Peter James
Professor
Differential proteomic analysis of HT29 Cl.16E cells line and intestinal epithelial cells by LC ESI/QTOF mass spectrometry.
Author
Summary, in English
Abstract in Undetermined
Intestinal epithelial cells (IECs) play a key role in Crohn's disease, a chronic inflammatory bowel disease which requires invasive examinations to be diagnosed. The comparison of the cellular protein expression profiles of Crohn's disease patients and healthy subjects is fundamental for the identification of proteins clinically relevant as new biomarkers or as drug targets. For this purpose a differential label-free nano-LC ESI/QTOF mass spectrometry (MS) approach combined with targeted MS/MS analysis has been developed and applied to isolated IECs.
We report here a study of the protein variations in IECs from healthy subjects (H) and Crohn's disease patients (CD). The method was previously validated using HT29 Cl.16E cell line, normal or treated with interferon-gamma as a model of inflammation.
Subcellular fractions proteins were extracted from HT29 and IECs and for each fraction monodimensional gel-electrophoresis was performed and the proteins subjected to tryptic digestion. The resulting peptides were analysed by LC ESI/QTOF MS and the obtained chromatographic runs were aligned with msInspect software. The peptides differently expressed were statistically evaluated using the Proteios Software Environment (ProSE) and identified by LC ESI/QTOF MS/MS analysis and database search.
The preliminary results obtained allowed the identification of many proteins involved in the inflammation processes.
Intestinal epithelial cells (IECs) play a key role in Crohn's disease, a chronic inflammatory bowel disease which requires invasive examinations to be diagnosed. The comparison of the cellular protein expression profiles of Crohn's disease patients and healthy subjects is fundamental for the identification of proteins clinically relevant as new biomarkers or as drug targets. For this purpose a differential label-free nano-LC ESI/QTOF mass spectrometry (MS) approach combined with targeted MS/MS analysis has been developed and applied to isolated IECs.
We report here a study of the protein variations in IECs from healthy subjects (H) and Crohn's disease patients (CD). The method was previously validated using HT29 Cl.16E cell line, normal or treated with interferon-gamma as a model of inflammation.
Subcellular fractions proteins were extracted from HT29 and IECs and for each fraction monodimensional gel-electrophoresis was performed and the proteins subjected to tryptic digestion. The resulting peptides were analysed by LC ESI/QTOF MS and the obtained chromatographic runs were aligned with msInspect software. The peptides differently expressed were statistically evaluated using the Proteios Software Environment (ProSE) and identified by LC ESI/QTOF MS/MS analysis and database search.
The preliminary results obtained allowed the identification of many proteins involved in the inflammation processes.
Department/s
- Department of Immunotechnology
Publishing year
2009
Language
English
Pages
865-873
Publication/Series
Journal of Proteomics
Volume
72
Issue
5
Document type
Journal article
Publisher
Elsevier
Topic
- Immunology in the medical area
Keywords
- Crohn's disease
- HT29 Cl.16E
- Intestinal epithelial cells
- Label-free mass spectrometry
- Proteomics
Status
Published
ISBN/ISSN/Other
- ISSN: 1874-3919