High-throughput multidimensional bioseparations for next-generation proteomics

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PROJECT SUMMARY Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The quality of the analysis is determined not only by the MS resolution and sensitivity but by the liquid-phase separation used to deliver sample to the instrument, with separations having larger peak capacities leading to more identified species and improved confidence in those identifications. Increasing the peak capacity of a separation currently requires a dramatic increase in the total analysis time, imposing a tradeoff between proteome coverage and measurement throughput. The combination of long analysis times and expensive instrumentation also results in a high cost per analysis, impeding studies with large numbers of samples and imposing a barrier to routine implementation in clinical diagnostics. Here, we propose to develop a separation method based on liquid chromatography (LC) followed by fast capillary electrophoresis (CE) to achieve ultrahigh peak capacity separations with short overall analysis times, thus dramatically decreasing the cost per sample. Rather than waste the majority of sample during transfer from LC to CE as past approaches have done, we will preconcentrate and focus the sample eluting from the LC column into a narrow band using a microfluidic valve-based electrokinetic preconcentrator recently developed in our laboratory. The focused band will then be injected into the CE separation column for rapid separation prior to MS analysis. We will analyze approximately 60 LC fractions by CE in 1 hour, with an overall peak capacity approaching 2,000. The resulting platform should provide an order of magnitude improvement in peak capacity per unit time over existing approaches and will enable an unprecedented combination of sample measurement throughput, sensitivity and cost per analysis. It will also broadly impact other biological analyses that will benefit from dramatically improved throughput and peak capacities, including selected reaction monitoring MS for targeted proteomics, as well as metabolomics and glycomics. The compatibility of the system with commercially available LC and MS instrumentation will promote broad applicability and implementation in the biomedical research community.