Published: December 1st, 2023
This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.
Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.
Tear proteomics has received attention to explore potential biomarkers for ocular diseases and conditions1,2,3,4,5,6, to access the pathogenesis of the ocular and systemic condition, as well as to exploit the advantage of non-invasive tear sample collection using Schirmer strips. The technological advancement of next-generation mass spectrometry has enabled protein quantification in microliter-scale tears with accuracy and precision not possible in the past. Sample preparation methods are not yet standardized. A robust and standardized sample preparation workflow is essential for the success of clinical application in tear protein biomarker research. The suspension trapping (S-Trap) sample preparation workflow was recently reported as an effective and sensitive sample preparation method for broad downstream proteomic analysis7,8. Yet, this strategy has not been well-reported in the analysis of human tear proteome, outperformed filter-aided sample preparation (FASP) and in-solution digestion in terms of enzymatic digestion efficiency and the greater number of protein identification by mass spectrometric analysis9. The S-Trap-based approach was demonstrated in the preparation of retinal tissue 10, formalin-fixed, paraffin-embedded (FFPE) tissue11, cells12, microorganism13, and liquid biopsies14,15.
This protocol describes an integrated quantitative workflow from clinical samples to enzymatically digested protein for the discovery of a non-invasive tear protein biomarker panel with a rapid, reproducible, and robust technical strategy. Briefly, tear fluid was collected using a standard Schirmer strip and immediately dried with an ophthalmic frame heater to prevent protein autolysis at room temperature. Embedded total proteins were extracted using 5% sodium dodecyl sulfate (SDS) lysis buffer according to the manufacturer's suggestion, followed by protein assay measurement. The extracted lysate then underwent standard reduction with dithiothreitol (DTT) and alkylation with iodoacetamide (IAA).
After acidification with phosphoric acid, suspension trapping column protein precipitation buffer containing 90% methanol and 100 mM triethylammonium bicarbonate (TEAB) was added to aggregate proteins. The sample was then transferred into a new suspension trapping micro column. Enzymatic digestion with done with sequencing grade trypsin at a 1:25 ratio (w/w, trypsin: protein) at 47 °C for 1 h. The resulting peptides were then eluted via centrifugation, sequentially with 50 mM TEAB, 0.2% aqueous formic acid (FA), and 50% acetonitrile (ACN) containing 0.2% FA. The eluted peptides were vacuum-dried and reconstituted in 0.1 % FA. The peptide concentration was measured and adjusted to 0.5 µg/µL for mass spectrometric analysis.
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
2. Preparation of chemicals and reagents
3. Protein extraction in Schirmer strip
4. Suspension-trapping sample preparation
5. Reconstitute peptides for mass spectrometry analysis
6. Sample Acquisition by Liquid Chromatography-Tandem Mass Spectrometry
This protocol allows tear sample collection with Schirmer Strips that are stored dry at room temperature before subsequent sample preparation for mass spectrometry analysis. Filter-aided sample preparation (FASP) workflow with suspension-trapping micro column enabled fast sample preparation in hours, compared to commonly adopted in-solution digestion procedures in days that require overnight incubation. The peptide recovery yield was significantly higher (p < 0.001) than the standard in-solution digestion protocol and with good reproducibility at a coefficient of variation (%CV) < 7%. A pool of tears samples was repeated in six technical replicates with 74.2 ± 5.0% peptide recovery and 52.8 ± 1.6% peptide recovery in samples prepared with in-solution procedures. A pooled tear sample with a protein amount of 36.3 µg was spiked onto the Schirmer strip and extracted as previously described, the extraction efficiency of protein was 81% (29.5 ± 6.8 µg, mean ± SD).
Both workflows loaded 3 µg of peptides on the MS, and the DDA search yielded a total of 1,183 ± 118 proteins (5,757 ± 537 distinct peptides) and 874 ± 70 proteins (4,400 ± 328 peptides) at 1% FDR in the suspension trapping group and in-solution group, respectively. Analysis of gene ontology (GO) using the PANTHER classification system revealed very similar proteomes for both approaches, with binding, catalytic activity, and molecular function regulation being their main molecular functions (Figure 4).
Figure 1: Bend the top of the Schirmer strip at the 0 mm mark before application. Please click here to view a larger version of this figure.
Figure 2: Position of the Schirmer strip during tear collection. Please click here to view a larger version of this figure.
Figure 3: Illustration of Schirmer strip handling before protein extraction. Please click here to view a larger version of this figure.
Figure 4: Pie chart illustrating the gene ontology analysis for the proteomes identified using the in-solution workflow and suspension trapping workflow, using the PANTHER classification system. Please click here to view a larger version of this figure.
To achieve accurate results using this method, power-free disposable gloves should be worn in all procedures from tear sample collection to avoid sample contamination. It is important to avoid bubbles and air gaps between the filter and sample solution in each step by utilizing micro-spinning columns. If the sample volume is larger than the capacity of the columns, it is recommended to repeat the process. This protocol has proven to be more efficient than the traditional in-solution protocol in terms of preparation time, protein recovery, and total protein identification. This is largely because the samples undergo most of the required procedures on the same column, unlike the in-solution method which involves multiple transferring steps such as acetone precipitation, digestion, and cleanup (desalting), which increases the likelihood of variations in the resulting data.
In addition to the tear samples collected with the microcapillary method16,17, this FASP workflow with suspension-trapping micro column provides an alternative sample preparation method that allows fast and robust tear sample preparation collected using Schirmer strips, with minimal material preparation and user-friendly steps to follow. This allows reproducible tear sample preparation for large cohort studies across multiple ocular diseases or conditions with improved peptide recovery and protein identification by MS over in-solution procedures. This reliable process can be utilized regularly in the preparation of tear biomarker samples for research and other clinical purposes. Most importantly, it requires minimal staff training for on-site collection and negates the need for sample storage in a freezer. Samples are dried onsite to minimize protein autolysis and degradation. Hence, this allows convenient shipment by mail to facilitate downstream analysis, as opposed to using micro-capillary tubes.
The authors have no conflicts of interest to declare.
This work was supported by the InnoHK initiative and the Hong Kong Special Administrative Region Government; Research Centre for SHARP Vision; and the Research Centre for Chinese Medicine Innovation (RCMI) at The Hong Kong Polytechnic University.
|9 mm Plastic Screw Thread Vials
|Acetonitrile, LCMS Grade
|Centrifuge MiniSpin plus
|DL-dithiothreitol (DTT), BioUltra
|Eppendorf Safe-Lock Tubes, 1.5 mL
|Formic acid, ACS reagent, ≥96%
|Frame Heater OPTIMONSUN Electronic
|Breitfeld & Schliekert GmbH
|Iodoacetamide (IAA), BioUltra
|Methanol, HPLC Grade
|Nunc Biobanking and Cell Culture Cryogenic Tubes, 2 mL
|Thermo Fisher Scientific
|Phosphoric acid, 85 wt.% in H2O
|Pierce Quantitative Colorimetric Peptide Assay
|Thermo Fisher Scientific
|Pierce Rapid Gold BCA Protein Assay Kit
|Thermo Fisher Scientific
|Quadrupole Time-of-Flight Mass Spectrometry
|Schirmer Ophthalmic Strips
|Entod Research Cell UK Ltd
|S-Trap Micro Column
|SureSTART 9 mm Screw Caps
|Triethylammonium bicarbonate (TEAB), 1 M
|Ultra-performance Liquid Chromatography
|UltraPure Sodium dodecyl sulfate (SDS)
|Thermo Fisher Scientific
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