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High-resolution mass spectrometry and partial de novo sequencing constitute a useful approach for determining the profile of chemokine secretion following the stimulation of human intestinal epithelial cells.
- Source :
-
Rapid communications in mass spectrometry : RCM [Rapid Commun Mass Spectrom] 2013 Oct 15; Vol. 27 (19), pp. 2179-87. - Publication Year :
- 2013
-
Abstract
- Rationale: Intestinal epithelial cells (IEC) secrete many chemokines in response to proinflammatory stimuli. We investigated their role in the mucosal inflammatory response in the intestine, by developing a non-targeted approach for analyzing the profile of peptides secreted by stimulated IEC, based on differential mass spectrometry analysis.<br />Methods: Lipopolysaccharide (LPS) was incubated with IEC as a proinflammatory stimulus. Differential peptidomic analysis was then carried out, comparing the profiles of IEC with and without LPS stimulation. A mass spectrometry procedure was developed, based on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach without enzymatic pretreatment of the peptides. Partial de novo sequencing was carried out by Fourier transform ion cyclotron resonance (FTICR), and the native peptides in the culture media were identified.<br />Results: A major ion (m/z 7862.51) detected after stimulation was identified as GRO alpha and a minor ion (m/z 8918.17) was identified as IL-8. ELISA-based comparisons gave results consistent with those obtained by MS. Surprisingly, GRO alpha was secreted in amounts 5 to 15 times higher than those for IL-8 in our cellular model. The truncated form of IL-8, resulting from activation, was detected and distinguished from the native peptide by MS, whereas this was not possible with enzyme-linked immunosorbent assay (ELISA).<br />Conclusions: Mass spectrometric analysis of culture media can be used to identify the principal peptides produced in response to the stimulation of IEC, and their metabolites. Mass spectrometry provides a comprehensive view of the chemokines and peptides potentially involved in gut inflammation, making it possible to identify the most appropriate peptides for further quantification.<br /> (Copyright © 2013 John Wiley & Sons, Ltd.)
- Subjects :
- Chemokine CXCL1 analysis
Chemokine CXCL1 chemistry
Chemokine CXCL1 metabolism
Chemokines chemistry
Chemokines metabolism
Dose-Response Relationship, Immunologic
Enzyme-Linked Immunosorbent Assay
Epithelial Cells drug effects
HT29 Cells
Humans
Interleukin-8 analysis
Interleukin-8 chemistry
Interleukin-8 metabolism
Intestinal Mucosa cytology
Lipopolysaccharides pharmacology
Peptides analysis
Peptides chemistry
Peptides metabolism
Proteome drug effects
Chemokines analysis
Chromatography, Liquid methods
Epithelial Cells metabolism
Intestinal Mucosa metabolism
Tandem Mass Spectrometry methods
Subjects
Details
- Language :
- English
- ISSN :
- 1097-0231
- Volume :
- 27
- Issue :
- 19
- Database :
- MEDLINE
- Journal :
- Rapid communications in mass spectrometry : RCM
- Publication Type :
- Academic Journal
- Accession number :
- 23996391
- Full Text :
- https://doi.org/10.1002/rcm.6680