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

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 QconCAT-based absolute quantification
of major ATI proteins while simultaneously quantifying the proteome
by label-free quantification (LFQ). We analyzed extracts of 60 varieties
of common wheat grown in replication and evaluated the reproducibility
and precision of the workflow for the quantification of ATIs. 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 validated inter-laboratory and cross-methodology
reproducibility of ATI quantification, which is essential in the context
of large-scale breeding projects. Additionally, we applied our workflow
to assess environmental effects on ATI expression, analyzing ATI content
and proteome of same varieties grown at different locations. Finally,
we explored the potential of combining QconCAT-based absolute quantification
with DIA-based LFQ proteome analysis for the generation of new hypotheses
or assay development.