Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.)

Abstract

Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples.

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

“…This modified Osborne fractionation is time consuming and alternatives with lower number of extraction steps were established to extract all or the majority of proteins ( van den Broeck et al, 2009 ; Henrottin et al, 2019 ; Jira and Münch, 2019 ). Furthermore, specific protocols were developed to extract ATIs ( Geisslitz et al, 2018 ; Call et al, 2019 ; Sielaff et al, 2021 ; Sagu et al, 2022 ) using extractions solvents containing Ambic and/or NaCl with a pH between 7 and 8 and without any organic solvents and reducing reagents. We revealed that in our sample set, the Osborne fractionation and the TEP extraction resulted in comparable protein content by RP-HPLC-UV.…”

“…Hence, the sequences of the isoforms being studied may often not be unique, which limits its clear identification and thus, their assignment to proteins. Furthermore, theoretical mass of proteins listed by databases and those measured often differ strongly, e.g., as shown for CM3 (reference mass without signal 10.3389/fpls.2022.974881 peptide 15.8 kDa and measured mass 15.5 kDa) by several studies (Capocchi et al, 2013;Call et al, 2019;Sagu et al, 2022).…”