Detection of cysteine residue numbers in wheat gluten proteins by MALDI-TOF

Abstract

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Abstract:The wheat seed storage protein plays a key role in determining processing quality.The disulfide bonds formed between cysteine residues in these proteins are critical in the formation of the unique rheological properties of wheat dough, which is the physical basis of bread making. Determining the number of cysteine residues in a particular protein, especially in high molecular weight glutenin subunits (HMW-GS)is an important task in evaluating wheat glutenin effects on end-product quality. In the current study, we established a fast method to accurately measure the number of cysteine residues in the HMW-GS. An alkylation reagent, 4-vinylpyridine (4-vp), was used to treat the proteins during extraction. For every cysteine residue in a protein, this treatment increases its molecular mass value by 105.14 Da, which can be accurately determined by MALDI-TOF equipment. Based on the changes of the molecular mass value caused by 4-vp treatment, the number of cysteine residue in a protein can be reliably determined. This method is also confirmed to be useful in studying non-glutenin proteins such as lupin seed storage proteins. It is expected that this method will speed up the process of selecting desirable HMW-GS in wheat breeding.

Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat

“…The increase in molecular mass of individual HMW-GS 1Dy12, 1By8, 1Bx7, and 1Dx2 in 4-vp treated samples was 737.2, 714.9, 401.6, and 425.7 Da, respectively, which is in good agreement with previous knowledge that y-type HMW-GS have 7 cysteine residues, and most x-type HMW-GS have 4 cysteine residues (with the exception of 1Dx5 and 1Bx20 that have 5 and 2 cysteines, respectively). Although the measured increase in molecular mass differs somewhat from the 4-vp molecular mass of 105.14 Da, it is within the instrument’s common error range of a 90–115 Da increase for one cysteine [ 42 ]. Taking together the results of Wang et al [ 42 ] and our results in this study, treatment with 4-vp showed enhanced resolution of mass spectra and could give useful information about the numbers of cysteine residues in the HMW-GS.…”

“…Although the measured increase in molecular mass differs somewhat from the 4-vp molecular mass of 105.14 Da, it is within the instrument’s common error range of a 90–115 Da increase for one cysteine [ 42 ]. Taking together the results of Wang et al [ 42 ] and our results in this study, treatment with 4-vp showed enhanced resolution of mass spectra and could give useful information about the numbers of cysteine residues in the HMW-GS.…”