Novel Blood Collection Method Allows Plasma Proteome Analysis from Single Zebrafish

Zebrafish
is an important model organism in biological research. One of the
least explored tissues of zebrafish is blood, because the existing
methods for isolating blood from this organism are tedious and irreproducible.
The small volume of blood collected by these methods also prohibits
many biochemical and cytological analyses. This technical obstacle
limits the utilization of zebrafish in many applications, particularly
in hematological research and plasma biomarker discovery. To overcome
this limitation, we have established a novel method of extracting
blood from zebrafish, based on the use of low centrifugal force to
collect blood from a wound. This method consistently recovers more
blood than traditional methods. Gel electrophoresis and flow cytometry
showed that composition of blood harvested by this method is indistinguishable
from traditional methods. The increase in yield enables us to perform
biochemical experiments on zebrafish blood. In particular, we have
demonstrated that quantitative proteomics can be performed on plasma
collected from single zebrafish. Here, we have compared, by using
shotgun proteomic analysis, the plasma proteomes of adult male and
female zebrafish. Twenty-seven gender-dependent plasma proteins are
identified and their biochemical importance discussed. Taken together,
this novel technique enables analyses that were previously difficult
to perform on zebrafish blood.

斑马鱼（Zebrafish）是生命科学研究中重要的模式生物。其体内探索程度最低的组织之一为血液，由于现有从该生物体中分离血液的方法繁琐且重复性差，且此类方法采集的血液体积过小，也阻碍了诸多生化与细胞学分析的开展。这一技术瓶颈限制了斑马鱼在诸多应用场景中的使用，尤其在血液学研究与血浆生物标志物发掘领域。为突破这一局限，本研究建立了一种全新的斑马鱼血液提取方法：基于低离心力从伤口处采集血液。该方法的血液回收率始终高于传统手段。凝胶电泳与流式细胞术检测结果显示，本方法采集的血液组分与传统方法无显著差异。回收率的提升使得我们能够开展斑马鱼血液的生化实验。尤为重要的是，我们证实可对单条斑马鱼采集的血浆进行定量蛋白质组学分析。本研究通过鸟枪蛋白质组学分析，对比了成年雌雄斑马鱼的血浆蛋白质组，共鉴定出27种性别依赖性血浆蛋白，并对其生化意义进行了探讨。综上，这一新技术使得此前难以开展的斑马鱼血液分析成为可能。