Protocol to extract tear fluid for proteomics using Schirmer strips

Summary

Schirmer strips are widely regarded as the gold standard for tear fluid collection. However, their use presents several challenges for proteomic analysis. Here, we present a protocol for extracting tear proteins from Schirmer strips. We describe steps for acquisition and handling of strips, extraction buffer preparation, strip preparation, and protein extraction. This protocol is designed to improve protein yield and facilitate proteomic workflows and is adaptable for various protein-based studies, particularly in the context of ocular disease research and diagnostics.

Graphical abstract

Highlights

• •Protocol for quantifying tear volume for proteomic analysis using Schirmer strips
• •Procedures for protein extraction through a diffusion-based workflow
• •Guidance on optimizing for high-yield protein recovery and minimizing protein loss

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Schirmer strips are widely regarded as the gold standard for tear fluid collection. However, their use presents several challenges for proteomic analysis. Here, we present a protocol for extracting tear proteins from Schirmer strips. We describe steps for acquisition and handling of strips, extraction buffer preparation, strip preparation, and protein extraction. This protocol is designed to improve protein yield and facilitate proteomic workflows and is adaptable for various protein-based studies, particularly in the context of ocular disease research and diagnostics.

Before you begin

Background

Produced by the lacrimal glands, tears are a complex fluid consisting of ∼98% water along with electrolytes, proteins, lipids, and mucins.¹ Beyond lubricating the ocular surface, they perform several critical physiological roles, including immune surveillance, antimicrobial defense and supplying oxygen and nutrition to the avascular corneal epithelium.² Tears are known to harbor inflammatory cytokines (e.g., IL-1, IL-6, IL-8, IFN-γ, TNF-α), anti-inflammatory proteins (e.g., IL-1Ra) and neuromodulators derived from neighboring blood vessels.³ Compared to other ocular fluids (e.g., aqueous and vitreous humor),⁴ tears offer a non-invasive and attractive medium for biomarker discovery (Figure 1). Proteomics has emerged as a valuable tool for identifying reproducible diagnostic biomarkers and potential therapeutic targets in a variety of ocular surface and systemic diseases.^5,6 However, limited tear sample volumes (∼10 μL), the complexity of tear film composition, and suboptimal sample handling resulting in sample loss, have hindered comprehensive proteome coverage.⁷ To date, only 794 core tear proteins have been robustly and reliably identified, highlighting the need for a dedicated protocol that minimizes sample loss and leverages more sensitive analytical tools for deeper tear proteome exploration.⁸

Figure 1.

Tear fluid, as well as the aqueous and vitreous humors, are potential reservoirs for identifying proteomic biomarkers: Schirmer strip-based tear fluid collection provides a non-invasive approach compared to the other two ocular fluids

Innovation

The tear proteome represents a promising source of biomarker discovery in various ocular diseases with mass spectrometry (MS) serving as an effective tool for protein identification and quantification.⁹ Nonetheless, improper handling and protein loss during extraction from Schirmer strips often limit tear proteome coverage.^9,10,11,12 TEARDROP (Tear Enrichment using Advanced Recovery via Deep Resolution of Proteins) addresses this technical gap through an optimized diffusion-based tear protein extraction workflow from standard-of-care Schirmer strips coupled with nanoparticle enrichment mass spectrometry. It focuses on key concepts relevant to the entire sample processing cycle, including standardized tear collection, appropriate storage conditions, optimized elution strategies, and downstream processing steps, supporting nanoparticle-based MS from Seer’s Proteograph platform.¹³ TEARDROP is ideal for discovery proteomic analyses and deep proteomic coverage of the tear proteome. It can be combined with other mass spectrometry proteomics such as liquid chromatography (LC)-scheduled multiple reaction monitoring (LC-sMRM)^9,14 or other high-throughput targeted methods for clinical applications.¹⁵

Institutional permissions

This study was approved by the Stanford Institutional Review Board and adheres to the tenets of the Declaration of Helsinki and the Health Insurance Portability and Accountability Act (HIPAA). Informed consent was obtained by all patients for the collection of tear fluid and subsequent analysis prior to participation in the study.

