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Open Access Research article

Attempting to distinguish between endogenous and contaminating cytokeratins in a corneal proteomic study

Mikkel Lyngholm1*, Henrik Vorum23, Kim Nielsen1, Niels Ehlers1 and Bent Honoré2

Author Affiliations

1 Department of Ophthalmology, Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus C, Denmark

2 Department of Medical Biochemistry, Aarhus University, Ole Worms Allé 3, Bldg. 1170, 8000 Aarhus C, Denmark

3 Department of Ophthalmology, Aalborg Hospital, Aarhus University Hospital, Hobrovej 18-22, 9100 Aalborg, Denmark

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BMC Ophthalmology 2011, 11:3  doi:10.1186/1471-2415-11-3

Published: 27 January 2011

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.