Phenol Red Thread-based Sampling Procedure for Untargeted Tear Fluid Lipidomics in Biomarker Discovery

Summary

This protocol describes a unique clinical protocol for collecting human tear fluid samples using phenol red threads and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based workflow to discover the tear lipidomic profile. The simple methyl tert-butyl ether (MTBE)/methanol biphasic separation method enables rapid tear lipid extraction with high recovery for tear biomarker discovery.

Abstract

Tear lipids are crucial for tear film stability and ocular surface health. Changes in tear composition could be associated with ocular and systemic diseases such as meibomian gland dysfunction and dyslipidemia. Profiling tear lipids may help biomarker discovery for disease diagnosis and management. However, common tear sampling methods present distinct limitations: Schirmer's strips frequently cause ocular irritation and discomfort due to their large contact area with the eye surface, while microcapillary tube collection could yield low reproducibility due to operator variability, especially when performed by different personnel. These limitations might compromise the accuracy and consistency of lipidomic data. This study introduces a minimally invasive phenol red thread (PRT)-based sampling method optimized for tear lipidomics. The thin structure of PRT minimizes the risk of ocular irritation and allows rapid and gentle tear collection. This user-friendly and easy-to-perform method is suitable for subjects with reduced tear volume or lower tolerance for foreign body sensation, and it enables more reproducible sample collection by reducing operator-dependent variability. Tear lipids collected by PRT were extracted using an optimized methanol/methyl tert-butyl ether (MTBE) phase separation protocol and analyzed by high-resolution LC-Orbitrap-IQX MS/MS with LipidSearch software. The workflow identified more than 700 unique tear lipid species, each characterized by specific fatty-acid-derived product ions. These results indicate that PRT-based sampling provides robust lipid recovery for tear lipidomic analysis. This minimally invasive and reproducible approach offers a practical platform for clinical and experimental tear lipid research. Ultimately, this could also facilitate biomarker discovery and disease monitoring.

Introduction

Tear film is the outermost barrier that protects the ocular surface from pathogens and maintains ocular homeostasis^1,2, while the lipid layer in tear film is the key component for preventing tear evaporation and maintaining tear film stability^3,4. Dysregulation of the tear lipid composition has been linked to various eye diseases, including dry eye disease (DED), meibomian gland dysfunction (MGD), and allergic conjunctivitis^5,6,7,8. As a result, detailed characterization of tear lipidomics has become a critical and trending research direction for a more comprehensive understanding of the molecular pathologies underlying these ocular conditions⁹.

While the tear lipidomic approach holds significant clinical importance, a critical challenge in tear lipidomics is establishing a standardized, reproducible collection and analysis workflow. Current tear sampling methods, Schirmer's strips and microcapillary tube, each present distinct limitations. Schirmer's strips have a large contact area with the ocular surface that often causes ocular irritation and reflex tearing^9,10. In comparison, microcapillary tube collection is highly dependent on operator technique and can yield inconsistent results when performed by different personnel¹¹. Additionally, improper use of these sampling tools may also carry a risk of ocular surface injury. These limitations could compromise the accuracy and reproducibility of downstream data^12,13. Furthermore, tear collection methods that require complex handling procedures may limit their accessibility and standardization across different laboratories and clinical settings. Therefore, there is a pressing need to establish a non-invasive, robust, and reproducible tear collection and processing workflow that can preserve the native tear lipid profile.

Recent studies have highlighted the utility of phenol red thread (PRT) for downstream LC-MS/MS workflows analyses in proteomic and metabolomic applications^14,15. PRT offers inherent advantages as a tear collection tool. Its thin structure, minimally invasive nature, and straightforward sampling procedure could minimize technical complexity and reduce operator-dependent variability, while being well-tolerated by most subjects^14,16. However, a standardized workflow specifically for PRT-based tear lipidomic analysis has not yet been established.

