Name: Protein Extraction from Human Tear Using Schirmer Strip
Uploaded: 2023-12-01T12:00:00
Duration: 1 min 19 s
Description: Watch this Scientific Journal Video about Protein Extraction from Human Tear Using Schirmer Strip at JoVE.com

protein extraction from human tear using schirmer strip

Niet-invasieve ontdekking van biomarkers voor traanproteomics met behulp van LC-MS

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.

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

Een monstervoorbereiding voor het vangen van eiwitten voor traanproteomics door middel van vloeistofchromatografie-tandem massaspectrometrie

Our research aims to demonstrate the use of a protein suspension trapping sample preparation, known as S trap for mass spectrometry-based research. This protocol employs a rapid, reproducible, and standardized sample preparation of human tear fluid sample collected using the for translational research in ophthalmology. Human tear fluid is a body fluid that can be collected for non-invasive molecular tests in ophthalmology.There is a lot of standardized methods of tear fluid collection and sample preparation, which are critical to successful clinical research of diseases and conditions. Non-standardized methods are difficult to compare and interpret between methodologies and therefore affect chemical outcomes. This protocols allows tear body fluid sampling and storage at room temperature.This helps with easier sample collections and shipping. It provides a rapid and robust peptide extractions for mass spectrometer analysis, which improves peptide recovery and identifications. This protocol has minimal sample preparation steps to minimize error and uncertainties.The fast and superior performance of protein identification and quantification by mass spectrometry will facilitate clinicians and researchers in the development of non-invasive molecular tests. Optimize the sample preparation will always be one of the critical steps. Regardless of the detection method, the clinical value of human tear fluid is yet to be fully investigated.The proposal sample preparation method would hopefully open the door for potential clinical application in the near future.

Watch this Scientific Journal Video about Protein Extraction from Human Tear Using Schirmer Strip at JoVE.com

Protein Extraction from Human Tear Using Schirmer Strip  

Eiwitextractie uit menselijke traan met behulp van Schirmer-strip

Begin met het knippen en weggooien van het voorste uiteinde van de Schirmer-strip met de gedroogde menselijke tranen voorbij de nul millimeter-markering. Snijd de strip vervolgens in intervallen van een millimeter en plaats de stukjes in een microcentrifugebuisje van 1,5 milliliter. Voeg vervolgens 100 microliter lysisbuffer toe aan de microcentrifugebuis.Draai het op 1000 RPM bij kamertemperatuur gedurende een uur in een thermomixer. Breng het supernatans na een korte centrifugatie over in een nieuwe microcentrifugebuis van 1,5 milliliter. Verplaats de strips met een schone pincet in pipetpunten van 200 microliter.Plaats de tips terug in dezelfde microcentrifugebuis en centrifugeer deze vervolgens voor maximale monsterterugwinning. Meet de eiwitconcentratie met behulp van bicinchoninezuur of een compatibele eiwittest.

Research

JoVE Journal

Medicine

Protein Extraction from Human Tear Using Schirmer Strip

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 ...