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Published (Version of Record)CC BY V4.0, Open Access
Journal article
Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi
Frontiers in plant science
2022
PMCID: PMC9465248
PMID: 36105703
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.
Details
- Title
- Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi
- Authors/Creators
- Sabrina GeisslitzShahidul IslamLukas BuckClemens Grunwald-GruberFrancesco SestiliFrancesco CamerlengoStefania MasciStefano D’Amico
- Publication Details
- Frontiers in plant science
- Publisher
- Frontiers Media
- Identifiers
- 991005621111907891
- Copyright
- © 2022 Geisslitz, Islam, Buck, Grunwald-Gruber, Sestili, Camerlengo, Masci and D’Amico
- Murdoch Affiliation
- Centre for Crop and Food Innovation
- Language
- English
- Resource Type
- Journal article
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- 1 Clinical & Life Sciences
- 1.249 Digestive System Disorders
- 1.249.1112 Celiac Disease
- Web Of Science research areas
- Plant Sciences
- ESI research areas
- Plant & Animal Science