Dataset Information

Comprehensive Characterization and Relative Quantification of ?-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS.

ABSTRACT: The ?-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker's asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60-80%)/trypsin (10-20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7-34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported.

SUBMITTER: Sagu ST

PROVIDER: S-EPMC7600490 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS.

Sagu Sorel Tchewonpi ST Zimmermann Lynn L Landgräber Eva E Homann Thomas T Huschek Gerd G Özpinar Haydar H Schweigert Florian J FJ Rawel Harshadrai M HM

Foods (Basel, Switzerland) 20201013 10

The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker's asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entrie ...[more]

PMID: 33066015

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Project description:Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods.

Dataset Information

Comprehensive Characterization and Relative Quantification of ?-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS.

Publications

Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS.

Similar Datasets

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