Single-cell proteomics workflows have considerably improved in sensitivity and reproducibility to characterize yet unknown biological phenomena. With the emergence of multiplexed single-cell proteomics, studies increasingly present single-cell measurements in conjunction with an abundant congruent carrier to improve precursor selection and enhance identifications. While these extreme carrier spikes are often >100-times more abundant than the investigated samples, undoubtedly the total ion current increases, but quantitative accuracy possibly is affected. We here focus on narrowly titrated carrier spikes (i.e., <20x) and assess their elimination for comparable sensitivity at superior accuracy. We find that subtle changes in the carrier ratio can severely impact measurement variability and describe alternative multiplexing strategies to evaluate data quality. Lastly, we demonstrate elevated replicate overlap while preserving acquisition throughput at improved quantitative accuracy with DIA-TMT and discuss optimized experimental designs for multiplexed proteomics of trace samples. This comprehensive benchmarking gives an overview of currently available techniques and guides conceptualizing the optimal single-cell proteomics experiment.

中文翻译：

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多重单细胞蛋白质组学的定量准确度和精密度

单细胞蛋白质组学工作流程在表征未知生物现象的灵敏度和重现性方面得到了显着提高。随着多重单细胞蛋白质组学的出现，越来越多的研究将单细胞测量与丰富的全等载体结合起来，以改善前体选择和增强识别。虽然这些极端的载流子尖峰通常比所研究的样品丰度 > 100 倍，但无疑总离子流会增加，但定量准确性可能会受到影响。我们在此重点关注精确滴定的载体尖峰（即 <20x），并评估它们的消除，以实现更高准确度的可比灵敏度。我们发现载波比的细微变化会严重影响测量可变性，并描述了用于评估数据质量的替代复用策略。最后，我们展示了增加的重复重叠，同时使用 DIA-TMT 以提高的定量精度保持采集吞吐量，并讨论了痕量样品多重蛋白质组学的优化实验设计。这种全面的基准测试概述了当前可用的技术，并指导了最佳单细胞蛋白质组学实验的概念化。