suspension-trapping sample preparation of human tear protein

使用 LC-MS 进行泪液蛋白质组学的非侵入性生物标志物发现

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.

作者聚焦：使用蛋白质组学研究的标准化方案推进泪液分析 

液相色谱-串联质谱法制备泪液蛋白质组学的蛋白质悬浮捕获样品

Watch this Scientific Journal Video about Suspension-Trapping Sample Preparation of Human Tear Protein at JoVE.com

Suspension-Trapping Sample Preparation of Human Tear Protein  

人泪液蛋白悬浮捕获样品制备

首先将人泪液蛋白样品归一化为 50 微克蛋白质。然后加入 200 毫摩尔二硫苏糖醇或 DTT，至终浓度为 20 毫摩尔 DTT，在 95 摄氏度下孵育 10 分钟。将蛋白质溶液冷却至室温后，加入 400 毫摩尔碘乙酰胺至终浓度为 40 毫摩尔，然后在室温下避光孵育 10 分钟。接下来，以 1 比 10 的比例加入 12% 磷酸水溶液，最终浓度为 1.2%。短暂涡旋混合物以混合溶液。在再次涡旋之前，以 1 比 6 的比例加入蛋白质结合缓冲液。打开悬浮捕获微量离心柱的盖子，将离心柱组装到两毫升微量离心管上以收集流出物。向色谱柱中加入多达 200 微升酸化蛋白裂解物混合物。将色谱柱以4000G离心20秒。接下来，加入 150 微升蛋白质结合缓冲液并像以前一样离心以洗涤悬浮蛋白质。将悬浮捕获微量柱组装到新的1.5毫升微量离心管上。小心地将 20 微升 TEAB 消解缓冲液添加到离心柱内的过滤器上，避免出现气泡和气隙。盖上悬浮捕获微离心柱的盖子，以防止蒸发。在 47 摄氏度的热混合器中孵育一小时，不要摇晃。用 40 微升 50 毫摩尔 TEAB 洗脱肽。然后以 4000 G 离心 20 秒。现在加入 40 微升 0.2% 甲酸并像以前一样离心。最后，加入 35 微升 0.2% 甲酸和 50% 乙腈中，然后再次离心。拉出三种洗脱液并使用真空离心机干燥。干燥后，将样品储存在 80 摄氏度以备将来分析。数据依赖性采集搜索在悬浮捕获组中共产生 1183 个正负 118 个蛋白质，在溶液内组中产生 874 个正负 70 个蛋白质（1%FDR）。基因本体分析揭示了两种方法相似的蛋白质组，主要功能是结合催化活性和分子功能调控。

suspension-trapping-sample-preparation-of-human-tear-protein

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Medicine

Suspension-Trapping Sample Preparation of Human Tear Protein

267 次观看. 首先将人泪液蛋白样品归一化为 50 微克蛋白质。然后加入 200 毫摩尔二硫苏糖醇或 DTT，至终浓度为 20 毫摩尔 DTT，在 95 摄氏度下孵育 10 分钟。将蛋白质溶液冷却至室温后，加入 400 毫摩尔碘乙酰胺至终浓度为 40 毫摩尔，然后在室温下避光孵育 10 分钟。接下来，以 1 比 10 的比例加入 12% 磷酸水溶液，最终浓度为 1.2%。短暂涡旋混合物以混合溶液。在再次涡旋之前，以 1 ?...

视频: Suspension-Trapping Sample Preparation of Human Tear Protein  

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 ...