Dataset Information

Validation of N-terminal Labeling Mass Spectrometry for Characterization of Partially Hydrolyzed Gluten Proteins

ABSTRACT: Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder which affects about 1% of people worldwide. The toxicity of partially hydrolyzed gluten (PHG) in fermented products such as beer is unknown due to the significant analytical challenges in PHG detection. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized for the in-depth analysis of PHG and validated using a test protease (trypsin) with known cleavage specificities. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated and native N-termini were confirmed by showing an MS1 peak area Log2 H:L with and without a significant difference (p < 0.01) from zero, respectively. Using the strategy, all abundant theoretical protease generated peptides in alpha/beta gliadins and gamma gliadins were identified. The validation indicated that the strategy can identify multiple protease cleavage sites in gluten and be used in subsequent analysis of yeast cleavage patterns in beer throughout brewing. This strategy can be further applied to characterize a broader partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Triticum Aestivum (wheat)

TISSUE(S): Seed

SUBMITTER: Wanying Cao

LAB HEAD: Melanie L. Downs

PROVIDER: PXD012296 | Pride | 2019-11-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20170918_VC-MD_WNNC.msf	Msf
	20170918_VC-MD_WNNC.msfView	Msf
	20170918_VC-MD_WNNC.pdResult	Other
	20170918_VC-MD_WNNC.pdResultView	Other
	20170918_VC-MD_WNNC_a.raw	Raw

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Publications

Evaluation of N-terminal labeling mass spectrometry for characterization of partially hydrolyzed gluten proteins.

Cao Wanying W Baumert Joseph L JL Downs Melanie L ML

Journal of proteomics 20191017

Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder that affects about 1% of people worldwide. The toxicity of partially hydrolyzed gluten (PHG) in fermented products is less well understood due to the significant analytical challenges in PHG characterization. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized for the in-depth analysis of PHG and vali ...[more]

PMID: 31629960

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Project description:Within the complex wheat flour proteome, the gluten proteins have attracted most of the attention because of their importance in determining the functional properties of wheat flour doughs and their roles in human health conditions such as celiac disease and food allergies. However, certain non-gluten proteins also trigger immunological responses but may be present in flour in low amounts or obscured by the more abundant gluten proteins in two-dimensional gels of total protein preparations. Non-gluten proteins were preferentially extracted from the flour with a dilute salt solution and separated by two-dimensional gel electrophoresis. Proteins in 172 gel spots were identified by tandem mass spectrometry after cleavage with trypsin or chymotrypsin. Fifty-seven different types of non-gluten proteins were identified, including 14 types that are known or suspected immunogenic proteins. The predominant proteins in 18 gel spots were gluten proteins. Transgenic wheat lines in which specific groups of gluten proteins were suppressed by RNA interference were used to estimate the amount of carry-over of gluten proteins in the salt-soluble protein fraction. Analysis of salt-soluble proteins from a transgenic line missing omega-1,2 gliadins demonstrated that certain omega-1,2 gliadins were present in large amounts in the salt-soluble fraction and obscured relatively small amounts of beta-amylase and protein disulfide isomerase. In comparison, analysis of a transgenic line in which alpha gliadins were absent revealed that glyceraldehyde-3 phosphate dehydrogenase was a moderately abundant protein that co-migrated with several alpha gliadins. The proteomic map of the non-gluten protein fraction of wheat flour developed in this study complements a proteomic map of the total flour proteins developed previously for the same cultivar. Knowing the identities of low abundance proteins in the flour as well as proteins that are hidden by some of the major gluten proteins on two-dimensional gels is critical for studies aimed at assessing the immunogenic potential of wheat flour and determining how the growth conditions of the plants affect the levels of specific immunogenic proteins in the flour.