Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.本研究开发了一种兼具加速在线蛋白质酶解与高分辨纳米液相色谱-质谱联用（LC-MS）功能的新型开管纳米蛋白质组学平台（open tubular nanoproteomic platform）。该平台采用极窄内径的开管色谱柱，因此特别适配样本量有限的实验场景。针对蛋白质的酶解步骤，将样本流经内径为20 µm的固定化胰蛋白酶+内肽酶Lys-C开管酶反应器（OTER）。所得肽段随后被捕获于整体式预柱中，并在线转移至内径为10 µm的多孔层开管（PLOT）液相色谱分离柱。研究以具备潜在诊断价值的Wnt/β-连环蛋白信号通路（Wnt-pathway）蛋白为对象，在120分钟内完成了小型细胞样本与肿瘤组织的酶解与靶向串联质谱（targeted-MS/MS）检测。例如，仅需10 ng样本（约1000个HCT15结肠癌细胞的蛋白提取物）即可识别出潜在生物标志物Axin1。在全谱鉴定模式下，本研究所用的OTER-PLOT装置可在240分钟的较短酶解-检测周期内，从HCT15细胞中鉴定出约1500种蛋白质，性能优于此前报道的在线酶解/分离系统。该平台可依托通用商用仪器与配件实现全自动化操作，且反应器与色谱柱制备简便、残留率极低。上述初步结果表明，该平台可为基于质谱的快速高灵敏蛋白质组学研究提供自动化解决方案，尤其适用于样本量有限的研究场景。