Dataset Information

Quantification of ATIs in Wheat by LC-MS/MS – LFQ IonTrap

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): Xevo G2-XS QTof

ORGANISM(S): Triticum Aestivum (wheat)

TISSUE(S): Endosperm

SUBMITTER: Stefano D'Amico

LAB HEAD: Clemens Grünwald-Gruber

PROVIDER: PXD034769 | Pride | 2022-10-14

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

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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.

			Action	DRS
	1A.dat	Other
	1A.mzML	Mzml
	1A.mzid	Mzid
	1B.dat	Other
	1B.mzML	Mzml