Acquisition and handling of Schirmer strip

Schirmer strips are used clinically to assess tear secretion in patients without the need for topical anesthesia. Based on the principle of capillary action, these strips allow the tears to be absorbed along their length. They are widely used as a diagnostic procedure to identify patients with suspected dry eyes or excessive tear production. Studies have successfully recovered a range of biomolecules such as cystatins,¹⁶ immunoglobulins,¹⁷ vitamin C,¹⁸ cytokines and matrix metalloproteinases^6,19,20 from Schirmer strips making them an ideal tool for tear collection in downstream proteomic studies. For this protocol, Schirmer strips were bent at the zero line and placed in the inferior cul-de-sac of patients for 5 min (Figures 2A and 2B). On average, 15 millimeters of each Schirmer strip absorbed tears.²¹ All patients were anonymized and did not receive anesthesia. After collection, strips were placed in cryogenic tubes on dry ice and subsequently snap-frozen at −80°C until reconstitution (Figures 2C and 2D). It is strongly recommended that the strips be processed immediately after thawing.

Figure 2.

Tear sample collection using Schirmer strips

(A) Remove the sterile schirmer strip from its packaging.

(B) Insert the strip into the lower conjunctival fornix for 5 min, then gently remove it.

(C) Place in a cryogenic tube and immediately keep on dry ice.

(D) Archive the sealed cryovial at −80°C until processing.

Extraction buffer preparation

Timing: 30 min

The buffer needs to be prepared before thawing the Schirmer strips. The extraction buffer consists of 100 mM Tris-EDTA (TE) with 0.5% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). The inclusion of EDTA disodium salt dihydrate helps protect protein degradation.

• 1.Prepare 50 mL of 100 mM TE buffer with 0.5% CHAPS and filter-sterilized before use; as outlined in the reagent tables (buffer formulations section).

CRITICAL: Store the prepared buffer at room temperature until use. This buffer remains stable at room temperature for approximately 3 to 6 months.

• 2.Dissolve all the reagents in 40 mL of deionized water, adjust the pH to 7.5 with HCl, and bring the final volume to 50 mL with deionized water.
• 3.Sterilize the solution by filtration through a 0.22 μm membrane in the Millipore Steriflip vacuum tube top filter and store the filtrate in the attached 50 mL tube.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Patient tear fluid in Schirmer strips	Spencer Center for Vision Research Clinic, CA, USA	N/A
Chemicals, peptides, and recombinant proteins
CHAPS	Research Products International, IL, USA	SKU: C41010–5.0
EDTA (0.5 M), pH 8.0	Research Products International, IL, USA	SKU: E57020–500.0
Ethanol 190 proof	Fisher Scientific, MA, USA	Cat. #: 04-355-723
Pierce BCA Protein Assay Kit	Fisher Scientific, MA, USA	Cat. #: PI23227
Pierce Quantitative Fluorometric Peptide Assay	Thermo Fisher Scientific, MA, USA	Cat. #: 23290
Proteograph XT Assay Kit	Seer, Inc., CA, USA	Part number: S55R1100
Tris-Base	Research Products International, IL, USA	SKU: T60040–100.0
Software and algorithms
BioVenn	Hulsen et al.22	https://www.biovenn.nl/index.php
DIA-NN	Demichev et al., 202023	https://github.com/vdemichev/diann
GraphPad Prism v.10	GraphPad Software, Inc.	https://www.graphpad.com/
Metascape	Zhou et al.24	https://metascape.org/
Nanoparticle-based MS platform	Seer, Inc., CA, USA	https://seer.bio
Orbitrap Astral	Thermo Fisher Scientific, Germany	Cat. #: BRE725600
Tecan Spark multimode microplate reader	Tecan System Inc., CA, USA	Article number: 93000001
Vanquish NEO nanoLC system	Thermo Fisher Scientific, Germany	Cat. #: VN-S10-A-01
Other
BD PrecisionGlide Needle 30 g × 1 in	BD, Franklin Lakes, NJ, USA	Cat #: 305128
Benchtop microcentrifuge 5418	Eppendorf, HH, Germany	Cat #: 5401000137
High-precision stainless-steel scissors	Fisher Scientific, MA, USA	Cat. #: 12000148
Incubator shaker	New Brunswick Scientific, NJ, USA	Product Code: NB-I24
Millipore Steriflip vacuum tube top 50 mL filter	MilliporeSigma, MA, USA	Cat #: SE1M179M6
Petri dish, 60/15 mm, with vents	Greiner Bio-One North America Inc., NC, USA	Cat #: 628102
Pipette 20–200 μL	Eppendorf, HH, Germany	SKU: 3123000055
Pipette 100–1,000 μL	Eppendorf, HH, Germany	SKU: 3123000063
Protein LoBind tube 0.5 mL	Fisher Scientific, MA, USA	Cat. #: 13698793
Precision balance	Mettler Toledo, OH, USA	Material #: 30315631
Sterile 1.5 mL conical screw cap with attached cap & silicon O-ring	Corning, NY, USA	Product #: 430909
Sterile microcentrifuge tubes 1.5 mL	Thermo Fisher Scientific, MA, USA	Cat #: 21402903
Straight tapered ultrafine point tweezers	Fisher Scientific, Waltham, MA, USA	Cat. #: 12000122
VWR Symphony ultrasonic cleaner	Avantor, PA, USA	Cat #: 97043986

