Complex Mixture Analysis

The analysis of protein complexes and complicated systems serves as one of the primary reasons for pushing microchip CE-ESI forward as a method for HX MS. In order to demonstrate the improved speed and separation power of CE-ESI, a complex mixture of 270 kDa was separated using both CE-ESI and LC-MS. Figure 3.5 illustrates the electropherogram (top) and chromatogram (bottom) of the sample. As illustrated by the figure, the CE separation is much faster and efficient compared to the LC separation. The CE run time is around 4 min, while the LC run time is near 10 min. For the CE electropherogram, the observed median peak width was 0.79 s (n = 36), resulting in a peak capacity of 195. For the LC chromatogram, the median peak width was 7.6 s (n = 47), resulting in an observed peak capacity of 77. This comparison illustrates the potential for CE-ESI as a platform for

HX MS experiments; the CE separation is over 2 times faster with more than double the peak capacity for a protein complex mixture.

Figure 3.5. Comparison of CE-ESI electropherogram (top) and LC-MS chromatogram (bottom) of 270 kDa complex mixture separation.

3.4 Conclusions

In this report, we have demonstrated that microchip CE-ESI is fast, reproducible, and offers increased resolving power for HX MS experiments. Microchip CE-ESI of a bovine hemoglobin pepsin digest resulted in vastly superior peak capacity (62 in less than 60 s) compared to a state of the art UPLC separation of the same sample (peak capacity of 31 in 7 min). Faster separation means higher deuterium recovery, all with improved separative performance. These innovations are possible due to implementing the CE-ESI monolithically on a microchip. In the simple tests shown here, the CE method observed similar amounts of deuterium uptake when compared against the LC method even though the temperature and time parameters were different. Future reports will describe both the operation of microchip CE-ESI at low temperatures as well as integrating digestion and sample processing directly on a microchip to further minimize experiment processing time and back exchange and increase automation. An additional benefit of integrating these functionalities onto a single microchip could be to reduce the overall sample consumption, which could provide a significant advantage in a sample limited setting. While the sequence coverage for the CE method left room for improvement, the results were attributed to the speed of the CE separation and the decreased sensitivity when compared to the LC method. Integrating pre- concentration and sample processing with microchip CE-ESI will be critical in improving the sensitivity of the method in order to increase the observed sequence coverage. The improved separation performance for complex mixtures was clear in the CE-ESI method; not only was the separation faster, but the peak capacity was higher. Even more complex systems could therefore be within reach, including large protein machines and systems. Overall, we believe that microchip CE-ESI presents a promising platform for HX MS. A fully integrated and

automated CE-ESI microchip could provide a very powerful tool for the HX MS analysis of complex mixtures, greatly expanding the scope of biological systems that can currently be analyzed.

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CHAPTER 4: LOW TEMPERATURE MICROCHIP CAPILLARY-