A novel chromatographic method allows on-line reanalysis of the proteome.

Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line coupling in LCMS is a major strength but also causes a principal limitation that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we developed a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly, and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched, and the portion stored in the capillary is analyzed ("replay run"). We describe a setup consisting of an analytical column, a splitting valve, and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system: nearly continuous MS utilization, duplicate analysis without requirement for additional sample, and targeting of important but undersampled features in the replay run.

Use of a microchip device coupled with mass spectrometry for ligand screening of a multi-protein target.

Nanoflow electrospray mass spectrometry has been applied previously to investigate noncovalent protein-protein and protein-ligand interactions. Here we evaluate a commercial microchip device for this application. We show that the microchip can be used to obtain mass spectra of the noncovalent tetramer transthyretin. The device showed a 10-fold increase in signal stability compared with a nanoflow capillary and a high level of nozzle-to-nozzle reproducibility. Binding of the natural ligand thyroxine was clearly observed, and a range of small molecules proposed as inhibitors of transthyretin amyloidosis were shown to be effective in stabilizing the tetramer. We propose that measuring the ability of small molecules to stabilize protein complexes using this automated microchip technology will enable high-throughput screening of multi-protein complexes by mass spectrometry.