Dataset Information

Side-by-side comparison of AP-MS, Tri-functional affinity probe, BioID, PUP-IT and SPIDER to identify CheZ-interacting proteins

ABSTRACT: Comparison of CheZ interactors’ ratio of two replicate experiments using five different methods, i.e., Affinity-Purification, Tri-functional affinity probe Sulfo-SBED and proximity labeling methods BioID, SPIDER, PUP-IT

ORGANISM(S): Escherichia Coli

SUBMITTER: Shengce Tao

PROVIDER: PXD037952 | iProX | Thu Nov 03 00:00:00 GMT 2022

REPOSITORIES: iProX

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Publications

Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Jiang He-Wei HW Chen Hong H Zheng Yun-Xiao YX Wang Xue-Ning XN Meng Qingfeng Q Xie Jin J Zhang Jiong J Zhang ChangSheng C Xu Zhao-Wei ZW Chen Zi-Qing ZQ Wang Lei L Kong Wei-Sha WS Zhou Kuan K Ma Ming-Liang ML Zhang Hai-Nan HN Guo Shu-Juan SJ Xue Jun-Biao JB Hou Jing-Li JL Liu Zhe-Yi ZY Niu Wen-Xue WX Wang Fang-Jun FJ Wang Tao T Li Wei W Wang Rui-Na RN Dang Yong-Jun YJ Czajkowsky Daniel M DM Pei JianFeng J Dong Jia-Jia JJ Tao Sheng-Ce SC

Science China. Life sciences 20230414 8

Protein-biomolecule interactions play pivotal roles in almost all biological processes. For a biomolecule of interest, the identification of the interacting protein(s) is essential. For this need, although many assays are available, highly robust and reliable methods are always desired. By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin (SA)-biotin system, we developed the Specific Pupylation as IDEntity Repor ...[more]

PMID: 37059927

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Project description:It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from non-specific interactors, preserving and isolating all unique interactions including those that are weak, transient or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact sub-micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under non-denaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry (SAC-MS). Here, we demonstrate that SAC-MS allows us to stabilize and elucidate local, distant and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.

Dataset Information

Side-by-side comparison of AP-MS, Tri-functional affinity probe, BioID, PUP-IT and SPIDER to identify CheZ-interacting proteins

Publications

Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Similar Datasets

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