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.

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.&#160;Collection of human tear fluid with Schirmer strip
<ol>
	<li>Wear gloves and sanitize to avoid sample contamination.</li>
	<li>Check that the inner packaging is intact, sterile, and not expired.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Ask the subject to look away from the position of the strip to be placed.</li>
	<li>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.</li>
	<li>Ask the subject to gently close the eyelids to minimize irritation.</li>
	<li>Collect tear fluid for about 5 min or preferably 15-20 mm tear sample was collected.</li>
	<li>Ask the subject to open both eyes, pull down the lower eyelid gently and remove the strips from the subject.</li>
	<li>Inspect the hydrated length on the Schirmer strip and record the collected amount in mm.</li>
	<li>Dry the strip with a standard, clean frame heater until the fluid is completely dry. 
	&#8203;NOTE: Excess heating that could potentially char the filter paper should be avoided.</li>
	<li>Insert each dried Schirmer strip in a labeled cryotube and store it preferably in a cool, dry place in the dark until further processing.</li>
</ol>
2.&#160;Preparation of chemicals and reagents
<ol>
	<li>Lysis Buffer (5% SDS, 50 mM TEAB, pH 7.5), 20 mL
	<ol>
		<li>Pipette 1 mL of 1 M TEAB and 80 &#181;L of 12% phosphoric acid. Vortex and sonicate briefly to remove bubbles. Top up to 20 mL with deionized water.</li>
		<li>Weigh 1 g of SDS and add to the solution with oscillation until dissolved.</li>
	</ol>
	</li>
	<li>Protein Precipitation Buffer (90% methanol, 100 mM TEAB, pH 7.1), 10 mL
	<ol>
		<li>Add 893 &#181;L of 1 M TEAB and 92 &#181;L of deionized water.</li>
		<li>Add 15 &#181;L of 85 wt. % phosphoric acid and 9 mL of methanol to the solution and vortex briefly.</li>
	</ol>
	</li>
	<li>Digestion Buffer (50 mM TEAB), 1 mL
	<ol>
		<li>Add 50 &#181;L of 1 M TEAB and top up to 1 mL with deionized water.</li>
	</ol>
	</li>
	<li>200 mM dithiothreitol (DTT) solution, 500 &#181;L
	<ol>
		<li>Weigh 15 mg of DTT and dissolve in 500 &#181;L of deionized water; vortex briefly. 
		NOTE: Prepare before use.</li>
	</ol>
	</li>
	<li>400 mM iodoacetamide (IAA) solution, 200 &#181;L
	<ol>
		<li>Weigh 15 mg of IAA and dissolve in 200 &#181;L of deionized water; vortex briefly. NOTE: Prepare before use and avoid light.</li>
	</ol>
	</li>
	<li>0.2% formic acid, 50% acetonitrile, 10 mL
	<ol>
		<li>Prepare 10 mL of 50% acetonitrile in deionized water.</li>
		<li>Add 20 &#181;L of formic acid and vortex briefly.</li>
	</ol>
	</li>
	<li>0.2% formic acid solution, 10 mL
	<ol>
		<li>Add 20 &#181;L of formic acid to 10 mL of deionized water and vortex briefly.</li>
	</ol>
	</li>
	<li>0.1% formic acid solution, 10 mL
	<ol>
		<li>Add 10 &#181;L of formic acid to 10 mL of deionized water; vortex briefly.</li>
	</ol>
	</li>
</ol>
3.&#160;Protein extraction in Schirmer strip
<ol>
	<li>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.</li>
	<li>Cut the strip in 1 mm intervals into a 1.5 mL microcentrifuge tube (Figure 3).</li>
