A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Subjects provided written informed consent before participation in the study. The study was approved by the Human Ethics Committee of The Hong Kong Polytechnic University and adhered to the tenets of the Declaration of Helsinki.

1. Collection of human tear fluid with Schirmer strip

1. Wear gloves and sanitize to avoid sample contamination.
2. Check that the inner packaging is intact, sterile, and not expired.
3. Bend the top of the Schirmer strip inward slightly at the 0 mm mark, as indicated near the semicircle tip of the strip (Figure 1).
4. Remove the strip from the inner package by holding the end of the strip, and avoid direct contact with the sample collection area from 0 to 25 mm.
5. Ask the subject to look away from the position of the strip to be placed.
6. Pull down the lower eyelid gently and hook the end of the strip in the lower fornix near the lateral canthus region (Figure 2). Repeat the same procedure on the other eye.
7. Ask the subject to gently close the eyelids to minimize irritation.
8. Collect tear fluid for about 5 min or preferably 15-20 mm tear sample was collected.
9. Ask the subject to open both eyes, pull down the lower eyelid gently and remove the strips from the subject.
10. Inspect the hydrated length on the Schirmer strip and record the collected amount in mm.
11. Dry the strip with a standard, clean frame heater until the fluid is completely dry.
    ​NOTE: Excess heating that could potentially char the filter paper should be avoided.
12. Insert each dried Schirmer strip in a labeled cryotube and store it preferably in a cool, dry place in the dark until further processing.

2. Preparation of chemicals and reagents

1. Lysis Buffer (5% SDS, 50 mM TEAB, pH 7.5), 20 mL
   1. Pipette 1 mL of 1 M TEAB and 80 µL of 12% phosphoric acid. Vortex and sonicate briefly to remove bubbles. Top up to 20 mL with deionized water.
   2. Weigh 1 g of SDS and add to the solution with oscillation until dissolved.
2. Protein Precipitation Buffer (90% methanol, 100 mM TEAB, pH 7.1), 10 mL
   1. Add 893 µL of 1 M TEAB and 92 µL of deionized water.
   2. Add 15 µL of 85 wt. % phosphoric acid and 9 mL of methanol to the solution and vortex briefly.
3. Digestion Buffer (50 mM TEAB), 1 mL
   1. Add 50 µL of 1 M TEAB and top up to 1 mL with deionized water.
4. 200 mM dithiothreitol (DTT) solution, 500 µL
   1. Weigh 15 mg of DTT and dissolve in 500 µL of deionized water; vortex briefly.
      NOTE: Prepare before use.
5. 400 mM iodoacetamide (IAA) solution, 200 µL
   1. Weigh 15 mg of IAA and dissolve in 200 µL of deionized water; vortex briefly. NOTE: Prepare before use and avoid light.
6. 0.2% formic acid, 50% acetonitrile, 10 mL
   1. Prepare 10 mL of 50% acetonitrile in deionized water.
   2. Add 20 µL of formic acid and vortex briefly.
7. 0.2% formic acid solution, 10 mL
   1. Add 20 µL of formic acid to 10 mL of deionized water and vortex briefly.
8. 0.1% formic acid solution, 10 mL
   1. Add 10 µL of formic acid to 10 mL of deionized water; vortex briefly.

3. Protein extraction in Schirmer strip

1. Cut and discard the front end beyond the 0 mm mark of the strip to remove potential contamination due to the contact of conjunctival tissues with clean stainless-steel scissors.
2. Cut the strip in 1 mm intervals into a 1.5 mL microcentrifuge tube (Figure 3).
3. Add 100 µL of lysis buffer to the microcentrifuge tube, and vortex at 1,000 rpm, room temperature for 1 h in a thermomixer.
4. Centrifuge briefly and transfer the supernatant to a new 1.5 mL microcentrifuge tube.
5. Move the strips into 200 µL pipette tips using clean forceps. Place the tips into the same tube from step 3.4 with holder and centrifuge at room temperature at 4,000 × g for 1 min further for maximum recovery of the remaining sample on the strip.
6. Measure protein concentration using bicinchoninic acid (BCA) or compatible protein assay.

