Differential proteomic analysis of HT29 Cl.16E and intestinal epithelial cells by LC ESI/QTOF mass spectrometry.
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.
Nanni
Paolo
P
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, Bologna 40126, Italy.
Mezzanotte
Laura
L
Roda
Giulia
G
Caponi
Alessandra
A
Levander
Fredrik
F
James
Peter
P
Roda
Aldo
A
D015415:Biomarkers; D002460:Cell Line; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D007422:Intestines / Q000166:cytology*; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013347:Subcellular Fractions
D015415:Biomarkers; D010455:Peptides; D011506:Proteins; D020543:Proteome