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High-Throughput Multidimensional Bioseparations for Next-Generation Proteomics


EMSL Project ID
49682

Abstract

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biological research including the study of microbiomes. 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 biological research community.

Project Details

Start Date
2016-12-06
End Date
2019-09-30
Status
Closed

Team

Principal Investigator

Ryan Kelly
Institution
Brigham Young University

Co-Investigator(s)

Daniel Orton
Institution
Pacific Northwest National Laboratory

Team Members

Maowei Dou
Institution
Environmental Molecular Sciences Laboratory

Kerui Xu
Institution
Environmental Molecular Sciences Laboratory

Ying Zhu
Institution
Environmental Molecular Sciences Laboratory

Roza Wojcik
Institution
Pacific Northwest National Laboratory

Rui Zhao
Institution
Environmental Molecular Sciences Laboratory

Related Publications

Dou M., C.D. Chouinard, Y. Zhu, G. Nagy, A.V. Liyu, Y.M. Ibrahim, and R.D. Smith, et al. 2019. "Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses." Analytical and Bioanalytical Chemistry 411, no. 21:5363–5372. PNNL-SA-146403. doi:10.1007/s00216-018-1452-5
Dou M., C. Tsai, P.D. Piehowski, Y. Wang, T.L. Fillmore, R. Zhao, and R.J. Moore, et al. 2019. "Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples." Analytical Chemistry 91, no. 15:9707-9715. PNNL-SA-141437. doi:10.1021/acs.analchem.9b01248
Liang Y., Y. Zhu, K.K. Hixson, M. Dou, K. Xu, R.K. Chu, and W.B. Chrisler, et al. 2018. "Spatially resolved proteomic profiling of <200 cells from tomato fruit pericarp by integrating laser-capture microdissection with nanodroplet sample preparation." Analytical Chemistry 90, no. 18:11106–11114. PNNL-SA-134717. doi:10.1021/acs.analchem.8b03005
Proteome Profiling of 1 to 5 Spiked Circulating Tumor Cells Isolated from Whole Blood Using Immunodensity Enrichment, Laser Capture Microdissection, Nanodroplet Sample Processing, and Ultrasensitive nanoLC–MS
Xu K., Y. Liang, P.D. Piehowski, M. Dou, R. Zhao, R.L. Sontag, and R.J. Moore, et al. 2019. "Benchtop-compatible sample processing workflow for proteome profiling of <100 mammalian cells." Analytical and Bioanalytical Chemistry 411, no. 19:4587-4596. PNNL-SA-146641. doi:10.1007/s00216-018-1493-9
Zhu Y., J. Podolak, R. Zhao, A.K. Shukla, R.J. Moore, G.V. Thomas, and R.T. Kelly. 2018. "Proteome Profiling of 1 to 5 Spiked Circulating Tumor Cells Isolated from Whole Blood Using Immunodensity Enrichment, Laser Capture Microdissection, Nanodroplet Sample Processing, and Ultrasensitive nanoLC-MS." Analytical Chemistry 90, no. 20:11756-11759. PNNL-SA-140551. doi:10.1021/acs.analchem.8b03268
Zhu Y., M. Dou, P.D. Piehowski, Y. Liang, F. Wang, R.K. Chu, and W.B. Chrisler, et al. 2018. "Spatially-resolved proteome mapping using automated, sacrificial liquid-mediated sample transfer from laser capture microdissection to nanodroplet sample preparation." Molecular & Cellular Proteomics. MCP 17, no. 9. PNNL-SA-132369. doi:10.1074/mcp.TIR118.000686
Zhu Y, PD Piehowski, R Zhao, J Chen, Y Shen, RJ Moore, AK Shukla, VA Petyuk, M Campbell-Thompson, CE Mathews, RD Smith, W Qian, and RT Kelly. 2018. "Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells." Nature Communications 9(1):882. doi:10.1038/s41467-018-03367-w