protein extraction from human tear using schirmer strip

LC-MS Kullanarak Gözyaşı Proteomikleri için İnvaziv Olmayan Biyobelirteç Keşfi

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.

Yazar Spotlight: Proteomik Araştırma için Standartlaştırılmış Bir Protokol Kullanarak Gözyaşı Sıvısı Analizini Geliştirme 

Sıvı Kromatografisi-Tandem Kütle Spektrometrisi ile Gözyaşı Proteomiği için Bir Protein Süspansiyonu-Tutucu Numune Hazırlama

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  

Schirmer Strip Kullanarak İnsan Gözyaşından Protein Ekstraksiyonu

Kurumuş insan gözyaşlarını içeren Schirmer şeridinin ön ucunu sıfır milimetre işaretinin ötesinde kesip atarak başlayın. Daha sonra şeridi bir milimetre aralıklarla kesin ve parçaları 1,5 mililitrelik bir mikro santrifüj tüpüne yerleştirin. Daha sonra, mikro santrifüj tüpüne 100 mikrolitre lizis tamponu ekleyin.Bir termo karıştırıcıda bir saat boyunca oda sıcaklığında 1000 RPM'de vorteksleyin. Kısa bir santrifüjlemeden sonra, süpernatanı yeni bir 1.5 mililitrelik mikro santrifüj tüpüne aktarın. Temiz forseps kullanarak şeritleri 200 mikrolitrelik pipet uçlarına taşıyın.Uçları aynı mikro santrifüj tüpüne geri yerleştirin, ardından maksimum numune geri kazanımı için santrifüjleyin. Bikinkoninik asit veya uyumlu bir protein tahlili kullanarak protein konsantrasyonunu ölçün.

protein-extraction-from-human-tear-using-schirmer-strip

Research

JoVE Journal

Medicine

Protein Extraction from Human Tear Using Schirmer Strip

467 Görüntüleme. Kurumuş insan gözyaşlarını içeren Schirmer şeridinin ön ucunu sıfır milimetre işaretinin ötesinde kesip atarak başlayın. Daha sonra şeridi bir milimetre aralıklarla kesin ve parçaları 1,5 mililitrelik bir mikro santrifüj tüpüne yerleştirin. Daha sonra, mikro santrifüj tüpüne 100 mikrolitre lizis tamponu ekleyin.Bir termo karıştırıcıda bir saat boyunca oda sıcaklığında 1000 RPM'de vorteksleyin. Kısa bir santrifüjlemeden sonra, süpernatanı yeni bir 1...

Video: 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 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 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 ...