Current mass spectrometry methods enable high-throughput proteomics of large sample amounts, but proteomics of low sample amounts remains limited in depth and throughput. To increase the throughput of sensitive proteomics, we developed an experimental and computational framework, called plexDIA, for simultaneously multiplexing the analysis of peptides and samples. Multiplexed analysis with plexDIA increases throughput multiplicatively with the number of labels without reducing proteome coverage or quantitative accuracy. By using three-plex non-isobaric mass tags, plexDIA enables quantification of threefold more protein ratios among nanogram-level samples. Using 1-hour active gradients, plexDIA quantified ~8,000 proteins in each sample of labeled three-plex sets and increased data completeness, reducing missing data more than twofold across samples. Applied to single human cells, plexDIA quantified ~1,000 proteins per cell and achieved 98% data completeness within a plexDIA set while using ~5 minutes of active chromatography per cell. These results establish a general framework for increasing the throughput of sensitive and quantitative protein analysis.

目前的质谱方法可以实现大样本量的高通量蛋白质组学，但低样本量的蛋白质组学在深度和通量方面仍然有限。为了提高敏感蛋白质组学的通量，我们开发了一个称为 plexDIA 的实验和计算框架，用于同时对肽和样品进行多重分析。使用 plexDIA 进行多重分析可成倍提高通量与标签数量，而不会降低蛋白质组覆盖率或定量准确性。通过使用三重非同量异位质量标签，plexDIA 能够对纳克级样品中三倍以上的蛋白质比率进行定量。使用 1 小时活性梯度，plexDIA 对标记的三重组的每个样本中的约 8,000 个蛋白质进行了定量，并提高了数据完整性，将样本中的缺失数据减少了两倍以上。 plexDIA 应用于单个人类细胞，对每个细胞约 1,000 个蛋白质进行定量，并在每个细胞使用约 5 分钟的活性色谱时在 plexDIA 组内实现了 98% 的数据完整性。这些结果建立了提高敏感和定量蛋白质分析通量的总体框架。