Dataset Information

Hybrid QconCAT-based Targeted Absolute and Data-independent Acquisition-based Label-free Quantification Enables In-depth Proteomic Characterization of Wheat ATI Extracts

ABSTRACT: In this study, we present a novel hybrid data-independent acquisition LC-MS approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method towards the quantitative proteome analysis of ATI extracts from wheat flour. The novel method is fast, robust and reproducible and provides accurate QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the non-targeted proteome by DIA-based label-free quantification. Using a set of 120 samples of full kernel flour derived from 60 varieties of common wheat grown in replication, we assess the repeatability and accuracy of the workflow for the quantification of ATI at the peptide and protein level. Applying the method to analyze different wheat species (i.e. common wheat, spelt, durum wheat, emmer and einkorn) and comparing the results to published data, we validate inter-laboratory and cross-methodology reproducibility of ATI quantification, which are essential in the context of large-scale breeding projects. Additionally, we apply our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of wheat species grown at different locations. Finally, we explore the potential of combining QconCAT-based absolute quantification with DIA-based untargeted proteome analysis and LFQ for the generation of new hypotheses or assay development.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Triticum Aestivum (wheat)

SUBMITTER: Malte Sielaff

LAB HEAD: Stefan Tenzer

PROVIDER: PXD021381 | Pride | 2021-04-22

REPOSITORIES: Pride

ACCESS DATA

Publications

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Sielaff Malte M Curella Valentina V Neerukonda Manjusha M Afzal Muhammad M El Hassouni Khaoula K Distler Ute U Schuppan Detlef D Longin C Friedrich H CFH Tenzer Stefan S

Journal of proteome research 20210128 3

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise Qcon ...[more]

PMID: 33507751

Similar Datasets

Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10^-18 moles to 10^-14 moles in a linear range), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 9 different tissues relating them with tissue functionality in the cardiocirculatory system.

Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10-18 moles to 10-14 moles in a linear range) (see Figure), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 14 different tissues relating it with tissue anatomical proximity and functional similarity.

Dataset Information

Hybrid QconCAT-based Targeted Absolute and Data-independent Acquisition-based Label-free Quantification Enables In-depth Proteomic Characterization of Wheat ATI Extracts

Publications

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure