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.

Keywords: affinity proteomics; complexomics; interactomics; macromolecular assemblies.

Figure 1.. Summary of the screening approach.

Cryomilled cell powders are distributed using a dispensing manifold and lysed with different extraction solutions (step 1); then samples are sonicated to disperse and homogenize the extracts, followed by centrifugal clarification (step 2); the clarified extracts are subjected to IP (step 3); finally, protein eluates are analyzed using MS (steps 4 and 5). Reproduced from [27].

Figure 2.. Example immunoprecipitation–mass spectrometry prescreen.

SDS-PAGE/Sypro Ruby stain of NCBP1-LAP co-immunoprecipitations with 24 different extraction solutions (left) and hierarchical clustering of the cognate MS data using log₂ LFQ intensity (right). Not detected proteins are indicated in gray. Six conditions were selected for subsequent quantitative screening: red numbers above lanes and table below the gel. LFQ: Label-free quantification; ND: Not detected. Reproduced from [27].