The use of in-strip digestion for fast proteomic analysis on tear fluid from dry eye patients

Abstract

Tear is an accessible fluid for exploring biomarkers of dry eye disease. This study describes a fast proteomic method by LC-Q-orbitrap-MS analysis with in-strip digestion and investigates the tear proteome of dry eye patients. Schirmer’s strips were used for collection of tear fluid from patients. These strips were cut into pieces and directly digested with trypsin before mass spectrometry analysis. The data showed that more than 50 proteins were found in tear fluid from dry eye patients. Gene Ontology (GO) annotation showed that most of proteins were transfer/carrier proteins, hydrolyses, enzyme modulators and signaling molecules. Targeted proteomics strategy revealed that 18 proteins were differentially expressed in dry eye patients. Furthermore, it was showed that the common post-translational modification in tear proteins is deamidation of Asn.

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“…Based on this interaction, the molecules are shown to be involved in potent biological pathway centering around cellular response to stimulus as part of regulatory mechanism towards homoeostasis. Previous reports in tear fluid proteome analysis in dry eye patients have shown similar findings [13,47].…”

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“…Topically and systemically administered drugs can be quantified in tear fluid to determine the clinical efficacy and dosing frequency from fitting of kinetic data. [211][212][213][214][215] Multiple "omics" approaches can also be utilized for analysis of the tear fluid including proteomics, 173,190,191,[216][217][218][219][220] lipidomics, 141,206,221,222 and metabolomics, 222…”

“…Schirmer tear strips are routinely used to measure tear volume for clinical assessment of dry eye disease in humans and veterinary species. 160,219,[252][253][254][255][256] The strips are made of Whatman no. 41 cellulose filter paper and possess specific characteristics to promote tolerance (5-mm width × 35-mm length, 0.22-mm thick, 20-25 µm porosity, foldable extremity for ease of insertion).…”

“…41 cellulose filter paper and possess specific characteristics to promote tolerance (5-mm width × 35-mm length, 0.22-mm thick, 20-25 µm porosity, foldable extremity for ease of insertion). 249,257 In addition to their conventional use for aqueous tear assessment, Schirmer strips can retrieve tear fluid for bioanalytical purposes in humans, 182,216,219,220,228,231,258 dogs, 190,209,211,214,215,240,259 and small laboratory species. 244,260,261 For instance, Schirmer strips were used for indepth characterization of proteomics in human 173 and canine tears, 190 and can also successfully recover specific analytes such as cytokines, 200,258 clusterin, 182 and xenobiotics.…”

“…For instance, the recovery of prednisone and prednisolone was maximized with a combination of centrifugation and elution in methyl tert-butyl ether, a solvent chosen over methanol and acetonitrile based on superior drug extraction from Schirmer strips ( Figure 4). 215 Of note, a comprehensive review of all reported protocols is beyond the scope of the present work, and further research is warranted to assess the potential benefits (or lack thereof) of extraction steps reported in the literature, such as cutting Schirmer strips into small pieces 173,219,262 or using ultrasonic agitation. 215,219,249 Ultimately, an optimized extraction protocol is important as it enhances the reliability of the data at hand, improving the sensitivity of the bioassay, and providing drug concentrations closer to "true" biological levels in the tear film.…”