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A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli

Nature Methods volume 5, pages 69–74 (2008)Cite this article

Abstract

Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.

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Figure 1: An E. coli proteome chip.
Figure 2: Design of the DNA probes.
Figure 3: Identification of YbcN and YbaZ.
Figure 4: Base-flipping assay of YbcN and YbaZ.

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Acknowledgements

We thank H. Mori (Nara Institute of Science and Technology, Japan) for providing the E. coli ORF collection, A. Osterman for help, C.L. Woodard for reviewing this manuscript, and D. McClellan for editorial assistance. This work was supported in part by the National Institutes of Health (grant GM071440 to C.H.; U54 RR020839 to H.Z.), the W. M. Keck Foundation (to C.H.), the Arnold and Mabel Beckman Foundation (to C.H.) and the Research Corporation (to C.H.).

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Author notes

  1. Chien-Sheng Chen and Ekaterina Korobkova: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences & High Throughput Biology Center, Johns Hopkins University School of Medicine, Broadway Research Building 333, 733 Broadway, Baltimore, 21205, Maryland, USA

    Chien-Sheng Chen, Jian Zhu, Sheng-Ce Tao & Heng Zhu

  2. Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, 20224, Taiwan

    Chien-Sheng Chen

  3. Department of Chemistry, The University of Chicago, 5735 South Ellis Avenue, Chicago, 60637, Illinois, USA

    Ekaterina Korobkova, Hao Chen, Xing Jian & Chuan He

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  1. Chien-Sheng Chen
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Contributions

C.-S.C. developed the high-throughput protein purification protocol, printed chips, performed chip assays, analyzed chip assay data and wrote the manuscript. E.K. made the DNA probes, analyzed chip assay data, and performed the base-flipping assays and electrophoretic mobility shift assays with the help of H.C. and X.J. J.Z. measured the Kd and helped purify proteins. S.-C.T. helped purify proteins and print chips. C.H. and H.Z. planned the project and wrote the manuscript.

Corresponding authors

Correspondence to Chuan He or Heng Zhu.

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Supplementary Figures 1–4, Supplementary Tables 1 and 2, Supplementary Methods and Supplementary Protocols (PDF 2413 kb)

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Chen, CS., Korobkova, E., Chen, H. et al. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat Methods 5, 69–74 (2008). https://doi.org/10.1038/nmeth1148

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