Table_1_Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi.DOCX

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.

小麦蛋白的定量分析仍是一项挑战，尤其在针对淀粉酶/胰蛋白酶抑制剂（ATIs）方面。通过对普通小麦品种Bobwhite和硬质小麦品种Svevo分别采用RNA干扰和基因编辑技术，选取了部分ATIs进行沉默，以降低ATIs的含量。随后，对对照组和沉默线进行消化处理，通过液相色谱-串联质谱（LC-MS/MS）技术，采用多种方法对ATIs组成的改变进行评估。首先，采用标记肽作为内标，运用稳定同位素稀释法（SIDA）进行靶向分析。此外，还进行了四种相对定量方法，具体包括iTRAQ标记和无标记定量（LFQ）结合数据依赖采集（DDA）与数据非依赖采集（DIA）。定量分析通过手动操作（Skyline和MASCOT）以及不同的蛋白质组学软件工具（PLGS、MaxQuant和PEAKS X Pro）进行。为了在蛋白质水平上表征小麦蛋白，还采用了高效液相色谱（HPLC）和凝胶电泳等互补技术。靶向SIDA方法能够定量所有ATIs，即使在低水平下，但也需要优化的提取方法。标记的iTRAQ方法表现出的性能并不明显。使用低分辨率设备（IonTrap）进行的LFQ显示，对于主要ATIs的结果相似，但低丰度ATIs如CM1则不可检测。使用Orbitrap系统进行DDA测量，并利用MaxQuant进行评估，显示相对定量依赖于小麦品种。将MaxQuant搜索结果的手动校对与Skyline相结合，显示出非常出色的性能。DIA方法与绝对定量相比，在分析流动中发现相似的结果，除了某些次要的ATIs未检测到。对应用方法的比较揭示了肽的选择是蛋白质定量分析的关键步骤。由于小麦蛋白质的高遗传复杂性、与其他谷物的紧密关系以及不完整、冗余的蛋白质数据库，小麦蛋白质组学面临着挑战，需要敏感、精确和准确的LC-MS/MS方法。