Materials and equipment

Buffer formulations (50 mL of TE with 0.5% CHAPS)

Tris-Base

Reagent	Final concentration	Amount
Tris-Base (MW. 121.1 g/mol)	10 mM	0.06 g

Note on storage condition: Store at room temperature for ∼1–2 years or as long as indicated by the manufacturer.

EDTA

Reagent	Final concentration	Amount
EDTA (0.5 M, pH 8.0)	1 mM	0.1 mL

Note on storage condition: Store at room temperature for ∼1–2 years or as long as indicated by the manufacturer.

CHAPS

Reagent	Final concentration	Amount
CHAPS	0.5% (w/v)	250 mg

Note on storage condition: Store at room temperature for ∼1–2 years or as long as indicated by the manufacturer.

500 μL collection tube making.

• •Using a sterile 25-gauge needle, puncture 3–4 holes in the bottom of a 500 μL microcentrifuge tube. Nest this tube inside a larger, capless 1.5 mL microcentrifuge tube.

Step-by-step method details

This section outlines detailed steps for processing tear fluid, with an emphasis on careful handling and storage to prevent contamination and degradation. Following extraction, the concentration of recovered proteins is determined using biochemical assays for downstream proteomic analysis.

Schirmer strip preparation

Timing: 15 min

Here, we describe steps for handling Schirmer strip after removal from −80°C cryogenic tube to avoid any changes in the sample.

Note: Schirmer strip can lose tear proteins during sample handling, therefore, strip that is not being cut should be stored securely in a cryogenic tube and always kept on ice. Prior to the procedure, prepare and organize sterile scissors and tweezers. These tools should be prewashed with 70% ethanol. To prevent cross-contamination, sterilize the tools with 70% ethanol after processing each strip and before handling the next one. Inspect each Schirmer strip for visible debris both before and after cutting and document any such observations.

• 1.Place one Schirmer strip in a 60 mm × 15 mm sterile petri dish plate after thawing from −80°C storage.
• 2.Hold a strip at the bottom (dry end) with sterile tweezers, and using sterilized scissors, cut the strip into five 5 mm segments, starting from the notch (see Figures 3A and 3B; troubleshooting 1).

Note: Tear production typically wets 15 mm or more of the Schirmer strip. Therefore, the first five 5 mm segments, spanning from the notch (−5 mm) to the 20 mm mark (total strip length = 20 mm) are collected for analysis.

• 3.Transfer the strip pieces into a 1.5 mL centrifuge tube using sterile tweezers.

