2) ◽  
pp. 128-129
Author(s):  
Suguru Kotani ◽  
Masaya Endo ◽  
Mahmudul Kabir ◽  
Kazutaka Mitobe
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Eds Analysis

scholarly journals An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiang Jin ◽  
Liping Zhu ◽  
Chengcheng Tao ◽  
Quanliang Xie ◽  
Xinyang Xu ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction

scholarly journals Relationship between Phenolic Compounds, Antioxidant Properties, and the Allergenic Protein Mal d 1 in Different Selenium-Biofortified Apple Cultivars (Malus domestica)

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2647
Author(s):  
Sabrina Groth ◽  
Christoph Budke ◽  
Timo Weber ◽  
Susanne Neugart ◽  
Sven Brockmann ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Antioxidant Properties ◽  
Total Phenolic ◽  
Allergenic Protein ◽  
Antibody Recognition ◽  
Allergenic Potential ◽  
Allergenic Proteins ◽  
Other Substances ◽  
Mal D 1 ◽  
Apple Cultivars

Notable parts of the population in Europe suffer from allergies towards apples. To address this health problem, the analysis of the interactions of relevant allergens with other substances such as phenolic compounds is of particular importance. The aim of this study was to evaluate the correlations between the total phenolic content (TPC), polyphenol oxidase (PPO) activity, antioxidant activity (AOA), and the phenolic compound profile and the content of the allergenic protein Mal d 1 in six apple cultivars. It was found that the PPO activity and the content of individual phenolic compounds had an influence on the Mal d 1 content. With regard to the important constituents, flavan-3-ols and phenolic acids, it was found that apples with a higher content of chlorogenic acid and a low content of procyanidin trimers and/or epicatechin had a lower allergenic potential. This is probably based on the reaction of phenolic compounds (when oxidized by the endogenous PPO) with proteins, thus being able to change the conformation of the (allergenic) proteins, which further corresponds to a loss of antibody recognition. When apples were additionally biofortified with selenium, the composition of the apples, with regard to TPC, phenolic profile, AOA, and PPO, was significantly affected. Consequently, this innovative agronomic practice seems to be promising for reducing the allergenic potential of apples.

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