title: Attempting to distinguish between endogenous and contaminating cytokeratins in a corneal proteomic study.
abstract: BACKGROUND
  The observation of cytokeratins (CK's) in mass spectrometry based studies raises the question of whether the identified CK is a true endogenous protein from the sample or simply represents a contaminant. This issue is especially important in proteomic studies of the corneal epithelium where several CK's have previously been reported to mark the stages of differentiation from corneal epithelial stem cell to the differentiated cell.
  
  
  METHODS
  Here we describe a method to distinguish very likely endogenous from uncertain endogenous CK's in a mass spectrometry based proteomic study. In this study the CK identifications from 102 human corneal samples were compared with the number of human CK identifications found in 102 murine thymic lymphoma samples.
  
  
  RESULTS
  It was anticipated that the CK's that were identified with a frequency of <5%, i.e. in less than one spot for every 20 spots analysed, are very likely to be endogenous and thereby represent a 'biologically significant' identification. CK's observed with a frequency >5% are uncertain endogenous since they may represent true endogenous CK's but the probability of contamination is high and therefore needs careful consideration. This was confirmed by comparison with a study of mouse samples where all identified human CK's are contaminants.
  
  
  CONCLUSIONS
  CK's 3, 4, 7, 8, 11, 12, 13, 15, 17, 18, 19, 20 and 23 are very likely to be endogenous proteins if identified in a corneal study, whilst CK's 1, 2e, 5, 6A, 9, 10, 14 and 16 may be endogenous although some are likely to be contaminants in a proteomic study. Further immunohistochemical analysis and a search of the current literature largely supported the distinction.
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus C, Denmark. mikkel.lyngholm@ki.au.dk
authors.forename: Mikkel
authors.initials: M
authors.lastname: Lyngholm
authors.forename: Henrik
authors.initials: H
authors.lastname: Vorum
authors.forename: Kim
authors.initials: K
authors.lastname: Nielsen
authors.forename: Niels
authors.initials: N
authors.lastname: Ehlers
authors.forename: Bent
authors.initials: B
authors.lastname: Honoré
mesh_terms: D000818:Animals; D003315:Cornea / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis* / Q000235:genetics; D008223:Lymphoma / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D013953:Thymus Neoplasms / Q000737:chemistry
chemical_list: D007633:Keratins