CRITICAL: If processing multiple strips simultaneously, always sterilize the scissors and tweezers with 70% ethanol between each strip to prevent cross-contamination.

• 4.Gently add 360 μL of 100 mM TE buffer containing 0.5% CHAPS to each tube containing strips pieces and place immediately on ice (4°C) (Figure 3C).

CRITICAL: Avoid storage on ice for more than 30 min to preserve protein integrity.

Figure 3.

Overview of the pipeline for sample processing post-collection

(A) The 1.5 mL screw-cap cryovials containing schirmer strips (ScS) were thawed and kept on ice.

(B) Using sterile forceps, each ScS was moved to a clean 60 mm Petri dish to facilitate precise cutting. The wetted portion of the ScS was cut into five equal 5 mm fragments with a sterile scissor to maximise extraction surface area.

(C) The pieces were transferred to a sterile 1.5 mL microcentrifuge tube and 360 μL pre-processing buffer was added to immerse the pieces completely.

(D) Tubes were sonicated in a 25°C water-bath sonicator for three cycles of 45 s on / 45 s off to dislodge proteins adsorbed onto strip fibres.

(E) Samples were placed on an orbital shaker and agitated at 1,200 × g for 1 h at 4°C to enhance protein diffusion into solution.

(F) The pieces were centrifuged at 1,000 × g for 1 min at 4°C. The supernatant was carefully transferred to a new sterile cryogenic tube without disturbing the pellet.

(G and H) Residual ScS pieces were transferred to a low-protein-binding 0.5 mL tube with a punched drain hole, nested inside the original tube and centrifuge again to recover remaining extract.

(I) All flow-throughs were combined in the previous cryogenic tube from step (F). Extracts were either processed immediately with proteomic workflow or flash-frozen in liquid nitrogen and stored at −80°C until proteomic analysis.

Protein extraction

Timing: 2–3 h

Here, we outline steps for protein extraction and elution from prepared Schirmer strips. The goal is to obtain sufficient protein yield with minimal sample loss to enable downstream analysis of tear fluid proteins. Be sure to record the final volume of each sample following elution for accurate documentation and comparison across samples.

• 5.Sonicate the samples for 45 s in a sonication water bath (VWR Symphony Ultrasonic Cleaner) at 25°C, followed by 45 s on pause (Figure 3D).

CRITICAL: Before powering on the unit, ensure the tank is filled with distilled water to the recommended level, and the temperature is set to 25°C. Place microcentrifuge tubes on floating foam tube racks to prevent direct contact with the tank base. Avoid over-sonication to minimize protein degradation (Troubleshooting 2). In some cases, (e.g., when dye debris is present), excessive sonication may lead to protein aggregation.

• 6.Repeat the step 6 two more times for a total of three sonication cycles (troubleshooting 3).

Note: Ensure strip pieces are fully submerged and not stuck to the wall of the microcentrifuge tube (troubleshooting 4).

• 7.Incubate the samples at 4°C for 1 h on an orbital shaker (New Brunswick I24 Incubator Shaker Series) at 1,200 × g (Figure 3E).
• 8.Centrifuge the tubes at 1,000 × g for 1 min at 4°C using a tabletop centrifuge.

Note: Pre-cool the centrifuge for 10 min at maximum speed before use to ensure it reaches 4°C.

• 9.Carefully transfer the supernatant to the new, labeled cryogenic tubes, avoiding contact with any precipitate (Figure 3F).

Note: A small black precipitate (tick mark dye) may be visible at the bottom of the tube. Do not disturb or pipette it.

• 10.Using the sterile tweezer, transfer the Schirmer strip pieces to a 500 μL microcentrifuge tube with a hole at the bottom and place this tube in a 1.5 mL microcentrifuge (Figure 3G).
• 11.Close the cap of a 500 μL microcentrifuge tub and microcentrifuge the tubes for another cycle at 1,000 × g for 1 min at 4°C (Figure 3H).
• 12.Discard the 500 μL tubes containing the strips. Collect all the flow-through into the same cryogenic tubes from step 10 (Figure 3I).

