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Molecular & Cellular Proteomics 5:737-748, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Tandem Affinity Tag for Two-step Purification under Fully Denaturing Conditions

Application in Ubiquitin Profiling and Protein Complex Identification Combined with in vivoCross-Linking^*,S

Christian Tagwerker^,,¶, Karin Flick, Meng Cui^||, Cortnie Guerrero^||, Yimeng Dou^**,, Bernhard Auer, Pierre Baldi^,**,, Lan Huang^||, and Peter Kaiser^,¶¶

From the Department of Biological Chemistry, School of Medicine, and ^|| Departments of Physiology & Biophysics and Developmental & Cell Biology, University of California Irvine, California 92697-1700, Institute of Biochemistry, University of Innsbruck, Peter-Mayr-Strasse 1a, A-6020 Innsbruck, Austria, and ^** School of Information and Computer Sciences, University of California, Irvine, California 92697-3435

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 M urea, by Ni²⁺ chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes.

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