Thermo Fisher Scientific, Dornier Str. 4, 82110 Germering, Germany.
IMP─Institute of Molecular Pathology, Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
Anal Chem. 2023 Dec 26;95(51):18673-18678. doi: 10.1021/acs.analchem.3c03058. Epub 2023 Dec 13.
This work demonstrates the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics analysis, including single-cell proteomics (SCP). Conditions were optimized on a 50 μm internal diameter (I.D.) column operated at 100 nL/min in the direct injection workflow to balance method sensitivity and sample throughput from 24 to 72 samples/day. Multiple data acquisition strategies were also evaluated for proteome coverage, including data-dependent acquisition (DDA), wide-window acquisition (WWA), and wide-window data-independent acquisition (WW-DIA). Analyzing 250 pg HeLa digest with a 10-min LC gradient (72 samples/day) provided >900, >1,800, and >3,000 protein group identifications for DDA, WWA, and WW-DIA, respectively. Total method cycle time was further reduced from 20 to 14.4 min (100 samples/day) by employing a trap-and-elute workflow, enabling 70% mass spectrometer utilization. The method was applied to library-free DIA analysis of single-cell samples, yielding >1,700 protein groups identified. In conclusion, this study provides a high-sensitivity, high-throughput nanoLC-MS configuration for sample-limited proteomics.
本研究展示了高通量纳升液相色谱-质谱联用技术（nanoLC-MS）和无标记定量（LFQ）在样品有限的蛋白质组学分析中的应用，包括单细胞蛋白质组学（SCP）。在直接进样工作流程中，我们在 50μm 内径（I.D.）柱上优化了条件，流速为 100nL/min，以平衡方法的灵敏度和从 24 到 72 个/天的样品通量。我们还评估了多种数据采集策略的蛋白质组覆盖范围，包括数据依赖性采集（DDA）、宽窗口采集（WWA）和宽窗口数据独立采集（WW-DIA）。用 10min 的 LC 梯度分析 250pg HeLa 酶解物（72 个/天），DDA、WWA 和 WW-DIA 分别可鉴定出>900、>1800 和>3000 个蛋白质组。通过采用阱和洗脱工作流程，进一步将总方法循环时间从 20 分钟缩短到 14.4 分钟（100 个/天），实现了 70%的质谱利用率。该方法应用于无文库的单细胞样品 DIA 分析，鉴定出了>1700 个蛋白质组。总之，本研究为样品有限的蛋白质组学提供了一种高灵敏度、高通量的纳升液相色谱-质谱联用技术配置。
