Capillary‐LC‐µESI‐MS/MS and Nano‐LC‐Nano ESI‐MS/MS Analysis Using a Single Binary Pump Capillary LC System: Applications in Proteomics

Abstract

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.

Keywords:

• Capillary‐LC‐µESI‐MS/MS
• Nano‐LC‐nano ESI‐MS/MS
• Column switching
• Two‐dimensional liquid chromatography
• Proteomics
• Human plasma

Acknowledgments

This work was supported in part by a Cancer Center Support Grant (CA016056‐27) awarded to RPCI from the National Cancer Institute. It was also supported by grants from the National Cancer Institute (CA109480), Human Proteome Organization, and Korean Ministry of Science and Welfare.