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Direct analysis of protein complexes using mass spectrometry

Nature Biotechnology volume 17, pages 676–682 (1999)Cite this article

Abstract

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

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Figure 1: Direct analysis of large protein complexes (DALPC).
Figure 2: Identifying proteins in the yeast ribosome complex using DALPC.
Figure 3: Identifying ribosomal proteins in a total yeast extract using DALPC.
Figure 4: Integrated DALPC.
Figure 5: Localizing YMR116p to the yeast 40S ribosomal subunit.
Figure 6: Localizing RACK1 to the human 40S ribosomal subunit.

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Acknowledgements

We thank D. Tabb, L. Hayes, G. Kleeman, E. Malone, and M. Olson for critical reading of the manuscript and T. Gatlin for assistance with the figures. Partially supported by NIH grant CA39053 (G.T.M and D.R.M). Supported by National Center for Research Resources Yeast Center grant RR11823, NIH grant GM52095 and NSF's Science and Technology Center grant BIR 8809710 to J.R.Y., and NIH postdoctoral fellowship grant T32 HG00035-03 to A.J.L.

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Author notes

  1. Andrew J. Link

    Present address: Millennium Predictive Medicine, Inc., One Kendall Square, Building 700, Cambridge, MA, 02139

Authors and Affiliations

  1. Department of Molecular Biotechnology, University of Washington, Seattle, 98195, WA

    Andrew J. Link, Jimmy Eng, David M. Schieltz, Edwin Carmack & John R. Yates III

  2. Department of Biochemistry, University of Washington, Seattle, 98195, WA

    Gregory J. Mize & David R. Morris

  3. Fred Hutchinson Cancer Research Institute, Seattle, 98109, WA

    Barbara M. Garvik

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Correspondence to John R. Yates III.

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Link, A., Eng, J., Schieltz, D. et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol 17, 676–682 (1999). https://doi.org/10.1038/10890

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