Easy and Accessible Workflow for Label-Free Single-Cell Proteomics
Easy and Accessible Workflow for Label-Free Single-Cell Proteomics
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DOI：
10.1021/jasms.3c00240
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发表时间：
2023-08-18
影响因子：
3.2
通讯作者：
Kelly, Ryan T. T.
中科院分区：
文献类型：
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作者：
Sanchez-Avila, Ximena;Truong, Thy;Kelly, Ryan T. T.
单细胞蛋白质组学(SCP)可以提供通过大规模蛋白质测量或其他基因组的单细胞图谱无法获得的信息。最大限度地扩大蛋白质组覆盖率通常需要用于样品处理和分离的定制仪器、消耗品和试剂，这限制了SCP仅限于少数专业实验室。商业平台已经可以用于SCP细胞分离和样品制备，但这些平台的高昂成本和操作所需的技术专业知识使许多感兴趣的实验室无法获得这些平台。在此，我们评估了新的HP D100单细胞分配器用于无标签SCP。该仪器具有较高的准确度和重复性，可用于200毫升至2毫升范围内的试剂配制。当在细胞分配和试剂分配后立即对孔板进行离心时，我们发现在仪器软件中确定的包含单个细胞的97%的孔确实提供了预期的单个细胞的蛋白质组覆盖。这种商业分配器与一步样品处理相结合，为SCP提供了一个非常快速和易于使用的工作流程，与基于纳米细胞的工作流程相比，蛋白质组覆盖范围没有减少，而且商业孔板还促进了使用未经修改的仪器进行自动采样。单细胞样本的分析使用家庭包装的30亩；米ID。Nano LC色谱柱以及市面上可买到的50亩/平方英寸色谱柱。柱子。商业专栏的结果是，识别出的蛋白质减少了35%。然而，与Well平板制备平台相结合，本工作流程为SCP样品的制备和分离提供了一个完全商业化和相对低成本的替代方案，这应该会极大地扩大SCP对其他实验室的可及性。
Single-cell proteomics (SCP) can provide informationthat is unattainablethrough either bulk-scale protein measurements or single-cell profilingof other omes. Maximizing proteome coverage often requires custominstrumentation, consumables, and reagents for sample processing andseparations, which has limited the accessibility of SCP to a smallnumber of specialized laboratories. Commercial platforms have becomeavailable for SCP cell isolation and sample preparation, but the highcost of these platforms and the technical expertise required for theiroperation place them out of reach of many interested laboratories.Here, we assessed the new HP D100 Single Cell Dispenser for label-freeSCP. The low-cost instrument proved highly accurate and reproduciblefor dispensing reagents in the range from 200 nL to 2 & mu;L. Weused the HP D100 to isolate and prepare single cells for SCP within384-well PCR plates. When the well plates were immediately centrifugedfollowing cell dispensing and again after reagent dispensing, we foundthat & SIM;97% of wells that were identified in the instrument softwareas containing a single cell indeed provided the proteome coverageexpected of a single cell. This commercial dispenser combined withone-step sample processing provides a very rapid and easy-to-use workflowfor SCP with no reduction in proteome coverage relative to a nanowell-basedworkflow, and the commercial well plates also facilitate autosamplingwith unmodified instrumentation. Single-cell samples were analyzedusing home-packed 30 & mu;m i.d. nanoLC columns as well as commerciallyavailable 50 & mu;m i.d. columns. The commercial columns resultedin & SIM;35% fewer identified proteins. However, combined with thewell plate-based preparation platform, the presented workflow providesa fully commercial and relatively low-cost alternative for SCP samplepreparation and separation, which should greatly broaden the accessibilityof SCP to other laboratories.