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

Our research aims to demonstrate the use of a protein suspension-trapping sample preparation, known as S-Trap, for mass spectrometry-based research. This protocol employs a rapid, reproducible, and standardized sample preparation of human tear fluid sample, collected using the Schirmer strip for translational research in ophthalmology. Human tear fluid is a biofluid that can be collected for non-invasive, individual molecular tests in ophthalmology.

There is a lack of standardized methods of tear fluid collection and sample preparation, which are critical to successful clinical research of diseases and conditions. Non-standardized methods are difficult to compare and interpret between methodologies, and therefore affect clinical outcomes. This protocol allows tear biofluid sampling and storage at room temperature.

This helps with easier sample collections and shipping. It provides a rapid and robust peptide extractions for mass spectrometry analysis, which improves peptide recovery and identifications. This protocol has minimal sample preparation steps to minimize error and uncertainties.

The fast and superior performance of protein identification and quantification by mass spectrometry will facilitate clinicians and researchers in the development of non-invasive molecular tests. Optimized sample preparation will always be one of the critical steps, regardless of the detection method. The clinical value of human tear fluid is yet to be fully investigated.

The proposed sample preparation method would hopefully open the door for potential chemical application in the near future. Begin by wearing gloves and sanitizing hands to prevent sample contamination. Check that the inner packaging of the Schirmer strip is sterile, unexpired, and intact.

Next, bend the top of the Schirmer strip slightly inward at the zero millimeter mark near the semicircle tip. Remove the strip by holding the end, avoiding direct contact with the zero to 25-millimeter sample collection area. Instruct the subject to look away, then gently pull down the lower eyelid.

Hook the strip end in the lower fornix near the lateral canthus region. After repeating the process for the other eye, ask the subject to close their eyelids gently to minimize irritation. Collect the tear fluid for approximately five minutes until a 15 to 20-millimeter tear sample is obtained.

Instruct the subject to open their eyes. Gently pull down the lower eyelid and remove the strips. Next, inspect the hydrated length on the strip and record the collected amount in millimeters.

Dry the strip using a standard, clean frame heater until the fluid evaporates. Once dried, place the Schirmer strip into a labeled cryo tube and store it in a cool dark place for further processing. Begin by cutting and discarding the front end of the Schirmer strip containing the dried human tears beyond the zero-millimeter mark.

Then cut the strip into one-millimeter intervals, and place the pieces in a 1.5-milliliter microcentrifuge tube. Next, add 100 microliters of lysis buffer to the microcentrifuge tube. Vortex it at 1000 RPM at room temperature for one hour in a thermomixer.

After brief centrifugation, transfer the supernatant to a new 1.5-milliliter microcentrifuge tube. Using clean forceps, move the strips into 200-microliter pipette tips. Place the tips back into the same micro centrifuge tube, then centrifuge it for maximum sample recovery.

Measure the protein concentration using bicinchoninic acid or a compatible protein assay. Begin by normalizing the human tear protein samples to 50 micrograms of protein. Then add 200 millimolar dithiothreitol, or DTT, to a final concentration of 20 millimolar DTT.

Incubate it at 95 degrees Celsius for 10 minutes. After cooling the protein solution to room temperature, add 400 millimolar iodoacetamide to a final concentration of 40 millimolar. Then incubate in the dark at room temperature for 10 minutes.

Next, add 12%aqueous phosphoric acid in a one-to-10 ratio, resulting in a 1.2%final concentration. Vortex the mixture briefly to mix the solution. Add protein-binding buffer in a one-to-six ratio before vortexing again.

Uncap the suspension-trapping MicroSpin column and assemble the spin column onto a two-milliliter microcentrifuge tube to collect flow through. Add up to 200 microliters of acidified protein lysate mixture into the column. Centrifuge the column at 4, 000 G for 20 seconds.

Next, add 150 microliters of protein-binding buffer and centrifuge as before to wash the suspended protein. Assemble the suspension-trapping microcolumn onto a new 1.5-milliliter microcentrifuge tube. Carefully add 20 microliters TEAB digestion buffer onto the filter inside the spin column, avoiding bubbles and air gaps.

Securely cap the suspension-trapping MicroSpin column to prevent evaporation. Incubate at 47 degrees Celsius for one hour in a thermomixer without shaking. Elute peptides with 40 microliters of 50 millimolar TEAB.

Then centrifuge it at 4, 000 G for 20 seconds. Now add 40 microliters of 0.2%formic acid and centrifuge as before. Lastly, add 35 microliters of 0.2%formic acid and 50%acetonitrile before centrifuging again.

Pull the three eluates and dry them using a vacuum centrifuge. Once dried, store the samples at minus 80 degrees Celsius for future analysis. The data-dependent acquisition search yielded a total of 1, 183 plus or minus 118 proteins in the suspension-trapping group, and 874 plus or minus 70 proteins at 1%FDR in the in-solution group.

Gene ontology analysis revealed similar proteomes for both approaches with main function as binding catalytic activity and molecular function regulation.