Dilute to Enrich for Deep Proteomics: Yolk-Depleted Carrier for a Limited Population of Embryonic (Frog) Cells

This project developed a proteomic methodology that deepens the detectable and quantifiable proteome from limited proteomes where abundant proteins pose interference. As proof-of-principle, this study used Xenopus laevis, where ~90% of the proteome is dominated by abundant yolk proteins in the early developing embryo. We pooled Xenopus embryos and depleted the proteome to ~30% yolk protein content to prepare a carrier proteome. This yolk-depleted carrier proteome digest was spiked into isobarically tagged proteome digests that were prepared from limited tissues and cells isolated by fluorescence-activated cell sorting (FACS). The resulting samples were analyzed using liquid chromatography (LC) high resolution mass spectrometry (HRMS). We demonstrated that the yolk depleted carrier dilutes abundant yolk peptides and boosts the overall depth of proteome coverage, including many biologically important non-yolk proteins present in low abundance.

针对丰度蛋白造成干扰的受限蛋白质组样本，本项目开发了一种蛋白质组学方法，可提升其可检测与定量蛋白质组的覆盖深度。本研究以非洲爪蟾（Xenopus laevis）作为原理验证模型——其早期发育胚胎中约90%的蛋白质组由丰度极高的卵黄蛋白主导。我们将非洲爪蟾胚胎混样后，对其蛋白质组进行卵黄蛋白去除处理，使卵黄蛋白占比降至约30%，以此制备载体蛋白质组。将该经卵黄去除处理的载体蛋白质组酶解产物掺入到由有限组织及通过荧光激活细胞分选（FACS）分离得到的细胞所制备的等压标记蛋白质组酶解产物中。随后采用液相色谱（LC）-高分辨质谱（HRMS）对所得样本进行分析。本研究证实，经卵黄去除的载体可稀释丰度较高的卵黄肽段，并提升整体蛋白质组的覆盖深度，涵盖大量丰度较低且具有重要生物学意义的非卵黄蛋白。