Instrumentation used for chromatography included a nano-LC Ultimate 3000 HPLC (Dionex, Amsterdam, Netherlands) and Switchos and Famos autosampler (LC-Packings, Amsterdam, The Netherlands). One microliter of tear digests were concentrated and desalted onto a micro C18 pre-column (500 μm × 2 mm; Michrom Bioresources, Auburn, CA) with H
2O:CH
3CN (98:2, 0.05% trifluoroacetic acid) at 15 μL/min for 4 minutes. The precolumn (10 port valve; Valco, Houston, TX), which was connected via a fused silica capillary (Upchurch Scientific, Oak Harbor, WA), was automatically switched into a nano-flow liquid chromatography (LC) system.
37 Solvents used in chromatography were H
2O:CH
3CN (98:2, 0.1% formic acid; Solvent A) and H
2O:CH
3CN (36:64, 0.1% formic acid; Solvent B). Samples were eluted at approximately 300 nL/min over 30 minutes. Selection reaction monitoring analyses were performed on a 4000 Qtrap (Applied Biosystems). The MS/MS system consisted of an ion spray (voltage 2.4 kV), curtain gas flow of 12 μL/min and nebulizing gas flow of 5 μL/min with positive ion mode, and was controlled by Analyst 1.5 software (Applied Biosystems) and interface heater temperature set at 150°C. Collision energies (CE) were optimized as described before (CE = 0.043 m/z + 4.756)
38 for maximum transmission of individual “signature peptides.”
39 A total of 60 SRM transitions (3 per peptide) were monitored during an individual sample analysis. Instrument parameters remained unchanged for each unlabelled/labelled pair. To ensure that maximum specificity was achieved with samples as complex as human tears, SRM transitions were acquired at unit resolution in the first and third quadruples. Dwell times of 25 or 50 ms were used for selected transitions and cycle times did not exceed 1 second.