A novel tandem affinity tag for two-step purification under fully denaturing conditions : applications in ubiquitin profiling / by Christian Tagwerker
ger: 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 stringe...
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Place / Publishing House: | 2006 |
Year of Publication: | 2006 |
Language: | English |
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Classification: | 42.03 - Methoden und Techniken der Biologie 42.13 - Molekularbiologie 42.15 - Zellbiologie |
Physical Description: | 144, XXIII Bl.; Ill., graph. Darst. |
Notes: | Auch erschienen in: Journal of Molecular and Cellular Proteomics, 2006, 5.4, 737-742 |
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Tagwerker, Christian aut <<A>> novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling by Christian Tagwerker 2006 144, XXIII Bl. Ill., graph. Darst. Auch erschienen in: Journal of Molecular and Cellular Proteomics, 2006, 5.4, 737-742 Innsbruck, Univ., Diss., 2006 ger: 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.<br />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.<br />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 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.<br />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. Ubiquitin s (DE-588)4314848-7 Proteomanalyse s (DE-588)4596545-6 Massenspektrometrie s (DE-588)4037882-2 AT-OBV UBISA. YWOAW MAG1-3 35298-C.Stip. 2221668790004498 |
language |
English |
format |
Thesis Book |
author |
Tagwerker, Christian |
spellingShingle |
Tagwerker, Christian A novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling Ubiquitin (DE-588)4314848-7 Proteomanalyse (DE-588)4596545-6 Massenspektrometrie (DE-588)4037882-2 |
author_facet |
Tagwerker, Christian |
author_variant |
c t ct |
author_role |
VerfasserIn |
author_sort |
Tagwerker, Christian |
title |
A novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling |
title_sub |
applications in ubiquitin profiling |
title_full |
A novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling by Christian Tagwerker |
title_fullStr |
A novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling by Christian Tagwerker |
title_full_unstemmed |
A novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling by Christian Tagwerker |
title_auth |
A novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling |
title_new |
A novel tandem affinity tag for two-step purification under fully denaturing conditions |
title_sort |
novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling |
publishDate |
2006 |
physical |
144, XXIII Bl. Ill., graph. Darst. |
callnumber-raw |
35298-C.Stip. |
callnumber-search |
35298-C.Stip. |
topic |
Ubiquitin (DE-588)4314848-7 Proteomanalyse (DE-588)4596545-6 Massenspektrometrie (DE-588)4037882-2 |
topic_facet |
Ubiquitin Proteomanalyse Massenspektrometrie |
illustrated |
Illustrated |
work_keys_str_mv |
AT tagwerkerchristian anoveltandemaffinitytagfortwosteppurificationunderfullydenaturingconditionsapplicationsinubiquitinprofiling |
status_str |
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<<A>> novel tandem affinity tag for two-step purification under fully denaturing conditions applications in ubiquitin profiling |
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42.03 - Methoden und Techniken der Biologie 42.13 - Molekularbiologie 42.15 - Zellbiologie |
basiskl_txtF_mv |
42.03 - Methoden und Techniken der Biologie 42.13 - Molekularbiologie 42.15 - Zellbiologie |
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