Gaizley Emily J, Chen Xiuyuan, Bhamra Amandeep, Enver Tariq, Surinova Silvia
UCL Cancer Institute, University College London, London, UK.
Commun Biol. 2025 Apr 26;8(1):666. doi: 10.1038/s42003-025-08068-x.
Understanding cellular diversity and disease mechanisms requires a global analysis of proteins and their modifications. While next-generation sequencing has advanced our understanding of cellular heterogeneity, it fails to capture downstream signalling networks. Ultrasensitive mass spectrometry-based proteomics enables unbiased protein-level analysis of low cell numbers, down to single cells. However, phosphoproteomics remains limited to high-input samples due to sample losses and poor reaction efficiencies associated with processing low cell numbers. Isobaric stable isotope labelling is a promising approach for reproducible and accurate quantification of low abundant phosphopeptides. Here, we introduce SPARCE (Streamlined Phosphoproteomic Analysis of Rare CElls) for multiplexed phosphoproteomic analysis of low cell numbers. SPARCE integrates cell isolation, water-based lysis, on-tip TMT labelling, and phosphopeptide enrichment. SPARCE outperforms traditional methods by enhancing labelling efficiency and phosphoproteome coverage. To demonstrate the utility of SPARCE, we analysed four patient-derived glioblastoma stem cell lines, reliably quantifying phosphosite changes from 1000 FACS-sorted cells. This workflow expands the possibilities for signalling analysis of rare cell populations.
了解细胞多样性和疾病机制需要对蛋白质及其修饰进行全面分析。虽然新一代测序技术增进了我们对细胞异质性的理解，但它无法捕捉下游信号网络。基于超灵敏质谱的蛋白质组学能够对低至单个细胞的少量细胞进行无偏倚的蛋白质水平分析。然而，由于样品损失以及处理少量细胞时反应效率低下，磷酸化蛋白质组学仍局限于高输入量样本。等压稳定同位素标记是一种对低丰度磷酸肽进行可重复且准确量化的有前景的方法。在此，我们介绍了用于对少量细胞进行多重磷酸化蛋白质组分析的SPARCE（稀有细胞简化磷酸化蛋白质组分析）方法。SPARCE整合了细胞分离、水相裂解、原位TMT标记和磷酸肽富集。SPARCE通过提高标记效率和磷酸化蛋白质组覆盖率，优于传统方法。为了证明SPARCE的实用性，我们分析了四种患者来源的胶质母细胞瘤干细胞系，可靠地量化了来自1000个经荧光激活细胞分选术分选的细胞的磷酸化位点变化。此工作流程扩展了对稀有细胞群体进行信号分析的可能性。
