Dataset Information

Dia-PASEF HeLa dilution series 0.2ng - 200ng on Bruker timsTOF Pro 2

ABSTRACT: Different sample preparation methods were tested for HeLa proteome analysis. A sample obtained using sodium deoxycholate-based lysis allowed identification of the highest number of proteins. For this sample, a dilution series was acquired in triplicates ranging from 0.2ng to 200ng. All measurements were performed on Bruker timsTOF Pro 2 operated in dia-PASEF mode and analysed library-free using DIA-NN 1.8.

INSTRUMENT(S): Bruker Daltonics timsTOF series

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Vadim Demichev

LAB HEAD: Markus Ralser

PROVIDER: PXD029836 | Pride | 2022-06-20

REPOSITORIES: Pride

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Project description:Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in the pathogenesis of CD, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent Nε-(γ-glutamyl)-lysine isopeptide crosslinks resulting in TG2-gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear. To gain more insight into the molecular structures of TG2-gluten peptide complexes, we developed a reciprocal proteomic analysis strategy, which facilitates the comprehensive identification of isopeptides in a complex protein hydrolysate. The workflow consists of four steps: (1) performance of the model reaction between TG2 and gluten peptide(s) followed by tryptic hydrolysis, clean-up and untargeted nLC-MS/MS analysis (2), prediction of tryptic TG2-derived lysine peptides that serve as potential isopeptide modifications (3) the search for isopeptides by configuring the TG2-modifications in MaxQuant and search against the α-gliadin fasta file and (4) the reciprocal search for isopeptides by configuring the gluten peptide(s) as modification in MaxQuant and search against the TG2 fasta file. After verification by manual data curation and visualization, this strategy enabled the identification of 26 different isopeptides involving 19 TG2 lysine residues, only six of which were known previously. Experiments with different TG2-gluten peptide molar ratios revealed that K-425, K-590, K-600 and K-649 were the most preferred lysine residues involved in isopeptide crosslinking. Further, expanding the model system to three gluten peptides allowed the localization of the preferred glutamine crosslinking sites. The described strategy is generally applicable to any hydrolysate containing isopeptides and these new insights into the structure of TG2-gluten peptide complexes may help clarify the role of extracellular TG2 in CD autoimmunity and in other inflammatory diseases.

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Dia-PASEF HeLa dilution series 0.2ng - 200ng on Bruker timsTOF Pro 2

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