Capillary-LC-μESI-MS/MS and nano-LC-nano ESI-MS/MS analysis using a single binary pump capillary LC system: Applications in proteomics

We describe the use of a single pump capillary-LC system (flow rate: 1-100 μL/min) to perform both capillary-LC-μESI-MS/MS and nano-LC-nano ESI-MS/MS (flow rate: 50-1000 nL/min) analysis and its applications in proteomics. A highly constant nanoflow (180 nL/min) delivery has been achieved by using a capillary binary-pump with electronic flow control and a flow splitter with compensation for the viscosity changes during gradient elution. A column switching technique was successfully used in nano-LC-nanoESI-MS/MS analysis to provide two flow paths, one for sample loading with a flow rate of 15 μL/min directly from the capillary pump, the other for nanoflow gradient elution with a flow rate of 180 nL/min after flow splitting. In comparison with the capillary-LC-μESI-MS/MS at a flow rate of 3 μL/min, this nano-LC-nano ESI-MS/MS analysis improved sensitivity by more than 50 times. It has been successfully used to identify protein spots from 2DGE, as well as protein bands from 1DGE, after in-gel tryptic digestion. Similar to any commercial nano-LC system, this novel nano-LC configuration can be used to perform MudPIT analysis, as well as offline capillary-SCX/nano-RPLC-nanoESI-MS/MS analysis. Our data has shown the unnecessary need for either a second pump or an expensive nano-pump to do nano-LC analysis as most commercial nano-LC system requires, and the increased sensitivity, functionality, and flexibility of a simple capillary-LC system, which is very useful to typical analytical laboratories for multiple-purpose usages of limited resources.