Abstract
Efforts to develop a liquid chromatography (LC)/mass spectrometry (MS) technology for ultra-sensitive proteomics studies (i.e., nanoscale proteomics) are described. The approach combines high-efficiency nanoscale LC (separation peak capacity of ≈103; 15-μm-i.d. packed capillaries with flow rates of 20 nL min−1, the optimal separation linear velocity) with advanced MS, including high-sensitivity and high-resolution Fourier transform ion cyclotron resonance MS, to perform both single-stage MS and tandem MS (MS/MS) proteomic analyses. The technology enables broad protein identification from nanogram-size proteomics samples and allows the characterization of more abundant proteins from sub-picogram-size samples. Protein identification in such studies using MS is demonstrated from <75 zeptomole of a protein. The average proteome measurement throughput is ~50 proteins h−1 using MS/MS during separations, presently requiring approximately 3 h sample−1. Greater throughput (~300 proteins h−1) and improved detection limits providing more comprehensive proteome coverage can be obtained by using the “accurate mass and time” tag approach developed in our laboratory. This approach provides a dynamic range of at least 106 for protein relative abundances and an improved basis for quantitation. These capabilities lay the foundation for studies from single or limited numbers of cells.
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Acknowledgments
We thank the US Department of Energy’s Office of Biological and Environmental Research and the National Cancer Institute (Grant CA 86340) for their support of portions of this research. Pacific Northwest National Laboratory is operated by the Battelle Memorial Institute for the US Department of Energy through Contract DE-ACO6–76RLO 1830.
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Shen, Y., Tolić, N., Masselon, C. et al. Nanoscale proteomics. Anal Bioanal Chem 378, 1037–1045 (2004). https://doi.org/10.1007/s00216-003-2329-8
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DOI: https://doi.org/10.1007/s00216-003-2329-8