Multiparameter Screen Optimizes Immunoprecipitation

Abstract

Immunoprecipitation (IP) coupled with mass spectrometry effectively maps protein–protein interactions when genome-wide, affinity-tagged cell collections are used. Such studies have recorded significant portions of the compositions of physiological protein complexes, providing draft ’interactomes’; yet many constituents of protein complexes still remain uncharted. This gap exists partly because high-throughput approaches cannot optimize each IP. A key challenge for IP optimization is stabilizing in vivo interactions during the transfer from cells to test tubes; failure to do so leads to the loss of genuine interactions during the IP and subsequent failure to detect. Our high-content screening method explores the relationship between in vitro chemical conditions and IP outcomes, enabling rapid empirical optimization of conditions for capturing target macromolecular assemblies.

Method summary

There is presently no way to know, a priori, the optimal experimental conditions to use to immunoprecipitate a target protein complex: different interactors and subpopulations respond diversely to experimental conditions, and these behaviors must be discovered empirically. We present a high-content screen to rapidly optimize immunoprecipitations.

Keywords: :

• affinity proteomics
• complexomics
• interactomics
• macromolecular assemblies

Supplementary data

Author contributions

Conceptualization: J LaCava. Methodology: S Xie, L Saba, H Jiang, M Oghbaie, J LaCava. Software: M Oghbaie, O Bringas. Investigation: S Xie, L Saba, H Jiang, L DiStefano. Resources: V Sherman. Writing (original draft): S Xie, L Saba, H Jiang, J LaCava. Writing (review and editing): S Xie, J LaCava. Supervision: J LaCava. Project administration: J LaCava. Funding acquisition: J LaCava.

Acknowledgments

The authors thank Qsonica (Newton, CT, USA) for their contribution to the development of sonication tools, with special acknowledgment to A Coppola for helpful discussions and for coordinating our sonication R&D efforts; L Martínez and I Piana of Sepmag (Barcelona, Spain) for helpful discussions regarding magnetic separations and for use of the Sepmag LAB system; and the National Center for Dynamic Interactome Research (www.ncdir.org) for financial and infrastructural support and productive scientific exchanges.

Financial disclosure

This work was supported in-part by National Institutes of Health grants R01GM126170, R01AG078925 and P41GM109824. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.