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

Abstract

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.

Influence of Roasting Temperature on the Detectability of Potentially Allergenic Lupin by SDS-PAGE, ELISAs, LC-MS/MS, and Real-Time PCR

“…Reduction, alkylation, and tryptic digestion were performed according to Geisslitz et al with some modifications [ 40 ]. Due to the higher protein content of lupin compared to wheat, a lower extract volume of 30 µL was used.…”

“…Separation and data acquisition were performed according to Geisslitz et al [ 40 ]. SONAR mode with a 35 Da window was applied for data-independent acquisition (DIA).…”

Simultaneous Determination of Animal Products from Ruminant, Pig, Poultry, and Fish in Feedingstuff by Targeted High-Resolution LC–MS/MS

“…Detailed setup of gradient separation and mass spectrometry settings are listed in Supplement Table 1. DIA was performed in the SONAR mode according to Geisslitz et al, 2022 . Instead of 10 Da, a 35 Da window was used to increase the sensitivity.…”

“…Two different extraction protocols were applied. For discovery proteomics, total extractable protein (TEP) extraction with 50% isopropanol, 1 N urea, 0.1 N TRIS, and 0.5% DTE (pH 8.2) was performed according to Geisslitz et al, 2022, 16 followed by protein precipitation. To 100 μL of extract, 400 μL of cold acetone was added; afterward, the extract was kept at −18 °C overnight, and the pellet was washed twice with 400 μL of cold acetone.…”

Identification, Characterization, and Expression of a β-Galactosidase from Arion Species (Mollusca)

“…After the reduction with DTT (15 mM in 100 mM ammonium bicarbonate pH 7.0) and carbamidomethylation with iodoacetamide (30 mM) [ 29 ], the sample was digested with trypsin (Promega, sequencing grade), followed by a C18-SPE clean-up. The sample was loaded onto a LC (Dionex UltiMate 3000 Rapid, ThermoFisher Scientific - Bonn, Germany) equipped with nano-C18 column, coupled to an Orbitrap (ThermoFisher Scientific—Bonn, Germany, performed at IMBA, Vienna).…”