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

Mikkel Lyngholm,Kim Nielsen,Niels Ehlers,Bent Honoré,Henrik Vorum

doi:10.1186/1471-2415-11-3

Mikkel Lyngholm, Kim Nielsen + Show 3 more

Open Access

https://doi.org/10.1186/1471-2415-11-3

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Abstract

BackgroundThe 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.MethodsHere 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.ResultsIt 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.ConclusionsCK'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.

Highlights

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 represents a contaminant
In the search for stem cell markers, we previously investigated the differences in protein expression between the central corneal epithelium and the limbal epithelium by a proteomic approach, in an effort to identify proteins either highly expressed or exclusively present in limbal epithelium [13]
The intermediate epithelium (8-10 mm ring) was discarded and the limbal fractions were considered to be the epithelium scraped outside the 10 mm ring since, the conjunctival epithelium was not able to be as loosend by this method

Summary

Introduction

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 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. One way to diminish the environmental contribution of CK’s is to filter all the liquids used for 2D-PAGE, including the reducing agents [11] and to have efficient laboratory protocols to maintain clean conditions in general [12]

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