Single-step protease cleavage elution for identification of protein-protein interactions from GST pull-down and mass spectrometry

Luo, Lin, King, Nathan P., Yeo, Jeremy C., Jones, Alun and Stow, Jennifer L. (2013) Single-step protease cleavage elution for identification of protein-protein interactions from GST pull-down and mass spectrometry. Proteomics, Accepted Article 1-15. doi:10.1002/pmic.201300315

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Author Luo, Lin
King, Nathan P.
Yeo, Jeremy C.
Jones, Alun
Stow, Jennifer L.
Title Single-step protease cleavage elution for identification of protein-protein interactions from GST pull-down and mass spectrometry
Journal name Proteomics   Check publisher's open access policy
ISSN 1615-9853
1615-9861
Publication date 2013-11-20
Year available 2013
Sub-type Article (original research)
DOI 10.1002/pmic.201300315
Volume Accepted Article
Start page 1
End page 15
Total pages 15
Place of publication Weinheim, Germany
Publisher Wiley - V C H Verlag
Collection year 2014
Language eng
Formatted abstract
The study of protein-protein interactions is a major theme in biological disciplines. Pull-down or affinity-precipitation assays using glutathione S-transferase (GST) fusion proteins have become one of the most common and valuable approaches to identify novel binding partners for proteins of interest (bait). Non-specific binding of prey proteins to the beads or to GST itself however, inevitably complicates and impedes subsequent analysis of pull-down results. A variety of measures, each with inherent advantages and limitations, can minimise the extent of the background. This technical brief details and tests a modification of established GST pull-down protocols. By specifically eluting only the bait (minus the GST tag) and the associated non-specific binding proteins with a simple, single-step protease cleavage, a cleaner platform for downstream protein identification with mass spectrometry is established. We present a proof of concept for this method, as evidenced by a GST pull-down/mass spectrometry case study of the small GTPase Rab31 in which: (i) sensitivity was enhanced, (ii) a reduced level of background was observed, (iii) distinguishability of non-specific contaminant proteins from genuine binders was improved and (iv) a putative new protein-protein interaction was discovered. Our protease cleavage step is readily applicable to all further affinity tag pull-downs.
Keyword GST pull-down
LC-MS/MS
PreScission protease
Protease cleavage elution
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Accepted manuscript online: 20 NOV 2013

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2014 Collection
Institute for Molecular Bioscience - Publications
 
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Citation counts: TR Web of Science Citation Count  Cited 5 times in Thomson Reuters Web of Science Article | Citations
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Created: Tue, 10 Dec 2013, 14:32:36 EST by Susan Allen on behalf of Institute for Molecular Bioscience