Characterization and comparative analysis of wheat high molecular weight glutenin subunits by SDS-PAGE, RP-HPLC, HPCE, and MALDI-TOF-MS.
High molecular weight glutenin subunits (HMW-GS) from 60 germplasms including 30 common wheat cultivars and 30 related species were separated and characterized by a suite of separation methods including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (RP-HPLC), high-performance capillary electrophoresis (HPCE), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Comparative analysis demonstrated that each methodology has its own advantages and disadvantages. The main drawback of SDS-PAGE was its overestimation of molecular mass and incorrect identification of HMW-GS due to its low resolution. However, it had the advantages of technical simplicity and low requirements of equipment; thus, it is suitable for large-scale and high-throughput HMW-GS screening for breeding programs, especially when the glutenin composition is clear in the breeding material. MALDI-TOF-MS clearly expressed many technical advantages among the four methods evaluated, including high throughput, high resolution, and accuracy; it was, however, associated with high equipment cost, thus preventing many breeding companies from accessing the technology. RP-HPLC and HPCE were found to be intermediate between SDS-PAGE and MALDI-TOF-MS. Both RP-HPLC and HPCE demonstrated higher resolution and reproducibility over SDS-PAGE but lower detection power than MALDI-TOF-MS. Results demonstrated that MALDI-TOF-MS is suitable for analyzing HMW-GS for routine breeding line screening and for identifying new genotypes.
Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, 100048 Beijing, China.
Liyan
L
Gao
Wujun
W
Ma
Jing
J
Chen
Ke
K
Wang
Jing
J
Li
Shunli
S
Wang
Frank
F
Bekes
Rudi
R
Appels
Yueming
Y
Yan
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005983:Glutens / Q000737:chemistry*; D008970:Molecular Weight; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014908:Triticum / Q000737:chemistry*
D005983:Glutens; C037302:glutenin