Nanowell-mediated two-dimensional liquid chromatography enables deep proteome profiling of <1000 mammalian cells.
Nanowell-mediated two-dimensional liquid chromatography enables deep proteome profiling of <1000 mammalian cells.
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DOI：
10.1039/c8sc02680g
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发表时间：
2018-09-14
期刊：
影响因子：
8.4
通讯作者：
Kelly RT
中科院分区：
文献类型：
--
作者：
Dou M;Zhu Y;Liyu A;Liang Y;Chen J;Piehowski PD;Xu K;Zhao R;Moore RJ;Atkinson MA;Mathews CE;Qian WJ;Kelly RT
小型化多维肽分离可以大大增加痕量样品蛋白质组分析的覆盖范围。多维肽分离可以大大增加蛋白质组分析的覆盖深度。然而，多维分离的一个主要挑战是需要大量生物样品，通常含有毫克量的蛋白质。我们开发了纳米孔介导的二维 (2D) 反相纳流液相色谱 (LC) 分离，用于低纳克样品的深入蛋白质组分析。首先使用高 pH LC 分离肽，并将流出物连接到 4 或 12 个纳米孔中。每个纳米孔的内容物在 LC 缓冲液中重构并收集，用于随后通过低 pH nanoLC-MS/MS 进行分离和分析。纳米孔平台最大限度地减少了离线 2D LC 分级分离中表面的肽损失，从而能够从仅 50 ng HeLa 消化物中自信地鉴定出超过 5800 种蛋白质。此外，结合最近开发的基于纳米孔的样品制备工作流程，我们展示了对来自小细胞群的超过 6000 个蛋白质组的深度蛋白质组分析，其中包括来自症状前 1 型糖尿病供体的~650 个 HeLa 细胞和 10 个单个人胰岛薄切片（~1000 个细胞）。
Miniaturized multidimensional peptide separations can greatly increase the coverage in proteome profiling for trace samples. Multidimensional peptide separations can greatly increase the depth of coverage in proteome profiling. However, a major challenge for multidimensional separations is the requirement of large biological samples, often containing milligram amounts of protein. We have developed nanowell-mediated two-dimensional (2D) reversed-phase nanoflow liquid chromatography (LC) separations for in-depth proteome profiling of low-nanogram samples. Peptides are first separated using high-pH LC and the effluent is concatenated into 4 or 12 nanowells. The contents of each nanowell are reconstituted in LC buffer and collected for subsequent separation and analysis by low-pH nanoLC-MS/MS. The nanowell platform minimizes peptide losses to surfaces in offline 2D LC fractionation, enabling >5800 proteins to be confidently identified from just 50 ng of HeLa digest. Furthermore, in combination with a recently developed nanowell-based sample preparation workflow, we demonstrated deep proteome profiling of >6000 protein groups from small populations of cells, including ∼650 HeLa cells and 10 single human pancreatic islet thin sections (∼1000 cells) from a pre-symptomatic type 1 diabetic donor.