Automated SPR-LC−MS/MS System for Protein Interaction Analysis

We have developed a novel automated system to analyze protein complexes by integrating a surface plasmon resonance (SPR) biosensor with highly sensitive nanoflow liquid chromatography-tandem mass spectrometry (LC−MS/MS). A His-tagged protein, which is also tagged with FLAG and biotinylated sequences, was expressed in mammalian cells. After purification by using the His tag from the cell lysate, the sample protein mixture was applied to an SPR biosensor and the protein complex was captured on the sensor chip. The automated SPR-LC−MS/MS was then performed: (1) two-step on-chip purification of the protein complex by using the FLAG and the biotinylated tags, (2) on-chip protease digestion of the complex, and (3) online nanoflow LC−MS/MS analysis of the resulting peptide fragments for protein identification. All of these processes could be monitored in real-time by the SPR biosensor. We validated the performance of the system using either FK506-binding protein 52 kDa (FKBP52) or ribosomal protein S19 (rpS19) as bait. Thus, the fully automated SPR-LC−MS/MS system appeared to be a powerful tool for functional proteomics studies, particularly for snapshot analysis of functional cellular complexes and machines.

本研究开发了一种新型自动化系统，用于蛋白质复合物的分析：该系统将表面等离子体共振（surface plasmon resonance, SPR）生物传感器与高灵敏度纳流液相色谱-串联质谱（nanoflow liquid chromatography-tandem mass spectrometry, LC−MS/MS）相结合。本研究在哺乳动物细胞中表达了一种带有His标签（His-tag）的蛋白质，该蛋白质同时带有FLAG标签（FLAG-tag）与生物素化序列（biotinylated sequences）。通过His标签从细胞裂解液（cell lysate）中纯化后，将样品蛋白质混合物施加至SPR生物传感器，使蛋白质复合物被捕获于传感器芯片表面。随后开展自动化SPR-LC−MS/MS流程，包含以下三个步骤：(1) 利用FLAG标签与生物素化标签对蛋白质复合物进行两步芯片上纯化；(2) 对复合物开展芯片上蛋白酶（protease）酶解；(3) 对酶解得到的肽段片段（peptide fragments）进行在线纳流LC−MS/MS分析，以实现蛋白质鉴定（protein identification）。上述所有流程均可通过SPR生物传感器进行实时监测。本研究以FK506结合蛋白52 kDa（FK506-binding protein 52 kDa, FKBP52）或核糖体蛋白S19（ribosomal protein S19, rpS19）作为诱饵蛋白，对该系统的性能进行了验证。综上，这套全自动化SPR-LC−MS/MS系统有望成为功能蛋白质组学（functional proteomics）研究的有力工具，尤其适用于功能性细胞复合物与细胞机器的快照式分析。