Affinity enrichment and identification of inositol poly- and pyrophosphate interactomes

David Furkert; Michal Nadler-Holly; Dorothea Fiedler

doi:10.1016/j.xpro.2020.100277

Affinity enrichment and identification of inositol poly- and pyrophosphate interactomes

STAR Protoc. 2021 Jan 14;2(1):100277. doi: 10.1016/j.xpro.2020.100277. eCollection 2021 Mar 19.

Authors

David Furkert^{1

2}, Michal Nadler-Holly¹, Dorothea Fiedler^{1

2}

Affiliations

¹ Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Straße 10, 13125 Berlin, Germany.
² Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, 12489 Berlin, Germany.

Abstract

This protocol describes an affinity enrichment approach from mammalian cell extracts to identify protein binding partners of inositol hexakisphosphate (InsP₆) and 5-diphosphoinositol pentakisphosphate (5PP-InsP₅), two important eukaryotic metabolites. The interactomes are annotated using mass spectrometry-based proteomics, and comparison against a control resin can uncover hundreds of protein targets. Quantitative analysis of InsP₆- versus 5PP-InsP₅-binding proteins highlights specific protein-ligand interactions. The approach is applicable to different cells and organisms and will contribute to a mechanistic understanding of inositol poly- and pyrophosphate signaling. For complete details on the use and execution of this protocol, please refer to Furkert et al. (2020).

Keywords: Mass spectrometry; Molecular/chemical probes; Proteomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

HCT116 Cells
HEK293 Cells
Humans
Inositol Phosphates / metabolism*
Mass Spectrometry*
Phytic Acid / metabolism*
Second Messenger Systems*

Substances

Inositol Phosphates
1-diphosphoinositol pentakisphosphate
Phytic Acid