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.
Hayano
Toshiya
T
Department of Bioengineering, United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
Yamauchi
Yoshio
Y
Asano
Kazunobu
K
Tsujimura
Teruko
T
Hashimoto
Setsuko
S
Isobe
Toshiaki
T
Takahashi
Nobuhiro
N
D001331:Automation; D001483:Base Sequence; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D017931:DNA Primers; D012269:Ribosomal Proteins; C040343:ribosomal protein S19; D022021:Tacrolimus Binding Proteins; C403485:tacrolimus binding protein 4