*2.2.1 Concept description*

Affinity purification involves isolating a target-of-interest in non-denaturing conditions to enable co-isolation of any binding partners that are stably attached. The most common implementation is immunoprecipitation, where an antibody, generally attached to a solid substrate like a bead, plate or column, is used to specifically recognize the target-of-interest. Any factors that are not stably bound to the protein of interest are washed away by flushing the solid substrate with buffer. Proteins that survive the washes are identified (**Figure 3**).

### *2.2.2 Concept pros*

Affinity purification allows for the direct isolation of a target-of-interest from complex mixtures. It is versatile and can be combined with many other approaches.

### *2.2.3 Concept cons*

To isolate the target-of-interest, there needs to be a reagent, like an antibody, that will specifically and tightly bind the target. Since such reagents are not always readily available, the target may need to be tagged and introduced exogenously, which can affect target behavior. Affinity purification for unbiased identification of binding partners requires either large tagged libraries or access to MS. Importantly, to be identified, binding partners need to survive cell lysis and washes. This has limited the applicability of this otherwise widely-utilized approach in the study of ePPIs. Cell lysis and target extractions typically require membrane solubilization, which can disrupt membrane protein-dependent interactions. In addition, affinity purification workflows often miss detection of low affinity interactions, which are typical of ePPIs.

## *2.2.4 Specific applications*

The most widely used version of this concept is affinity purification followed by binding partner identification using mass spectrometry (AP/MS) (**Table 2**). MS allows for the unbiased identification of interactors in their endogenous form in virtually any cell type or tissue. Traditionally, AP/MS studies were mostly restricted to one or a few targets-of-interest. However, recent technological advances, dominated

#### **Figure 3.**

*Affinity purification isolates a protein and any stably interacting protein from a complex solution such as a cell or tissue lysate. Shown here is the most common implementation where an antibody is used to isolate the target protein, followed by unbiased identification of binding proteins using MS. the antibody is attached to a solid substrate to allow for physical manipulations of the target protein.*


#### **Table 2.**

*Affinity purification techniques covered in this section. These approaches differ primarily in their readout method.*

by the BioPlex project, have driven the development of a systematic pipeline that enables high-throughput AP/MS. Such efforts have already resulted in an interaction network with nearly 120,000 interactions identified for over 14,000 proteins in HEK293 cells [18].

A second and more recently developed method is the luminescence-based mammalian interactome mapping (LUMIER). In this approach, a library of epitope-tagged (specifically FLAG-tagged) constructs are co-transfected with a targetof-interest fused to Renilla luciferase. An anti-FLAG antibody is then used to pull down the tagged protein and binding is assayed by reading out luciferase activity of the immunoprecipitate [19]. Though in principle this approach offers increased sensitivity, this technique requires that both the target-of-interest and the library are tagged and expressed using artificial constructs. Thus, the applicability of this method for unbiased screening greatly depends on the accessibility to large libraries of tagged constructs.

While these approaches excel at identifying soluble interactions, ePPIs struggle to survive the processing steps and are noticeably underrepresented in both the LUMIER and even the much more comprehensive BioPlex dataset. One way to address some of the challenges associated to ePPIs is to combine affinity purification with cross-linking, turning transient ePPIs into permanent covalent linkages. By using cross-linking in combination with mass spectrometry (XL-MS), ePPIs can be identified in an unbiased manner. While cross-linking stabilizes weak interactions, XL-MS still has associated challenges like the presence of unproductive crosslinks or combinatorial database search space. To overcome these, newly developed cross-linkers used for XL-MS can include affinity tags or MS cleavable moieties [20]. However, these increase cross-linker size and the chances of cross-linking nearby, non-interacting proteins. Existing cross-linking reagents also primarily target reactive amines, limiting the number of protein interactions that can be captured. Cross-linking protein complexes also tends make them less soluble [21]. This is worsened by the fact that ePPIs often involve membrane proteins which already present solubility challenges that complicates down-stream processing. Since extracellular proteins are often heterogeneously post-translationally modified [22], they can be challenging to identify in mass databases for MS experiment. Overall, XL-MS represents one of the few techniques that does not have a bias against ePPIs over soluble PPIs.

While most cross-linking approaches select for general features of proteins like reactive amine groups, alternative strategies have been developed that specifically target cell-surface receptors using trifunctional cross-linkers. These methods typically have one moiety that covalently attaches the cross-linker to the target protein-of-interest, a soluble protein that ranges from peptides, to antibodies or even complex entities such as viral particles. A second moiety links to glycosylated receptor proteins bound to or near the target, and a third moiety enables purification. Three molecules and associated workflows have been described: TRICEPS [23], followed by ASB [24] and HATRIC [25]. These approaches have the potential to enable unbiased study of targets of diverse nature in physiologically relevant settings such as cells expressing endogenous receptors, thus offering an attractive option for ePPI discovery.