CRITICAL: To reduce sample loss, recover as much fluid as possible in this step.

• 13.Determine the protein concentration in each sample using Pierce BCA Protein Assay Kit.

Note: Use the microplate procedure with BSA standards ranging from 20–2000 μg/mL and incubate at 37°C for 30 min. Do not dilute the protein prior to quantification. Use the extracted samples immediately for analysis (e.g., direct digestion or mass spectrometry).

Optional: If not being used immediately, store samples at −80°C for future use.

Expected outcomes

Our protocol is designed to consistently quantify sufficient tear volume for proteomic analysis. With 360 μL of buffer added for elution, the final sample volume typically ranges from 250 to 320 μL, with protein concentrations between 0.2 to 0.4 mg/mL. By incorporating additional processing steps such as 1 h incubation with gentle shaking, combined with centrifugation and elution washes, protein recovery was significantly improved, resulting in the detection of over 5,000 proteins (representative data, Figure 4A).

Figure 4.

Comparison of TEARDROP method and conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) for tear proteome profiling

For conventional methods, each Schirmer strip was transferred to 0.1 μm filter unit and 450 μL of 100 mM ammonium bicarbonate in 50 mM NaCl was added. Filter units were incubated at 25°C for 4 h at 300 rpm. Samples were then centrifuged at 5,500 rcf for 5 min and flow through was collected and dried to completion in speed vac before processing to LC-MS/MS.

(A) Venn diagram showing the overlap of proteins identified using TEARDROP workflow (purple) versus conventional method (teal).

(B) Boxplot comparing the total number of proteins identified per sample by each method. The TEARDROP approach yielded significantly higher proteome coverage (∗∗∗∗p < 0.0001; Mann-Whitney test).

(C) Bar graph showing the top enriched biological processes among the proteins uniquely identified by the TEARDROP method using Metascape analysis. Enrichment significance is indicated as –log₁₀(P), calculated using gene ontology over-representation analysis.

(D) Protein abundance with wide dynamic range and resolution from the TEARDROP method and lower sensitivity and narrower proteomic depth from the conventional method.

(E) Enriched pathways from 20% bottom of low abundance protein using Metascape analysis.

(F) Protein-protein interaction (PPI) network from B cell activation pathway, yellow highlighted proteins seen in TEARDROP method.

Notably, proteins are susceptible to denaturation during the procedure due to their chemical and physical instability. Therefore, a general rule of thumb is to extract as much protein as possible. We compared our TEARDROP method with the conventional protocol. Representative results are shown in Figure 4. The TEARDROP method identified approximately 6-fold more proteins than the conventional approach, demonstrating its markedly enhanced sensitivity for tear proteome profiling.

Quantification and statistical analysis

Protein concentrations were determined spectrophotometrically using the Pierce BCA Protein Assay Protein concentrations were determined spectrophotometrically using the Pierce BCA Protein Assay Kit, with absorbance measured at 562 nm using SparkControl (Tecan). Extracted proteins were processed with the Proteograph XT Assay and analyzed with a Vanquish NEO nanoLC system coupled with a Orbitrap Astral.^25,26 Raw MS data was processed using the DIA-NN.

Venn diagram (Figure 4A) were generated using the BioVenn website.²² Overrepresentation analysis was performed using Metascape²⁴ to identify enriched biological pathways (Figures 4C and 4E). In the Metascape output, the top enriched pathways are represented as bar plots with color coding. A higher -log10(P) value indicates greater statistical significance, and darker bars correspond to more significantly enriched pathways.

