Dataset Information

Quantitation of ATIs in Wheat by LC-MS/MS – iTRAQ

ABSTRACT: 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).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Triticum Durum Triticum Aestivum (wheat)

TISSUE(S): Endosperm

SUBMITTER: Sabrina Geisslitz

LAB HEAD: Katharina Scherf

PROVIDER: PXD034478 | Pride | 2022-10-13

REPOSITORIES: Pride

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Publications

Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi.

Geisslitz Sabrina S Islam Shahidul S Buck Lukas L Grunwald-Gruber Clemens C Sestili Francesco F Camerlengo Francesco F Masci Stefania S D'Amico Stefano S

Frontiers in plant science 20220829

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 ...[more]

PMID: 36105703

Similar Datasets

Project description:Wheat (Triticum aestivum ssp. aestivum) contributes to 20% of the human protein supply, delivers essential amino acids and is of fundamental importance for bread and pasta quality. Wheat proteins are also involved in adverse human reactions like celiac disease, wheat allergy and the non-celiac wheat sensitivity (NCWS). Using LC-MS-based LFQ proteomics of aqueous flour extracts we determined 756 proteins across 150 wheat varieties grown in three environments. However, only 303 proteins were stably expressed across all environments in at least one variety underlining the large influence of environmental conditions on the expression of many proteins. Moreover, only 89 proteins were comparably expressed by all 150 varieties, with high coefficients of variation for the other proteins. Heritability (h) ranged from 0-1 with 114 proteins having h² > 0.6. Therefore, the expression of the variable proteins should be amenable to targeted manipulation across the wheat supply chain by varietal choice and breeding. Our study provides a first approach towards a fast and high-throughput methodology for quantifying these proteins which is required to breed wheats with the desired properties. Amylase trypsin inhibitors (ATIs) appear as a potential trigger for NCWS inducing intestinal and extra-intestinal inflammation. Studies on the prevalence and genetic architecture of ATI proteins in wheat are lacking so far. Large differences in the content and composition of 8 ATIs in the different varieties were identified by QconCAT-assisted quantification. The ATI proteins had low coefficients of correlations with quality traits commonly analyzed in wheat breeding. However, heritability was quite low except for ATI 0.28 and ATI CM2. A genome wide association mapping revealed a complex genetic architecture built up on many small but few medium and two major quantitative trait loci (QTL). The latter were on chromosome 3B for ATI 0.19-like and 6B for ATI 0.28 explaining 70.6 and 68.7% of the genetic variance, respectively. Using the wheat reference genome sequence, seven potential candidate genes behind the medium and major QTL were described with only one showing polymorphism based on exome capture analysis. Consequently, wheat breeding could contribute to a reduction of ATI contents in wheat products if incidence of ATI on human health is further confirmed.

Project description:To better undersand the effects of drought stress on wheat developing seeds, the transcription profile of early developing wheat seeds under control and drought stress conditions were comparatively analyzed by using the Affymetrix wheat geneChip. Drought stress is a major yield-limiting factor for wheat. Wheat yields are particularly sensitive to drought stress during reproductive development. Early seed development stage is an important determinant of seed size, one of the yield components. We specifically examined the impact of drought stress imposed during postzygotic early seed development in wheat. We imposed a short-term drought stress on plants with day-old seeds and observed that even a short-duration drought stress significantly reduced the size of developing seeds as well as mature seeds. Drought stress delayed the developmental transition from syncytial to cellularized stage of endosperm. Coincident with reduced seed size and delayed endosperm development, a subset of genes associated with cytoskeleton organization was misregulated in developing seeds under drought-stressed. Several genes linked to hormone pathways were also differentially regulated in response to drought stress in early seeds. Notably, drought stress strongly repressed the expression of wheat storage protein genes such as gliadins, glutenins and avenins as early as 3 days after pollination. Our results provide new insights on how some of the early seed developmental events are impacted by water stress, and the underlying molecular pathways that can possibly impact both grain size and quality in wheat. Winter wheat cultivar Redland, PI 502907 (Triticum aestivum L.) was used for this study. Seedlings were vernalized at 4°C for 6 weeks and then transplanted to a one gallon pot of soil-sand mixture (3:1, v/v) and grown in a growth chamber under the following conditions: relative humidity, 50–70%; 16-h light/8-h dark photoperiod; 21°C daytime temperature and 18°C nights. Plants were watered regularly twice daily at the rate of 100ml/ per pot. Because, wheat has an asynchronous fertilization pattern for ovlues in the inflorescence, each floret needs to be specifically marked for timing the fertilization and stress induction. After spikes developed, unfertilized ovules were monitored and observed for the fertilization process. Closed wheat spikes with anthers outside were marked as fertilized. Drought stress was imposed 24h after the fertilization (HAF). Drought stress treatment was initiated by discontinuing watering on the drought treatment plants while control plants were regularly watered twice daily. Stress treatment was applied at 48 HAF and relieved at 96 HAF. The microarray study focuses on 24 HAF to 72 HAF in control and drought stress conditions. We started to impose drought stress 24HAF.

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