This protocol introduces an integrated workflow for reproducible tear lipidomics: a user-friendly PRT-based tear collection method adapted for lipid analysis, an optimized methyl tert-butyl ether (MTBE)/methanol lipid extraction protocol with defined solvent-to-sample ratios and storage controls^17,18, and a modified lipid identification method using high-resolution LC-MS/MS. This standardized approach enables a consistent tear lipidomic profiling for downstream biomarker discovery and disease characterization. Also, it is particularly well-suited for subjects with low tear volume or reduced tolerance for foreign body sensation (Figure 1).

Figure 1: Schematic overview of the Phenol Red Thread (PRT)-based workflow for human tear lipidomics. Tear fluid is collected from subjects utilizing the PRT approach, which enables consistent sample acquisition with minimal discomfort. Sample processing involves sequential MTBE/methanol biphasic extraction, SpeedVac-mediated concentration, and reconstitution of tear lipid extracts for liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipidomic profiles are subsequently acquired via LC-MS/MS and subjected to annotation and identification using LipidSearch software. Please click here to view a larger version of this figure.

Protocol

The study was approved by the Institutional Review Board (IRB) of The Hong Kong Polytechnic University. The subjects provided written informed consent before participation in the study. The reagents and the equipment used are listed in the Table of Materials.

1. Phenol Red Thread (PRT)-based collection of human tear fluid

1. Wear gloves and disinfect the workstation to prevent sample contamination.
2. Make sure the PRT package is sealed and not expired before opening it.
3. Hold the thread by the end opposite the bent hook. Always avoid touching the bent hook half of the PRT.
4. Ask the subject to look at the superior nasal direction.
5. Pull down the subject's lower eyelid gently and insert the PRT with the bent hook positioned in the lower temporal palpebral conjunctiva near the outer canthus of the subject's eye (Figure 2).

Figure 2: Position of the phenol red thread during tear collection. Please click here to view a larger version of this figure.

6. Ask the subject to close their eyes gently and tilt his/her head slightly forward to prevent the PRT from touching his/her facial skin.
   NOTE: Tear absorbed would gradually induce a red dye front, indicating the sampled region of PRT due to the pH-sensitive phenol red. To ensure enough tear fluid for lipidomic analysis, collecting tear volume equivalent to 50 mm of PRT is preferred, typically completed within 2 min.
7. Pull down the subject's lower eyelid gently and pull out the sampled PRT. Avoid touching the sampled region.
8. Record the length of the sampled region of the PRT and transfer the PRT into a new sample tube with the aid of sterile forceps.
   NOTE: If the sampled PRT length is <50 mm, kindly ask the subject to rest for 1-3 min and repeat the collection with the same eye. Transfer each sampled PRT to a separate sample tube.
9. Label and cap the sample tubes properly.
10. Clean the gloves and forceps thoroughly with methanol and clean paper wipes to remove any tear residuals or contaminants, and then let them dry.
11. Repeat steps 1.2-1.10 for the other eye.
12. Seal the sample tubes with parafilm and immediately store the sample at -20°C for temporary storage or -80°C for long-term storage. Avoid unnecessary freeze-and-thaw cycles and protect the sampled PRT from light.

2. Processing of sampled PRT

1. Pre-wash the micro-scissors with methanol before processing the PRTs.
2. Discard the bent hook end (i.e., first 3 mm) of the PRTs (Figure 3).

Figure 3: Physical appearance of the phenol red thread (PRT). (A) Unused PRT with bent hook end. (B) Used PRT with the indicated section cut and collected for lipid extraction. Please click here to view a larger version of this figure.

3. Cut the sampled region of the PRT (i.e., region above the phenol red dye front) into 2 mm pieces and transfer these pieces to a clean 1.5 mL organic solvent-tolerant microcentrifuge tube.

3. MTBE/methanol biphasic separation for lipid extraction from sampled PRT

CAUTION: MTBE and methanol are flammable and volatile. Use in a well-ventilated area or fume hood. Avoid open flames and sparks. Wear gloves and a lab coat. Wash hands after handling.

