Comparison of Capillary and Schirmer Strip Tear Fluid Sampling Methods Using SWATH-MS Proteomics Approach

Abstract

The purpose of this study was to examine the protein profile differences between capillary and Schirmer strip tear fluid samples. Methods: Both capillary and Schirmer strip tear samples were collected from 31 healthy participants at the same visit, and the samples were analyzed with nanoflow liquid chromatography coupled with time-of-flight mass spectrometer (NanoLC-MSTOF), implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). Sample type-specific and combined spectral libraries were used to evaluate the differences between the sample types in protein expression levels and biological functions. Results: In proportion, more extracellular proteins connected to immune response were quantified from the capillary samples while Schirmer strip samples contained more intracellular proteins. The sample types yielded similar counts of quantified proteins when a combined spectral library including both sample types was implemented. The differential expression analysis between the sample types identified proteins increased in the capillary samples (e.g., immunoglobulins) and Schirmer strip samples (e.g., heatshock proteins, annexins, and S100 proteins). Conclusions: Tear proteomics data originating from the same participants vary depending on whether the sample is collected with capillary or Schirmer strip, although there is also overlap between the two sample types when a combined spectral library is implemented in the SWATH-MS analysis. In discovery-based proteomics research of tear fluid, appropriate sampling method should be chosen carefully based on the research focus. Translational Relevance: Currently, there is no consensus on how the tear fluid sampling methods affect the resulting proteomics data, and hence, identification of the most suitable sampling methods for clinical researchers with varying research interests is important.

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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…”

“…A microcapillary glass tube (1-10 µl) placed in contact with the inferior lacrimal lake is the most commonly reported technique to collect tear fluid. This method directly samples tear fluid by capillary action and is extensively described in humans, 182,216,220,[226][227][228][229][230][231][232][233][234][235][236][237][238][239] dogs, 183,191,[240][241][242][243] rabbits, 188,237,238,241,244 mice, 245,246 and rats. 183,202,237,247 Other direct techniques (seldom reported) involve micropipettes, 248 polypropylene tubing, 183 or polytetrafluoroethylene tubing.…”

“…Another critical limitation of direct sampling is the low volume of tear fluid retrieved, generally ≤5 µl 182,183,220,226,227,230,[232][233][234]237,239,[244][245][246] -as such, the small sample collected may be grossly insufficient in some individuals, 182,242 may require excessive dilution that renders the target analyte undetectable, 243 and does not take into account possible losses (e.g., transfer and storage) or the need to repeat certain assays in duplicates.…”

“…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.…”

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“…A detailed description of the sample preparation steps has been previously described [ 17 , 18 ]. In brief, samples were flushed from capillaries with 0.5% sodium dodecyl sulphate (SDS) and protein concentration was measured with DC protein assay kit (Bio-Rad laboratories Inc, Hercules, USA).…”