	<li>Add 100 &#181;L of lysis buffer to the microcentrifuge tube, and vortex at 1,000 rpm, room temperature for 1 h in a thermomixer.</li>
	<li>Centrifuge briefly and transfer the supernatant to a new 1.5 mL microcentrifuge tube.</li>
	<li>Move the strips into 200 &#181;L 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 &#215; g for 1 min further for maximum recovery of the remaining sample on the strip.</li>
	<li>Measure protein concentration using bicinchoninic acid (BCA) or compatible protein assay.</li>
</ol>
4.&#160;Suspension-trapping sample preparation
<ol>
	<li>Normalize samples to 50 &#181;g of protein based on the protein assay result.</li>
	<li>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 &#176;C for 10 min.</li>
	<li>Cool down the protein solution to room temperature.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Uncap the Suspension Trapping Micro Spin Column and assemble the spin column onto a 2 mL microcentrifuge tube to collect flowthrough.</li>
	<li>Add up to 200 &#181;L of acidified protein lysate mixture into the column.</li>
	<li>Centrifuge the column at 4,000 &#215; g for 20 s. Protein particulates were trapped; repeat until the samples are loaded to the column.</li>
	<li>Add 150 &#181;L of protein binding buffer and centrifuge at 4,000 &#215; g for 20 s to wash the suspended protein. Repeat 3x.</li>
	<li>Assemble the Suspension Trapping Micro Column onto a new 1.5 mL microcentrifuge tube.</li>
	<li>Add 20 &#181;L 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.</li>
	<li>Cap the Suspension Trapping Micro Spin Column to prevent evaporation. Incubate at 47 &#176;C for 1 h in a thermomixer; do not shake or vortex during digestion.</li>
	<li>Elute peptides with 40 &#181;L of 50 mM TEAB by centrifugal force at 4,000 &#215; g for 20 s.</li>
	<li>Add 40 &#181;L of 0.2% FA and centrifuge at 4,000 &#215; g for 20 s.</li>
	<li>Add 35 &#181;L of 0.2% FA, 50% acetonitrile, and centrifuge at 4,000 &#215; g for 20 s.</li>
	<li>Pool three eluates and dry with a vacuum centrifuge.</li>
	<li>Store at -80 &#176;C ultra-low temperature freezer until further analysis.NOTE: The protocol can be paused here.</li>
</ol>
5.&#160;Reconstitute peptides for mass spectrometry analysis
<ol>
	<li>Reconstitute the sample with 12 &#181;L of 0.1% FA, vortex, and centrifuge briefly.</li>
	<li>Measure peptide concentration with a Peptide Assay Kit.</li>
	<li>Normalize peptide concentration to 0.5 &#181;g/&#181;L in 0.1% FA.</li>
	<li>Transfer the peptide mixture to an autosampler vial and ready for mass spectrometry analysis.</li>
</ol>
6.&#160;Sample Acquisition by Liquid Chromatography-Tandem Mass Spectrometry
<ol>
	<li>Load 3 &#181;g of the peptide with isocratic loading buffer (0.1% FA, 5% ACN) to a trap-column (C18, 5 &#181;m, 100 &#181;m, 20 mm) at a flow rate of 2 &#181;L/min for 15 min.</li>
	<li>Fractionate the peptides using a reversed-phase nano analytical column (C18, 5 &#181;m, 100 &#181;m, 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.</li>
	<li>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.</li>
	<li>Analyze raw data with the referenced software or other compatible engines with a reference database in FASTA from the publicly accessible UniProt database.</li>
</ol>

