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Submitted on November 10, 2005
Revised on January 12, 2006
Accepted on January 23, 2006

A tandem-affinity tag for two-step purification under fully denaturing conditions: Application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking

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

Deparment of Biological Chemistry, University of California, Irvine, Irvine, CA 92697-1700

Corresponding Author: pkaiser{at}uci.edu

Tandem-affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomics 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 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 8M 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 particular 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 non-specific 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 proteomics analyses to the description of weakly or transiently associated protein complexes.


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