4. Suspension-trapping sample preparation

1. Normalize samples to 50 µg of protein based on the protein assay result.
2. Add 200 mM DTT in a ratio of 1:10 (v/v, DTT: sample) to a final concentration of 20 mM DTT and incubate at 95 °C for 10 min.
3. Cool down the protein solution to room temperature.
4. Add 400 mM IAA in a ratio of 1:10 (v/v, IAA: sample) to a final concentration of 40 mM IAA and incubate in the dark at room temperature for 10 min.
5. Add 12% aqueous phosphoric acid in a ratio of 1:10 (v/v, acid: sample) to a final concentration of 1.2% phosphoric acid and vortex briefly.
6. Add protein binding buffer in a ratio of 1:6 (v/v, sample: buffer) and vortex briefly.
   NOTE: In this step, colloidal protein particulate should be formed, and the solution will appear translucent if the protein amount is sufficient.
7. Uncap the Suspension Trapping Micro Spin Column and assemble the spin column onto a 2 mL microcentrifuge tube to collect flowthrough.
8. Add up to 200 µL of acidified protein lysate mixture into the column.
9. Centrifuge the column at 4,000 × g for 20 s. Protein particulates were trapped; repeat until the samples are loaded to the column.
10. Add 150 µL of protein binding buffer and centrifuge at 4,000 × g for 20 s to wash the suspended protein. Repeat 3x.
11. Assemble the Suspension Trapping Micro Column onto a new 1.5 mL microcentrifuge tube.
12. Add 20 µL of 50mM TEAB digestion buffer containing protease at a ratio of 1:25 (w/w, trypsin: protein) onto the filter inside the spin column, avoiding bubbles and air gap between the filter and digestion solution.
13. Cap the Suspension Trapping Micro Spin Column to prevent evaporation. Incubate at 47 °C for 1 h in a thermomixer; do not shake or vortex during digestion.
14. Elute peptides with 40 µL of 50 mM TEAB by centrifugal force at 4,000 × g for 20 s.
15. Add 40 µL of 0.2% FA and centrifuge at 4,000 × g for 20 s.
16. Add 35 µL of 0.2% FA, 50% acetonitrile, and centrifuge at 4,000 × g for 20 s.
17. Pool three eluates and dry with a vacuum centrifuge.
18. Store at -80 °C ultra-low temperature freezer until further analysis.NOTE: The protocol can be paused here.

5. Reconstitute peptides for mass spectrometry analysis

1. Reconstitute the sample with 12 µL of 0.1% FA, vortex, and centrifuge briefly.
2. Measure peptide concentration with a Peptide Assay Kit.
3. Normalize peptide concentration to 0.5 µg/µL in 0.1% FA.
4. Transfer the peptide mixture to an autosampler vial and ready for mass spectrometry analysis.

6. Sample Acquisition by Liquid Chromatography-Tandem Mass Spectrometry

1. Load 3 µg of the peptide with isocratic loading buffer (0.1% FA, 5% ACN) to a trap-column (C18, 5 µm, 100 µm, 20 mm) at a flow rate of 2 µL/min for 15 min.
2. Fractionate the peptides using a reversed-phase nano analytical column (C18, 5 µm, 100 µm, 300 mm) at a flow rate of 350 nL/min in a 155 min separation gradient. Use mobile phase A comprising a mixture of 0.1% formic acid (v/v), 5% ACN (v/v) in water and mobile phase B containing 0.1% FA (v/v), 98% ACN (v/v) in water. Use the following gradient: 0-0.5 min: 5% B, 0.5-90 min: 10% B, 90-120 min: 20% B, 120-130 min: 28% B, 130-135 min: 45% B, 135-141 min: 80% B, 141-155 min: 5% B.
3. Perform data-dependent acquisition (DDA) with a TOF-MS scan range between 350 to 1800 m/z with an accumulation time of 250 ms and a dynamic target exclusion time of 18 s. Then, carry out an MS/MS scan at 100 to 1,800 m/z in high-sensitivity mode with an accumulation time of 50 ms for the top 50 precursors per cycle with a threshold of 125 cps for MS/MS signal with a charge state from +2 to +4.
4. Analyze raw data with the referenced software or other compatible engines with a reference database in FASTA from the publicly accessible UniProt database.