The ability of mass spectrometry to detect histatin in tear samples collected on Schirmer strips suggests that this method may be useful for determining the abundance of histatin and other tear film proteins in dry eye patients.
Conclusion: We have identified histatin as a component of tear film following elution from Schirmer strips. Sample retention time was identical to that of the histatin standard. Results: Mass spectral data positively identified the presence of histatin in tear samples eluted from Schirmer strips. Samples were analyzed by RP-HPLC and electrospray mass spectrometry. Following centrifugation, the supernatant was removed, evaporated, and reconstituted in de-ionized water. An equal volume of an acid pretreatment was added to remove higher molecular weight proteins. Tears were eluted from dried Schirmer strips in a methanol: water mixture (75:25) with 0.1% formic acid.
Methods: Tears were collected from normal volunteers. We therefore explored the possibility of extracting tears directly from Schirmer strips for histatin analysis. Utilization of tear samples directly from Schirmer strips collected as part of routine ocular surface exams would be particularly advantageous in the acquisition of tear samples without repeated or extraneous sampling methods, especially in patients with low tear volumes. Tear sampling and low protein concentrations create specific challenges in the assessment of tear film. Cationic gel electrophoresis coupled with both tryptic hydrolysis and mass spectrometry demonstrated that mass spectral results were consistent with the presence of histatin in fractionated "whole" tears. Using RT-PCR, we have shown that histatin, like MUC7 and defensins, is present in ocular tissue. Purpose: Antimicrobial proteins, including MUC7, defensins, and histatins, are products of the oral mucosa and participate in the non-immune defense system that protects the oral cavity from opportunistic infection.
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