Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat
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...
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doaj-e8c977dcea6d459598de02275786ebb42020-11-25T03:26:02ZengMDPI AGMolecules1420-30492020-09-01254347434710.3390/molecules25184347Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in WheatYou-Ran Jang0Kyoungwon Cho1Se Won Kim2Susan B. Altenbach3Sun-Hyung Lim4Jae-Ryeong Sim5Jong-Yeol Lee6National Institute of Agricultural Science, RDA, Jeonju 54874, KoreaDepartment of Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, KoreaNational Institute of Agricultural Science, RDA, Jeonju 54874, KoreaUSDA-ARS, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USADivision of Horticultural Biotechnology, Hankyong National University, Anseong 17579, KoreaNational Institute of Agricultural Science, RDA, Jeonju 54874, KoreaNational Institute of Agricultural Science, RDA, Jeonju 54874, KoreaBecause 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 1Bx7<sup>OE</sup> 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.https://www.mdpi.com/1420-3049/25/18/4347high-molecular-weight glutenin subunitsMALDI-TOF-MSwheat flour quality |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
You-Ran Jang Kyoungwon Cho Se Won Kim Susan B. Altenbach Sun-Hyung Lim Jae-Ryeong Sim Jong-Yeol Lee |
spellingShingle |
You-Ran Jang Kyoungwon Cho Se Won Kim Susan B. Altenbach Sun-Hyung Lim Jae-Ryeong Sim Jong-Yeol Lee Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat Molecules high-molecular-weight glutenin subunits MALDI-TOF-MS wheat flour quality |
author_facet |
You-Ran Jang Kyoungwon Cho Se Won Kim Susan B. Altenbach Sun-Hyung Lim Jae-Ryeong Sim Jong-Yeol Lee |
author_sort |
You-Ran Jang |
title |
Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat |
title_short |
Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat |
title_full |
Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat |
title_fullStr |
Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat |
title_full_unstemmed |
Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat |
title_sort |
development of an optimized maldi-tof-ms method for high-throughput identification of high-molecular-weight glutenin subunits in wheat |
publisher |
MDPI AG |
series |
Molecules |
issn |
1420-3049 |
publishDate |
2020-09-01 |
description |
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 1Bx7<sup>OE</sup> 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. |
topic |
high-molecular-weight glutenin subunits MALDI-TOF-MS wheat flour quality |
url |
https://www.mdpi.com/1420-3049/25/18/4347 |
work_keys_str_mv |
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