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 that 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 specificity. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated neo N-termini were identified by exhibiting an MS1 Log2 H:L peak area ratio with a significant difference (p < 0.01) from zero and native N-termini with no significant difference from zero (p > 0.01). Using this strategy, all abundant, theoretical, protease-generated peptides in alpha/beta gliadins and gamma gliadins were identified. This study is the first study that modified and validated TAILS analysis for the analysis of partially hydrolyzed gluten proteins. The validation indicated that the strategy can identify multiple protease cleavage sites in gluten and may be a useful analytical technique in subsequent analysis of yeast cleavage patterns in beer throughout brewing. This strategy also may be further applied to characterize a broader range of partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.

ORGANISM(S): Wheat

SUBMITTER: Wanying Cao

PROVIDER: PXD013679 | panorama | Wed Nov 20 00:00:00 GMT 2019

REPOSITORIES: PanoramaPublic

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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:Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. The NS2B-NS3 viral protease cleaves the viral polyprotein. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. Moreover, while presence of NS2B/NS3 cleavage inhibits the activity of unlinked ZIKV NS2B-NS3 protease, no inhibition of protease activity was observed for unlinked DENV NS2B-NS3 full length with NS2B/NS3 cleavage site. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of flexible NS2B and NS3 N- and C-termini. We identified a second cis-cleavage site prior to NS2B C terminus and captured the crystal structure of self-cleaved NS2B47NS3 protease in post cleavage state. We report that the cleaved NS2B C-terminus occupy the neighbouring NS2B-NS3 molecule identifying a different mechanism where DENV NS2B C-terminus modulates NS2B-NS3 protease activity via trans-interaction in contrast to the cis-interacting NS2B C-terminus from the unlinked ZIKV protease. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the solvent-exposed beta-barrel of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.

Dataset Information

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

Publications

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

Similar Datasets

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