Abstract
Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.
Highlights
Unraveling the complexity and dynamic behavior of proteinprotein (PPIs)1 and protein-nucleic acid interactions (PNIs) that serve as central hubs for coordinated cellular events is one of the major objectives in cell biology and especially in proteome research [1]
In contrast to Tandem Affinity Purification (TAP) [35], the two-step coimmunoprecipitation (TIP) and bridged TIP (bTIP) approaches do not require the transgenic expression of the bait protein and can be performed in virtually all cell systems, including primary human cells
The opportunity to work under native expression conditions reduces the risk for nonspecific interactions resulting from alterations in protein homeostasis [36] or unintended activation of pathways such as apoptosis induction triggered by forced expression of CD95/FAS or caspase-8 [37,38,39]
Summary
Co-IP, coimmunoprecipitation; ChIP, chromatin immunoprecipitation; TAP, tandem affinity purification; PPI, protein-protein interaction; PNI, protein-nucleic acid interaction; TIP, two-step coimmunoprecipitation; bTIP, bridged two-step coimmunoprecipitation; bTChIP, bridged two-step chromatin immunoprecipitation; LC/MS, liquid chromatography and mass spectrometry; LFQ, label free quantification; FC, fold change; Ig(s), immunoglobulin(-s); ab(s), antibody(-ies); mAb(s), monoclonal antibody(-ies); pAb(s), polyclonal antibody(-ies); qPCR, quantitative polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; CD95(L), cluster of differentiation 95 (ligand); IKK, Inhibitor of kappa B Kinase; DISC, death inducing signaling complex; APO-1, apoptosis antigen 1; FADD, FAS-associated protein with death domain. Tandem Affinity Purification (TAP) results in cleaner samples that can be used for identification by mass spectrometry, or for other downstream applications [7,8,9,10,11,12,13,14,15]. The epitope tags need to be artificially added to the bait of interest, which in some cases may alter its functions and interactions. This prevents the application of TAP to biological material (e.g. patient samples) that cannot be genetically manipulated. In contrast to conventional single-step co-IP, TIP resulted in highly specific complex purifications as well as identification and quantification of all yet known DISC core components by MS. Genetic ablation of PPM1G significantly sensitized cells to CD95/FAS-induced apoptosis, indicating that it represents a novel CD95/FAS-associated negative regulator for the induction of apoptosis by this prototypic cell death receptor
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