HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture (SILAC) and isobaric tandem mass tag (TMT) labeling enabled up to 28 single cells to be analyzed in a single liquid chromatography–mass spectrometry (LC–MS) analysis, in addition to carrier, reference, and negative control channels. A custom nested nanowell chip was used for nanoliter sample processing to minimize sample losses. Using a 145-min total LC–MS cycle time, ∼280 single cells were analyzed per day. This measurement throughput could be increased to ∼700 samples per day with a high-duty-cycle multicolumn LC system producing the same active gradient. The labeling efficiency and achievable proteome coverage were characterized for multiple analysis conditions.

近年来，基于质谱的单细胞蛋白质组学（mass spectrometry-based single-cell proteomics, SCP）技术取得了显著进展，灵敏度得到大幅提升，但较低的测量通量仍是该领域的主要瓶颈。此前，研究人员已分别将同量异位素标记与同位素标记方法应用于SCP，通过多重化策略提升检测通量。本文中，我们将两类标记策略相结合，实现了通量的倍增效应。细胞培养氨基酸双工稳定同位素标记（stable isotope labeling of amino acids in cell culture, SILAC）与同量异位素串联质量标签（isobaric tandem mass tag, TMT）的联用方案，可在单次液相色谱-质谱联用（liquid chromatography–mass spectrometry, LC–MS）分析中完成最多28个单细胞的检测，同时兼容载体、参考及阴性对照通道。我们采用定制化嵌套式纳米孔芯片开展纳升级样品处理，以最大程度降低样品损耗。在单次LC–MS循环周期为145分钟的条件下，单日可完成约280个单细胞样品的分析；若搭配可生成同等活性洗脱梯度的高占空比多柱液相色谱系统，单日检测通量可提升至约700个样品。本研究针对多种分析条件，对标记效率与可实现的蛋白质组覆盖度进行了系统表征。