Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting

Microflow liquid chromatography tandem mass spectrometry (μLC–MS/MS) is becoming a viable alternative to nanoflow LC–MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 μL/min for high-throughput applications. On the basis of the analysis of ∼38 000 samples measured on two instruments over the past two years, we now show that the approach is extremely robust. Up to 1500 analyses were performed within one month, and >14 000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, tissues, and human body fluids, which were analyzed with or without prior peptide fractionation or stable isotope labeling. We show that the μLC–MS/MS system is capable of measuring 2600 proteins from undepleted human plasma and ∼5000 proteins from crude human urine in 1 day, demonstrating its potential for in-depth as well as high-throughput clinical application.微流液相色谱串联质谱法（μLC–MS/MS）正逐步成为蛋白质组分析领域中可替代纳流液相色谱串联质谱法（nanoflow LC–MS/MS）的可靠技术。我们近期已证实，采用内径1 mm × 150 mm色谱柱、流速50 μL/min的该系统在高通量应用场景中具备应用潜力。基于过去两年间在两台仪器上完成的约38000个样本的分析数据，本研究进一步证明该分析方法具有极佳的稳健性：单月可完成至多1500次分析，单根色谱柱可完成超过14000个样本的检测且色谱性能无衰减。本次分析的样本涵盖细胞系、组织以及人体体液的蛋白质组，部分样本在检测前进行了肽段分级或稳定同位素标记，部分则未做上述处理。实验结果显示，该μLC–MS/MS系统可在单日完成未去除高丰度蛋白的人血浆中2600种蛋白质的检测，以及未经处理的人尿液中约5000种蛋白质的检测，充分展现了其在深度分析与高通量临床应用中的潜力。