title: 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.
abstract: BACKGROUND
  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.
  
  
  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.
  
  
  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).
  
  
  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.
authors.affiliation: INSERM ERL U 1057, UMR 7203, F-75012, Paris, France. mamaubert@yahoo.fr
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Maubert
authors.forename: Elodie
authors.initials: E
authors.lastname: Quévrain
authors.forename: Estelle
authors.initials: E
authors.lastname: Capton
authors.forename: Jean Pierre
authors.initials: JP
authors.lastname: Grill
authors.forename: Ginette
authors.initials: G
authors.lastname: Thomas
authors.forename: Marie
authors.initials: M
authors.lastname: Bachelet
authors.forename: Dominique
authors.initials: D
authors.lastname: Rainteau
authors.forename: Germain
authors.initials: G
authors.lastname: Trugnan
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Tabet
authors.forename: Joëlle
authors.initials: J
authors.lastname: Masliah
authors.forename: Carlos
authors.initials: C
authors.lastname: Afonso
mesh_terms: D054360:Chemokine CXCL1 / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D018925:Chemokines / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D016209:Interleukin-8 / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000187:drug effects; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D054360:Chemokine CXCL1; D018925:Chemokines; D016209:Interleukin-8; D008070:Lipopolysaccharides; D010455:Peptides; D020543:Proteome