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Characterization of the small intestinal lesion in celiac disease by label-free quantitative mass spectrometry

ABSTRACT: This study describes a comparison of global proteome expression in small intestinal tissue collected from celiac disease patients at different stages of disease. We have analyzed sections from FFPE biopsy blocks and identified more than 4500 protein without sample pre-fractionation. We have compared protein expression in two sample cohorts that were chosen retrospectively from patients participating in research protocols at our facility. First we compared biopsies from 10 patients collected at the time of diagnoses (untreated CD) with biopsies collected 1 year later following gluten free diet (treated CD). We observed overall good agreement between differential expression of proteins and histological changes that occur in the intestine. We identified biological pathways that are known to operate in the disease lesion. We also observed a strong signal for neutrophil infiltration. By use of a dedicated immunoglobulin variable gene family database we found that differential expression of IGHV5-51 distinguished our untreated from treated CD patients. We also compared protein expression in 4 treated CD patients that developed histological changes in the small intestine in response to a 3 day gluten challenge. Although these patients develop an intestinal lesion resembling the lesion observed in untreated patients, we observed differential expression of proteins involved in acute tissue remodeling that were not seen when we compared UCD to TCD samples. Our study presents a proof-of-concept to demonstrate that analysis of material from FFPE tissue block material can provide a comprehensive overview of many disease processes that occur in the celiac small intestine. Changes in protein expression will reveal information on disease state that may not be observed by histological evaluation

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Small Intestine

DISEASE(S): Celiac Disease

SUBMITTER: Jorunn Stamnaes

LAB HEAD: Ludvig M. Sollid

PROVIDER: PXD008992 | Pride | 2018-11-21

REPOSITORIES: Pride

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Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry.

Tutturen Astrid E V AEV Dørum Siri S Clancy Trevor T Reims Henrik M HM Christophersen Asbjørn A Lundin Knut E A KEA Sollid Ludvig M LM de Souza Gustavo A GA Stamnaes Jorunn J

The American journal of pathology 20180422 7

Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens ...[more]

PMID: 29684362

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Project description:Background & Aims: Traditional celiac disease diagnostics based on histomorphometric evaluations are liable to misinterpretations due to the common technical flaws e.g. wrong orientation of the biopsy and subjective errors are relatively common e.g. interobserver errors. We envisioned that there is a need for molecular histomorphometric tool that obviates these error sources yielding an objective ratio instead. Gene expression determine the state of a tissue, so the changes in expression may be associated with the severity of lesions during CD and can be used to classify it. Methods: 15 CD patients, who have been at least one year on gluten-free diet, were enrolled. All participants were biopsied before and after the gluten challenge (10 weeks, 4 grams of gluten daily). 6 healthy non-CD individuals were included as controls. Biopsies were taken on PAXgene biological fixative and embedded in paraffin and Vh/Cd ratio was assessed. RNA was extracted from the same sections and subjected to genome-wide 3’RNA-sequencing. Sequencing data was used to determine differentially expressed genes and regression model, that successfully describes the mucosal damage, was created and tested in independent material. Results: 167 differentially expressed genes were identified in healthy vs. treated CD comparisons with 117 genes downregulated and 50 genes upregulated. 415 differentially expressed genes were identified in Post gluten challenge to Treated CD comparisons with 195 genes downregulated and 220 genes upregulated. 119 genes whose expression highly correlates to Vh/Cd ratio (Spearman’s rank correlation coefficient, |rho|>0.7) were identified. Gene ontology analyses show that genes involved in cellular response to cytokines, including interferons, were over-represented. Stepwise regression allowed us computationally cut down the number of genes, that describe Vh/Cd ratio changes, to 7 and IELs number to 5. Created models describe 98.9% of observed Vh/Cd and 97.5% of IELs number variabilities; there is a strong correlation between the model’s predicted and observed ratios. Conclusions: Adoption of molecular histomorphometry, with our selected set of target genes, is quantitative and reliable way of estimating gluten-induced mucosal injury and inflammation. By including this technology one can overcome the typical shortcomings common in celiac disease diagnostics based on traditional histomorphometry analyses alone. Likewise, molecular histomorphometry is a promising instrument when incorporated in clinical trials where assessing drug efficacy on mucosal health is paramount. In addition, despite deemed healthy, based on traditional histomorphometric analyses, celiac patients on gluten free diet have significantly distinctive molecular histomorphometric pattern when compared to healthy controls.

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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