1. Place the samples on ice.
2. Add 232 µL of ice-cold methanol (MS grade) to each sample and vortex for 15 s.
3. Sonicate the samples in a pre-cooled ultrasonic cleanser for 15 min.
4. Add 774 µL of MTBE (HPLC grade) and 194 µL of deionized water to each sample and vortex for 15 s. The resultant volume ratio of MTBE:methanol:water in the sample should be 4:1.2:1 (v/v/v).
5. Sonicate the samples again in a pre-cooled ultrasonic cleanser for 15 min.
6. Incubate the sample in the thermomixer for 8 h at 4 °C, with shaking at 1,200 rpm.
7. Let the mixture sit at room temperature for 10 min.
8. Centrifuge the resultant mixtures at 10,000 × g for 10 min at 4 °C.
9. After centrifugation, the mixtures are separated into two phases (Figure 4).
   NOTE: Upon completion of phase separation, inspect the sample for two distinct layers: the upper organic phase (MTBE-rich) should appear transparent with a slight milky opacity, while the lower aqueous phase exhibits a yellow coloration attributable to the presence of phenol red indicator. Proceed only if these phase characteristics are observed, as shown in Figure 4.

Figure 4: Phase separation of lipid extraction using MTBE-methanol-water: upper organic phase and lower aqueous phase. Please click here to view a larger version of this figure.

10. Collect 700 µL of the upper organic fraction containing lipids carefully and transfer to a pre-chilled new 1.5 mL microcentrifuge tube.
11. Discard the lower aqueous phase containing residual MTBE and methanol as hazardous chemical waste. Collect waste in labeled, sealed containers and dispose of it according to institutional hazardous waste protocols and local regulations. Do not pour solvents down the drain.
12. Dry the lipid extracts using a refrigerated SpeedVac concentrator for 4 h at 4 °C.
    NOTE: To ensure complete drying, periodically check the sample dryness starting at 2 h. Extend the drying time as necessary until samples are fully dried.
13. Store the dried lipid extracts at -20 °C for short-term storage or -80 °C for long-term storage.

4. Reconstitution of extracted tear lipid for LC- MS/MS analysis

1. Reconstitute the dried lipid extracts with 50 µL of the ice-cold methanol (MS grade)/chloroform (HPLC grade) mix (1:1, v/v) as the solvent (1 µL of solvent per 1 mm of PRT used) and follow by sonication in a pre-cooled ultrasonic cleanser for 15 min.
2. Centrifuge the reconstituted lipid extracts at 14,000 × g and 4 °C for 10 min.
3. Carefully transfer 40 µL of the supernatant to an autosampler glass vial with a glass insert installed.
4. Tightly cap the vial with a non-slit septum open top cap and ready for LC-MS/MS analysis.

5. Sample acquisition by LC-MS/MS

1. For Liquid Chromatography, set the column chamber and sampler temperature at 50 °C and 4 °C, respectively.
2. For each injection, load 5 µL of the sample onto a reverse-phase LC column (C18; 1.7 µm, 100 mm × 2.1 mm) and fractionate the lipids at a flow rate of 0.3 mL/min in a 24 min separation gradient.
   1. Use mobile phase A comprising a mixture of 60:40 acetonitrile (MS grade):water (v/v) with 5 mM ammonium formate (MS grade) and 0.1% (v/v) formic acid (MS grade) and mobile phase B containing 90:10 isopropanol (MS grade):acetonitrile (v/v) with 5 mM ammonium formate and 0.1% (v/v) formic acid.
   2. Use the following gradient: 0-2 min: 40% B; 2-2.5 min: 40% B; 2.5-3 min: 58% B; 3-18 min: 99% B; 18-20 min: 99% B; 20-20.1 min: 40% B; 20.1-24 min: 40% B.
3. For the Orbitrap Mass Spectrometer, set the spray voltage to +3.5 kV for positive mode or -2.5 kV for negative mode.
4. Adjust the sheath gas flow rate to 45 Arb and the auxiliary gas flow rate to 10 Arb.
5. Set the sweep gas flow rate to 2 Arb.
6. Set the ion transfer tube temperature to 300 °C and the vaporizer temperature to 320 °C.
7. Use data-dependent acquisition (DDA) mode.
8. For MS1, set the resolution to 120,000, scan range to 100-2000 m/z, standard AGC target, auto injection time, dynamic exclusion to 6 s, and intensity threshold at 5.0e4.
9. For MS2, set the resolution to 15,000, auto scan range, standard AGC target, dynamic injection time, isolation window at 1.6 m/z, collision energy type as higher-energy collisional dissociation (HCD), and normalized collision energy (NCE) to stepped values of 15, 30, and 40.
10. Analyze raw data with LipidSearch software or other compatible platforms.

