Department of Bioengineering, Northeastern University, Boston, MA, USA.
Barnett Institute, Northeastern University, Boston, MA, USA.
Nat Protoc. 2021 Dec;16(12):5398-5425. doi: 10.1038/s41596-021-00616-z. Epub 2021 Oct 29.
Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.
许多生物系统由不同的单细胞组成。这种多样性需要对单细胞进行功能和分子分析。单细胞蛋白质分析长期依赖于亲和试剂，但新兴的质谱方法（无论是无标记还是多重标记）已经能够对每个细胞进行 >1000 种蛋白质的定量分析，同时提高了蛋白质定量的特异性。在这里，我们描述了使用等压载体来增强肽序列鉴定的单细胞蛋白质组学（SCoPE2）方案。单细胞通过 FACS 或 CellenONE 分离到多微孔板中，并通过最小化蛋白质组学样品制备（mPOP）进行裂解，然后用等压质量标签（TMT 或 TMTpro）对其肽进行标记，用于多重分析。SCoPE2 提供了一种具有成本效益的单细胞蛋白质定量方法，可以使用广泛可用的设备完全自动化，并扩展到数千个单细胞。SCoPE2 使用廉价的试剂，适用于任何可以处理成单细胞悬浮液的样品。SCoPE2 工作流程使用仅标准商业设备，每 24 小时可分析约 200 个单细胞。我们强调实现定量蛋白质分析所需的实验步骤和基准。
