Comparison of MALDI-TOF-MS and RP-HPLC as rapid screening methods for wheat lines with altered gliadin compositions

Authors: JANG, YOU-RAN - National Institute For Agricultural Science & Technology; CHO, KYOUNGWON - Chonnam National University; KIM, SEWON - National Institute For Agricultural Science & Technology; SIM, JAE-RYEONG - National Institute For Agricultural Science & Technology; LEE, SU-BIN - National Institute For Agricultural Science & Technology; KIM, BEOM-GI - National Institute For Agricultural Science & Technology; Gu, Yong; Altenbach, Susan; LIM, SUN-HYUNG - Hankyong National University; GOO, TAE-WON - Dongguk University; LEE, JONG-YEOL - National Institute For Agricultural Science & Technology

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/13/2020
Publication Date: 12/4/2020
Citation: Jang, Y., Cho, K., Kim, S., Sim, J., Lee, S., Kim, B., Gu, Y.Q., Altenbach, S.B., Lim, S., Goo, T., Lee, J. 2020. Comparison of MALDI-TOF-MS and RP-HPLC as rapid screening methods for wheat lines with altered gliadin compositions. Frontiers in Plant Science. 11. Article 600489. https://doi.org/10.3389/fpls.2020.600489.
DOI: https://doi.org/10.3389/fpls.2020.600489

Interpretive Summary: The gliadins are complex groups of proteins that make it possible to produce a wide range of food products from wheat flour. Because some of these proteins are also responsible for food allergies and celiac disease, there has been considerable interest in reducing the amounts of gliadins likely to be most harmful using either breeding or biotechnology approaches. Such efforts require efficient, high-throughput analytical methods for selecting wheat lines that are missing specific gliadins. The large numbers of very similar proteins in each gliadin subclass make it challenging to develop such techniques. In this paper, two optimized high-throughput methods, MALDI-TOF-MS, that separates proteins on the basis of molecular weight, and RP-HPLC, that separates proteins on the basis of hydrophobicity, were used to analyze gliadins from the reference wheat cultivar Chinese Spring. Both aneuploid lines missing specific chromosomes as well as all gene sequences for gliadins are available from this cultivar. The results indicate that MALDI-TOF-MS does not provide sufficient resolution of gliadins and that RP-HPLC is the preferred high-throughput method.

Technical Abstract: Wheat gliadin proteins inflame celiac disease in humans. Here, we compared analytical methods to explore the genetic complexity of gliadins in the bread wheat (Triticum aestivum) cultivar 'Chinese Spring’ (CS) using aneuploid lines for chromosomes 1 and 6. We optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) and evaluated the quality of the resulting gliadin profiles using newly available genome sequences for gliadin genes. Using MALDI-TOF-MS, two major peaks in the 30.3–32.5 kDa range included as many as 23 a/'-gliadins out of the 44 total predicted CS gliadin proteins. Thus, MALDI-TOF-MS does not provide sufficient resolution for effective gliadin profiling. By contrast, we successfully improved the resolution of RP-HPLC to obtain 34 separate peaks in the CS gliadin fraction. By comparing the gliadin profile of CS with those of the aneuploid lines, we assigned 26 peaks to chromosomes 1 and 6: seven '- and eight '-gliadins encoded by chromosome 1, and eleven a-gliadins encoded by chromosome 6. These results indicate that RP-HPLC is an excellent analytical method to screen wheat lines, especially those that have lost epitopes for wheat-related diseases in classical breeding or biotechnology approaches