Results

Composite tear samples were collected from 16 healthy volunteers at three independent visits spaced two weeks apart (R1, oldest batch; R2, R3 collected sequentially two weeks after each). Using LipidSearch with stringent criteria (i.e., Signal-to-Noise Ratio ≥100, grade C or above, and ion intensity ≥30,000), we identified 773, 890, and 1,025 unique lipid species in R1-R3, respectively (Figure 5A). Positive ion mode identified 1,302 lipid species with 26.0% Grade A (i.e., both lipid class an...

Discussion

Advantages of PRT-based tear sampling and lipid extraction
The PRT-based tear lipidomics workflow presented here provides a minimally invasive and practical sampling method for comprehensive tear lipid profiling. Compared to Schirmer strips, which require prolonged contact and larger sample volumes, PRT sampling is rapid, can be completed within 2 min, well-tolerated by diverse populations, including those with reduced tear volume or heightened ocular sensitivity, and yields tear samples compatible...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile (ACN), LC-MS grade	RCI Labscan	LM1005
ACQUITY UPLC CSH C18 Column	Waters Corporation	186005297	130Å, 1.7 µm, 2.1 mm X 100 mm
Ammonium formate, LC-MS grade	Sigma-Aldrich	55674
Autosampler Glass Vial	Well Rich Scientific	2ML-9-V1002	2mL Clear Glass 12*32mm Flat Base 9-425 Screw Thread Vial with Label (WITH writing pad )
Blue 9-425 Open Top Ribbed Screw Cap	Well Rich Scientific	9-SP1003	with 9mm,White PTFE/Red Silicone Septa 1mm
Chloroform, HPLC grade	Duksan Reagents	1271
Eppendorf Safe-Lock Tubes, 1.5 mL	Eppendorf	30120086
Formic Acid (FA),  LC-MS grade	ThermoFisher Scientific	A117-50
Glass micro-insert	Well Rich Scientific	2ML-N2002	250 µl insert, clear glass, conical base with polyspring, size: 5.8*28.5mm
Isopropanol, LC-MS grade	RCI Labscan	LM1162
LipidSearch Software	ThermoFisher Scientific	OPTON-30879	Version 5
Methanol,  LC-MS grade	RCI Labscan	LM1115
Phenol Red Thread  (PRT)	Tianjin Jingming New Technology Development Co., Ltd	20192160086
Refrigerated Centrifuge	ThermoFisher Scientific	75007200
Refrigerated CentriVap Centrifugal Concentrator and CentriVap Cold Traps	Labconco	16108335
Screw Cap Micro Tubes	ThermoFisher Scientific	3488
tert-Butyl methyl ether (MTBE), HPLC grade	Duksan Reagents	1070
Thermo Scientific Dionex UltiMate 3000 HPLC	ThermoFisher Scientific	ULTIM3000RSLCNANO
Thermo Scientific Orbitrap IQ-X Tribrid MS	ThermoFisher Scientific	FSN05-10001
ThermoMixer C	Eppendorf	5382000015
Ultrasonic Cleanser	Crest Ultrasonic	P500D-45
Vortex Mixer	Benchmark	BV1003