Development of an optimized MALDI-TOF-MS method for high-throughput identification of high-molecular-weight glutenin subunits in wheat

Authors: JANG, YOU-RAN - National Institute For Agricultural Science & Technology; CHO, KYOUNGWON - Chonnam National University; KIM, SE WON - National Institute For Agricultural Science & Technology; Altenbach, Susan; LIM, SUN-HYUNG - Hankyong National University; SIM, JAE-RYEONG - National Institute For Agricultural Science & Technology; LEE, JONG-YEOL - National Institute For Agricultural Science & Technology

Submitted to: Molecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/2020
Publication Date: 9/22/2020
Citation: Jang, Y., Cho, K., Kim, S., Altenbach, S.B., Lim, S., Sim, J., Lee, J. 2020. Development of an optimized MALDI-TOF-MS method for high-throughput identification of high-molecular-weight glutenin subunits in wheat. Molecules. 25(18). https://doi.org/10.3390/molecules25184347.
DOI: https://doi.org/10.3390/molecules25184347

Interpretive Summary: High-molecular-weight glutenin subunits (HMW-GS) are a complex group of proteins accumulated in developing wheat grains. These proteins make an important contribution to the economic value of wheat because they are critical for the unique viscoelastic properties that make it possible to produce a wide range of products from the flour, including bread, noodles, tortillas and various baked goods. Because different HMW-GS can have large effects on the functional properties of the flour and its suitability for different products, it is critical to be able to identify individual HMW-GS for wheat breeding programs. This manuscript describes the optimal conditions for the separation of HMW-GS by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Analyses can be completed in about one minute per sample and require only minimal amounts of protein and solvents. The optimized method was validated using 24 standard wheat varieties grown in countries throughout the world as well as 38 Korean wheat cultivars and promises to be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.

Technical Abstract: Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.