Mass Spectrometric Detection of Keratins in Tear Fluid.

Purpose: Keratin contamination is a common problem in mass spectrometry proteomic analyses, particularly in bottom-up mass spectrometry. The purpose of this study was to determine the protein contaminants introduced during the proteomic analysis of tear fluid.

Methods: Human tear fluid samples were collected using Schirmer strips. Proteomic analyses were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on blank Schirmer strips and tear fluid samples, with empty vials serving as controls for assessing environmental contaminant proteins.

Results: We detected 26 contaminant proteins (18 keratins and 8 non-keratins). 98.2% of the total protein contamination can be attributed to the 9 keratins, including KRT10 (23.6%), KRT1 (23.5%), KRT2 (15.7%), KRT14 (7.6%), KRT16 (7.0%), KRT5 (6.1%), KRT9 (5.9%), KRT6B (4.6%), and KRT6A (4.3%). A comparison to the proteomic profile of blank Schirmer strips and controls (empty vials) found a strong correlation (R=0.9753), indicating that these proteins were not from the blank Schirmer strips but are environmental contaminants. On the other hand, several keratins including KRT19, KRT13, KRT4, KRT7, KRT15, KRT8 and KRT18 were present in tear fluid, but either not detected or were negligible in blank strips. Another set of keratins, including KRT5, KRT6A, KRT14, KRT16, and KRT17, were identified as components of tear fluid as well as environmental contaminants.

Conclusions: This study revealed nine major contaminant keratins in the mass spectrometry analysis. Several other keratins were identified as constituents of tear fluid. Background subtraction is necessary for the accurate analysis of tear fluid using mass spectrometry.