Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis.
Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis.
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DOI：
10.1002/jev2.12184
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发表时间：
2022-03
影响因子：
16
通讯作者：
Yang L
中科院分区：
文献类型：
--
作者：
Correll VL;Otto JJ;Risi CM;Main BP;Boutros PC;Kislinger T;Galkin VE;Nyalwidhe JO;Semmes OJ;Yang L
从患者样品中分离和随后的细胞外囊泡（EV）的分子分析是理解囊泡生物学和促进生物标志物发现的广泛使用的策略。尿液中表达的前列腺分泌物是肿瘤近端流体，其作为用于液体活检方案的潜在前列腺癌（PCa）生物标志物的来源已受到显著关注。标准EV分离方法如差示超离心（dUC）共分离蛋白质污染物，其在典型的质谱（MS）方案中掩盖低丰度蛋白质。使前列腺分泌物的分析进一步复杂化的是，尿调节素，也称为Tamm-Horsfall蛋白（THP），在尿液中以高浓度存在。THP可以形成在纯化过程中捕获EV的聚合物，从而降低产率。用二硫苏糖醇（DTT）破坏THP聚合物网络可以释放捕获的EV，但较小的THP纤维在随后的超离心过程中与EV共分离。为了解决这些挑战，我们在这里描述了一种dUC方法，该方法结合了THP聚合物还原和碱洗，以改善EV分离并耗尽THP和其他常见蛋白质污染物。当应用于尿液中的人前列腺分泌物时，我们实现了已知前列腺和前列腺癌相关EV驻留蛋白的相对富集。我们的方法为尿EV的全球蛋白质组学分析提供了一种有前途的策略。
The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co‐isolate protein contaminants that mask lower‐abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm‐Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co‐isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer‐associated EV‐resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs.
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DOI：
10.1083/jcb.93.1.97
发表时间：
1982-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Fujiki Y;Hubbard AL;Fowler S;Lazarow PB
通讯作者：
Lazarow PB
影响因子：
254.7
作者：
Jemal, Ahmedin;Siegel, Rebecca;Thun, Michael J.
通讯作者：
Thun, Michael J.
影响因子：
4.6
作者：
Fujita K;Kume H;Matsuzaki K;Kawashima A;Ujike T;Nagahara A;Uemura M;Miyagawa Y;Tomonaga T;Nonomura N
通讯作者：
Nonomura N
影响因子：
3.3
作者：
Drake, Richard R.;White, Krista Y.;Fuller, Thomas W.;Igwe, Elena;Clements, Mary Ann;Nyalwidhe, Julius O.;Given, Robert W.;Lance, Raymond S.;Semmes, O. John
通讯作者：
Semmes, O. John
影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias