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Comparative Study
. 2011 Jan 27:11:3.
doi: 10.1186/1471-2415-11-3.

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

Mikkel Lyngholm  1 , Henrik Vorum, Kim Nielsen, Niels Ehlers, Bent Honoré
Affiliations
Comparative Study

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

Mikkel Lyngholm et al. BMC Ophthalmol. .

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.

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Figures

Figure 1
Figure 1
2D-gels (A:12%; B:6%) from the limbal fraction shows the significant spots (marked with the SSP number). The approximate pI and molecular mass is indicated on the axes. SSP No. 0103 and 1205 were not expressed on this gel (B:6%).
Figure 2
Figure 2
Immunohistochemical staining against various antigens. (A) CK 15 and (B) CK 19 appear in the limbal epithelium. (C) CK 3/CK 12 stainings in central corneal epithelium.
Figure 3
Figure 3
Peptide sequences from the Mascot search results. A: CK 19 identified in a 39-kDa spot by 7 peptides (Matched peptides shown in bold red). It is most likely an endogenous protein although a contaminating protein cannot be excluded. B: Two peptides (24 amino acids) identify CK 3 from a spot focused around 28 kDa. This could be an endogenous protein or it could be due to contamination of the samples. C: Two peptides (20 amino acids) identify CK 4 from a spot focused around 60 kDa. This could be an endogenous protein or it could be a contamination of the samples. D: CK 9 identified by the matched peptides throughout the whole protein. Since the sample originates from a spot focused around 10 kDa in the gel the protein is regarded as a contaminant.
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