Non-Invasive Biomarker Discovery for Tear Proteomics Using LC-MS

<ol><li>Ma, J. Y. W., Sze, Y. H., Bian, J. F., Lam, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+role+of+mass+spectrometry+proteomics+in+tear+biomarker+discovery+for+multifactorial+ocular+diseases+%28Review%29%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Medicine%22%5BJournal%5D)">Critical role of mass spectrometry proteomics in tear biomarker discovery for multifactorial ocular diseases (Review).</a> International Journal of Molecular Medicine. 47 (5), 83(2021).</li>
<li>Tse, J. S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+clinical+data+and+tear+proteomics+to+assess+efficacy%2C+ocular+surface+status%2C+and+biomarker+response+after+orthokeratology+lens+wear%5BTitle%5D)+AND+%22Translational+Vision+Science+%26+Technology%22%5BJournal%5D)">Integrating clinical data and tear proteomics to assess efficacy, ocular surface status, and biomarker response after orthokeratology lens wear.</a> Translational Vision Science & Technology. 10 (11), 18(2021).</li>
<li>Cheung, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+tear+proteome+dataset+in+response+to+daily+wear+of+water+gradient+contact+lens+using+SWATH-MS+approach%5BTitle%5D)+AND+%22Data+in+Brief%22%5BJournal%5D)">Human tear proteome dataset in response to daily wear of water gradient contact lens using SWATH-MS approach.</a> Data in Brief. 36, 107120(2021).</li>
<li>Tse, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Data+on+assessment+of+safety+and+tear+proteome+change+in+response+to+orthokeratology+lens+-+Insight+from+integrating+clinical+data+and+next+generation+proteomics%5BTitle%5D)+AND+%22Data+in+Brief%22%5BJournal%5D)">Data on assessment of safety and tear proteome change in response to orthokeratology lens - Insight from integrating clinical data and next generation proteomics.</a> Data in Brief. 29, 105186(2020).</li>
<li>Zhan, X., Li, J., Guo, Y., Golubnitschaja, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry+analysis+of+human+tear+fluid+biomarkers+specific+for+ocular+and+systemic+diseases+in+the+context+of+3P+medicine%5BTitle%5D)+AND+%22The+EPMA+Journal%22%5BJournal%5D)">Mass spectrometry analysis of human tear fluid biomarkers specific for ocular and systemic diseases in the context of 3P medicine.</a> The EPMA Journal. 12 (4), 449-475 (2021).</li>
<li>Ponzini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry-based+tear+proteomics+for+noninvasive+biomarker+discovery%5BTitle%5D)+AND+%22Mass+Spectrometry+Reviews%22%5BJournal%5D)">Mass spectrometry-based tear proteomics for noninvasive biomarker discovery.</a> Mass Spectrometry Reviews. 41 (5), 842-860 (2022).</li>
<li>HaileMariam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((S-Trap%2C+an+ultrafast+sample-preparation+approach+for+shotgun+proteomics%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">S-Trap, an ultrafast sample-preparation approach for shotgun proteomics.</a> Journal of Proteome Research. 17 (9), 2917-2924 (2018).</li>
<li>Ding, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Urine+proteomics%3A+evaluation+of+different+sample+preparation+workflows+for+quantitative%2C+reproducible%2C+and+improved+depth+of+analysis%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">Urine proteomics: evaluation of different sample preparation workflows for quantitative, reproducible, and improved depth of analysis.</a> Journal of Proteome Research. 19 (4), 1857-1862 (2020).</li>
<li>Ludwig, K. R., Schroll, M. M., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+in-solution%2C+FASP%2C+and+S-Trap+based+digestion+methods+for+bottom-up+proteomic+studies%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">Comparison of in-solution, FASP, and S-Trap based digestion methods for bottom-up proteomic studies.</a> Journal of Proteome Research. 17 (7), 2480-2490 (2018).</li>
<li>Sze, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-pH+reversed-phase+fractionated+neural+retina+proteome+of+normal+growing+C57BL%2F6+mouse%5BTitle%5D)+AND+%22Scientific+Data%22%5BJournal%5D)">High-pH reversed-phase fractionated neural retina proteome of normal growing C57BL/6 mouse.</a> Scientific Data. 8 (1), 27(2021).</li>
<li>Marchione, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HYPERsol%3A+High-quality+data+from+archival+FFPE+tissue+for+clinical+proteomics%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">HYPERsol: High-quality data from archival FFPE tissue for clinical proteomics.</a> Journal of Proteome Research. 19 (2), 973-983 (2020).</li>
<li>Chhuon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+S-Trap-based+approach+to+the+analysis+of+T+cell+lipid+raft+proteome%5BTitle%5D)+AND+%22Journal+of+Lipid+Research%22%5BJournal%5D)">A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome.</a> Journal of Lipid Research. 61 (11), 1512-1523 (2020).</li>
<li>Hayoun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+sample+preparation+methods+for+fast+proteotyping+of+microorganisms+by+tandem+mass+spectrometry%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Evaluation of sample preparation methods for fast proteotyping of microorganisms by tandem mass spectrometry.</a> Frontiers in Microbiology. 10, 1985(2019).</li>
<li>Templeton, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+SPEED%2C+S-Trap%2C+and+in-solution-based+sample+preparation+methods+for+mass+spectrometry+in+kidney+tissue+and+plasma%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Comparison of SPEED, S-Trap, and in-solution-based sample preparation methods for mass spectrometry in kidney tissue and plasma.</a> International Journal of Molecular Sciences. 24 (7), 6290(2023).</li>
<li>Ding, Z., Wang, N., Ji, N., Chen, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomics+technologies+for+cancer+liquid+biopsies%5BTitle%5D)+AND+%22Molecular+Cancer%22%5BJournal%5D)">Proteomics technologies for cancer liquid biopsies.</a> Molecular Cancer. 21 (1), 53(2022).</li>
<li>Nättinen, J., Aapola, U., Jylhä, A., Vaajanen, A., Uusitalo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+capillary+and+Schirmer+strip+tear+fluid+sampling+methods+using+SWATH-MS+proteomics+approach%5BTitle%5D)+AND+%22Translational+Vision+Science+%26+Technology%22%5BJournal%5D)">Comparison of capillary and Schirmer strip tear fluid sampling methods using SWATH-MS proteomics approach.</a> Translational Vision Science & Technology. 9 (3), 16(2020).</li>
<li>Bachhuber, F., Huss, A., Senel, M., Tumani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+biomarkers+in+tear+fluid%3A+from+sampling+to+preanalytical+processing%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Diagnostic biomarkers in tear fluid: from sampling to preanalytical processing.</a> Scientific Reports. 11 (1), 10064(2021).</li>
</ol>

The authors have no conflicts of interest to declare.

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.

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&#39;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 &#176;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 &#181;g/&#181;L for mass spectrometric analysis.