Limitations

A well-known drawback of Schirmer strips is their ability to collect only marginal volumes of tear fluid, making them inadequate for latest high-sensitivity, high-resolution proteomics technologies. Our method addresses this technical limitation by using an optimized extraction buffer and mechanical recovery techniques, which together enable high-yield sample collection and improved extraction efficiency facilitating the analysis of tear fluid in greater detail. TE buffer was selected for protein extraction from Schirmer strips due to its ability to maintain a stable pH environment (via Tris) and to chelate divalent cations through EDTA, minimizing proteolytic activity and preserving protein integrity throughout a 1-h incubation process. Besides, a two-step centrifugation procedure was employed to maximize tear protein extraction from the Schirmer strips: first collecting the eluted fluid, then recovering any remaining fluid from the strips. In parallel, proper handling and storage of samples collected in the clinic are critical for maintaining Schirmer strips stabilization, as environmental factors such as humidity, temperature, and atmospheric pressure can affect tear fluid integrity. By implementing standardized procedures under controlled conditions, we aim to reduce variability and preserve both the quality and quantity of the tear proteome for downstream analysis.

Troubleshooting

Problem 1

Schirmer strips may retain residues from cosmetic products or environmental debris present near the patient’s ocular surface (step-by-step method details: schirmer strips preparation; step 3).

Potential solution

Remove the initial 1–2 mm of the strip (where contact with eyelid margins is highest) prior to extraction. Any portion of the strip showing discoloration or visible residue should be excluded from processing. The remaining sections should be handled carefully during sectioning to avoid contamination.

Problem 2

Over-sonication of samples can lead to heat buildup, resulting in protein degradation (step-by-step method details: protein extraction; step 6).

Potential solution

If the system has the heating capacity, ensure it is set at the appropriate temperature before operation, or adjust the ultrasonic power to suit the samples being processed. In such cases, cooling coils can be employed to prevent heat buildup in the water bath.

Problem 3

Paper fibers from the Schirmer strip may detach during sonication and remain suspended in the collected tear fluid (step-by-step method details: protein extraction; step 7).

Potential solution

Gently centrifuge the eluate post-extraction to pellet any detached fibers, then carefully transfer the clear supernatant to a new tube. Using Schirmer strips is important to minimize fiber shedding.

Problem 4

If any pieces of the strip remain dry or are not fully saturated during sonication, protein extraction may be incomplete, resulting in reduced sample recovery (step-by-step method details: protein extraction; step 7).

Potential solution

Gently centrifuge the eluate post-extraction to pellet any detached fibers, then carefully transfer the clear supernatant to a new tube. Using Schirmer strips is important to minimize fiber shedding.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Vinit B. Mahajan (vinit.mahajan@stanford.edu).

Technical contact

Further inquiries about the technical specifics of the protocol should be directed to the technical contact, Antoine Dufour (antoine.dufour@ucalgary.ca).

Materials availability

This study did not generate new unique reagents. All stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

All data generated in this paper will be shared by the lead contact by request. Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

Acknowledgments

V.B.M. is supported by NIH grants (R01EY031952, R01EY030151, R01EY024665, R01EY025225, and P30EY026877), Stanford ChEM-H IMA, the Stanford Center for Optic Disc Drusen, and Research to Prevent Blindness, United States. A.D. is supported by the STARS award by the Arthritis Society of Canada, the Canadian Institutes of Health Research (CIHR, Canada) (grant no. 449589), and the Natural Science and Engineering Council of Canada (NSERC, Canada) (grant no. DGECR-2019-00112). V.B.M. and P.M. are supported by the Alan and Irene Adler Ocular Research Initiative and the Mills and Margaret Cox Macula Society Research/Retina Research Foundation, United States. Y.J.S. is supported by BrightFocus Foundation’s Macular Degeneration Research program. We thank the Seer Inc. team for processing the tear samples. Biorender software was used to create the figure under an academic license.

Author contributions

Study concept and design, P.M. and V.B.M.; acquisition of data, G.Y., G.-H.N., and Y.J.S.; data analysis and interpretation, G.-H.N., M.C., Y.J.S., S.H.L., T.-C.Y., A.D., and V.B.M.; drafting of the manuscript, G.-H.N. and M.C.; critical revision of the manuscript, S.H.L., A.D., and V.B.M.; obtained funding, P.M. and V.B.M.; administrative, technical, and material support, P.M. and V.B.M.; study supervision, P.M., A.D., and V.B.M.

Declaration of interests

The authors declare no competing interests.