title: Single-step protease cleavage elution for identification of protein-protein interactions from GST pull-down and mass spectrometry.
abstract: The study of protein-protein interactions is a major theme in biological disciplines. Pull-down or affinity-precipitation assays using 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 MS is established. We present a proof of concept for this method, as evidenced by a GST pull-down/MS case study of the small guanosine triphosphatase (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.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Lin
authors.initials: L
authors.lastname: Luo
authors.forename: Nathan P
authors.initials: NP
authors.lastname: King
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Yeo
authors.forename: Alun
authors.initials: A
authors.lastname: Jones
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Stow
mesh_terms: D000818:Animals; D001709:Biotechnology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D020691:rab GTP-Binding Proteins
chemical_list: D011993:Recombinant Fusion Proteins; C544741:Rab22B protein, mouse; D020691:rab GTP-Binding Proteins
keywords: GST pull-down; LC-MS/MS; PreScission protease; Protease cleavage elution; Technology