PDF(Pubmed)
Abstract：
BACKGROUND: The active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood-brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins.
METHODS: Here we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice.
RESULTS: Using the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture.
CONCLUSIONS: The proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.
METHODS: Here we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice.
RESULTS: Using the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture.
CONCLUSIONS: The proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.
摘要：
背景：分子从血液进入大脑的主动转运受到受体的调节，运输商,和存在于血脑屏障（BBB）的内皮细胞腔表面的其他细胞表面蛋白。然而,由于难以以允许有效纯化这些相对低丰度的细胞表面蛋白的方式标记这些蛋白质,因此存在于BBB的腔内皮细胞表面上的蛋白质的蛋白质组学谱分析已被证明具有挑战性。
方法：这里我们描述了一种新的基于灌注的标记工作流程：体内糖捕获。该工作流程依赖于通过灌注温和氧化剂对腔血管表面存在的聚糖的氧化，随后通过将其氧化的聚糖与酰肼珠共价连接来分离糖蛋白。分离的蛋白质的基于质谱的鉴定能够高置信度地鉴定大鼠和小鼠中的内皮细胞表面蛋白质。
结果：使用开发的工作流程，从大鼠BBB中鉴定出347种蛋白质，在小鼠中鉴定出224种蛋白质，两个物种中总共有395种蛋白质。这些蛋白质包括许多具有转运蛋白活性的蛋白质（73种蛋白质），细胞粘附蛋白（47种蛋白质），和跨膜信号受体（31种蛋白质）。为了识别相对于整个大脑在血管中富集的蛋白质，我们建立了血管富集评分，并表明具有高血管富集评分的蛋白质参与血管发育功能，结合整合素，和细胞粘附。使用公开可用的单细胞RNAseq数据，我们表明，通过体内糖捕获鉴定的蛋白质更有可能在内皮细胞中被scRNAseq检测到，而不是在任何其他细胞类型中。此外，通过scRNAseq在内皮细胞中检测到的近50%的编码细胞表面蛋白的基因也通过体内糖捕获鉴定。
结论：在这项工作中通过体内糖捕获鉴定的蛋白质代表了迄今为止在腔BBB表面上最完整和特异性的蛋白质谱分析。鉴定的蛋白质反映了抗体开发的可能靶标，以改善治疗性蛋白质进入大脑的穿越，并将有助于我们对BBB转运机制的进一步了解。
方法：这里我们描述了一种新的基于灌注的标记工作流程：体内糖捕获。该工作流程依赖于通过灌注温和氧化剂对腔血管表面存在的聚糖的氧化，随后通过将其氧化的聚糖与酰肼珠共价连接来分离糖蛋白。分离的蛋白质的基于质谱的鉴定能够高置信度地鉴定大鼠和小鼠中的内皮细胞表面蛋白质。
结果：使用开发的工作流程，从大鼠BBB中鉴定出347种蛋白质，在小鼠中鉴定出224种蛋白质，两个物种中总共有395种蛋白质。这些蛋白质包括许多具有转运蛋白活性的蛋白质（73种蛋白质），细胞粘附蛋白（47种蛋白质），和跨膜信号受体（31种蛋白质）。为了识别相对于整个大脑在血管中富集的蛋白质，我们建立了血管富集评分，并表明具有高血管富集评分的蛋白质参与血管发育功能，结合整合素，和细胞粘附。使用公开可用的单细胞RNAseq数据，我们表明，通过体内糖捕获鉴定的蛋白质更有可能在内皮细胞中被scRNAseq检测到，而不是在任何其他细胞类型中。此外，通过scRNAseq在内皮细胞中检测到的近50%的编码细胞表面蛋白的基因也通过体内糖捕获鉴定。
结论：在这项工作中通过体内糖捕获鉴定的蛋白质代表了迄今为止在腔BBB表面上最完整和特异性的蛋白质谱分析。鉴定的蛋白质反映了抗体开发的可能靶标，以改善治疗性蛋白质进入大脑的穿越，并将有助于我们对BBB转运机制的进一步了解。
