The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.

中文翻译：

---

通过新兴的单细胞蛋白质组技术对细胞异质性进行深入分析。


全面分析功能蛋白质组细胞异质性的能力对于增进对细胞行为、生物体发育和疾病机制的理解至关重要。通过平均整个群体的生物反应进行的传统批量测量常常会丢失细胞变异的信息。单细胞蛋白质组技术对于理解和辨别细胞异质性变得越来越重要。基于流式细胞术和荧光显微镜的成熟的单细胞蛋白质分析方法由于标记抗体的荧光团的光谱重叠而受到低多重能力的限制。质谱 (MS)、微芯片和基于重复染色的单细胞蛋白质组学技术的最新进展使得能够以高通量、增加多重性和提高灵敏度来评估细胞异质性。在这篇综述中，描述了这些先进技术在单细胞蛋白质分析中的原理、发展、优点和局限性，以及它们在研究细胞异质性方面的生物学应用。最后，讨论了剩余的挑战、可能的策略和未来的机遇，这些挑战将促进单细胞蛋白质组技术在细胞生物学和医学研究中的改进和广泛应用。