<table><tbody><tr><td>9 mm Plastic Screw Thread Vials</td><td>Thermo Scientific</td><td>C4000-11</td><td></td></tr><tr><td>Acetonitrile, LCMS Grade</td><td>Anaqua</td><td>AC-1026</td><td></td></tr><tr><td>Centrifuge MiniSpin plus</td><td>Eppendorf</td><td>5453000097</td><td></td></tr><tr><td>DL-dithiothreitol (DTT), BioUltra</td><td>Sigma-Aldrich</td><td>43815</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL</td><td>Eppendorf</td><td>30120086</td><td></td></tr><tr><td>Formic acid, ACS reagent, &amp;ge;96%</td><td>Sigma-Aldrich</td><td>695076</td><td></td></tr><tr><td>Frame Heater OPTIMONSUN Electronic</td><td>Breitfeld &amp;amp; Schliekert GmbH</td><td>1203166</td><td></td></tr><tr><td>Iodoacetamide (IAA), BioUltra</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>Methanol, HPLC Grade</td><td>Anaqua</td><td>MA-1292</td><td></td></tr><tr><td>Nunc Biobanking and Cell Culture Cryogenic Tubes, 2 mL</td><td>Thermo Fisher Scientific</td><td>368632</td><td></td></tr><tr><td>Phosphoric acid, 85 wt.% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>345245</td><td></td></tr><tr><td>Pierce Quantitative Colorimetric Peptide Assay</td><td>Thermo Fisher Scientific</td><td>23275</td><td></td></tr><tr><td>Pierce Rapid Gold BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>A53225</td><td></td></tr><tr><td>Quadrupole Time-of-Flight Mass Spectrometry</td><td>Sciex</td><td>TripleTOF 6600</td><td></td></tr><tr><td>Schirmer Ophthalmic Strips</td><td>Entod Research Cell UK Ltd</td><td>I-DEW Tearstrips</td><td></td></tr><tr><td>S-Trap Micro Column</td><td>Protifi</td><td>C02-micro-80</td><td></td></tr><tr><td>SureSTART 9 mm Screw Caps</td><td>Thermo Scientific</td><td>CHSC9-40</td><td></td></tr><tr><td>Triethylammonium bicarbonate (TEAB), 1 M</td><td>Sigma-Aldrich</td><td>18597</td><td></td></tr><tr><td>Ultra-performance Liquid Chromatography</td><td>Eksigent</td><td>NanoLC 400&amp;nbsp;</td><td></td></tr><tr><td>UltraPure Sodium dodecyl sulfate (SDS)</td><td>Thermo Fisher Scientific</td><td>15525017</td><td></td></tr></tbody></table>

a protein suspension-trapping sample preparation for tear proteomics by liquid chromatography-tandem mass spectrometry

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.

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 &#60; 0.001) than the standard in-solution digestion protocol and with good reproducibility at a coefficient of variation (%CV) &#60; 7%. A pool of tears samples was repeated in six technical replicates with 74.2 &#177; 5.0% peptide recovery and 52.8 &#177; 1.6% peptide recovery in samples prepared with in-solution procedures. A pooled tear sample with a protein amount of 36.3 &#181;g was spiked onto the Schirmer strip and extracted as previously described, the extraction efficiency of protein was 81% (29.5 &#177; 6.8 &#181;g, mean &#177; SD).
Both workflows loaded 3 &#181;g of peptides on the MS, and the DDA search yielded a total of 1,183 &#177; 118 proteins (5,757 &#177; 537 distinct peptides) and 874 &#177; 70 proteins (4,400 &#177; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64617/64617fig01.jpg" /> 
Figure 1: Bend the top of the Schirmer strip at the 0 mm mark before application. <a href="https://www.jove.com/files/ftp_upload/64617/64617fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64617/64617fig02.jpg" /> 
Figure 2: Position of the Schirmer strip during tear collection. <a href="https://www.jove.com/files/ftp_upload/64617/64617fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64617/64617fig03.jpg" /> 
Figure 3: Illustration of Schirmer strip handling before protein extraction. <a href="https://www.jove.com/files/ftp_upload/64617/64617fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64617/64617fig04.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/64617/64617fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Advancing Tear Fluid Analysis Using a Standardized Protocol for Proteomics Research at JoVE.com

Author Spotlight: Advancing Tear Fluid Analysis Using a Standardized Protocol for Proteomics Research 

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.

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

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...

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Research

JoVE Journal

Medicine

2.3K Views.  The Hong Kong Polytechnic University. Ons onderzoek heeft tot doel het gebruik aan te tonen van een monstervoorbereiding voor het vangen van eiwitten, bekend als S-trap, voor onderzoek op basis van massaspectrometrie. Dit protocol maakt gebruik van een snelle, reproduceerbare en gestandaardiseerde monstervoorbereiding van menselijk traanvloeistofmonster dat is verzameld met behulp van translationeel onderzoek in de oogheelkunde. Menselijk traanvocht is een lichaamsvloeistof die kan worden verzameld voor niet-invasieve moleculaire...

Video: Auteur in de schijnwerpers: Bevordering van traanvochtanalyse met behulp van een gestandaardiseerd protocol voor proteomics-onderzoek 

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end ...

Chemistry

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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

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