Sample preparation is one of the most critical issues in clinical proteomics. While tandem mass spectrometry (MS/MS) is capable of identifying the structures of coeluting peptides in electrospray-mass spectrometric (ESI-MS) analyses, it still is not be effective enough for peptide digests resulting from complex protein mixtures. As a result of the heterogeneity of proteins derived from cell populations, comprehensive techniques have been developed for multidimensional electrophoretic sample pretreatment to achieve adequate separation of complex protein mixtures. Also, rapid and accurate identification of proteins and their posttranslational modification is necessary because these molecular signals might be time sensitive. Although 2D gel electrophoresis has been used as the primary method for protein separation from complex mixtures, its laborious and time-consuming steps involving protein transfer and extraction from the gel can result in sample loss and make it a less favorable technique.

This project’s focus is developing a multi-dimensional protein/peptide microfluidic separation component for the integration of heterogeneous (micro) analytic system, possibly with different buffer requirements. This is one of the critical issues which have been a main technical barrier for developing true micro Total Analysis Systems.

Figure 1 Multi-dimensional biomolecule separation device connected with trap column.

Figure 2 Using two sets of microvalves (red), we can clamp the fluidic channel (yellow) in two different ways. Initially when the left microvalves are closed, we can do isoelectric focusing on the right channel. When we close the right microvalves and open the left microvalves, we can perform capillary gel electrophoresis on the left channel.

Here, we developed a new method to integrate IEF, a charge-based separation, and CE or capillary gel electrophoresis (CGE) with high ionic strength buffers in a microfluidic system. A three-step process was used to achieve 2D electrophoresis on a chip. First, a high-resolution pH gradient from pH 3 to 10 for IEF was established within a short channel. As target proteins moved into the peak transfer region, one set of microfluidic valves was closed for the isolation of selected protein peaks. Second, the peak transfer region was connected to the second-dimension separation channel by opening another set of valves. To prevent intermixing between two separation buffers (ampholyte and CE buffer), we built microfluidic valves in this device (shown in Figure 2) that can isolate a group of isoelectric-focused proteins, preventing peak interdiffusion and band dispersion during the peak transfer process. Finally, the trapped proteins in the peak transfer region were sent into a second-dimension capillary channel for further separation. We already successfully demonstrate 2D on-chip protein separation and rapid sample preparation for MS/MS analysis using this device.

References

1. Wang, Y.-C., Choi, M. H. & Han, J. "Two-dimensional protein separation with advanced sample and buffer isolation using microfluidic valves," Analytical Chemistry 76, 4426-4431 (2004). (doi)
2. Choi, M. H., Wang, Y. -C. & Han, J. "On-chip isoelectric focusing coupled to micro liquid chromatography in blood proteomics," Proceedings of the MicroTAS 2004 Symposium, Malmo, Sweden, vol. 2, pp. 255-257. (poster presentation)
3. Wang, Y.-C., Choi, M. H. & Han, J. "On-chip IEF peak manipulation for 2D protein separation and MS coupling," Proceedings of the MicroTAS 2003 Symposium, Squaw Valley, CA, pp. 955-958. (poster presentation)