Protein-Protein Interactions
Cellular proteins are directly responsible for adaptation to disease-mediated changes. Because of the connectivity between proteins, the impact of a disease-related mutation is not restricted to a specific gene product. Instead, it affects the entire
network and can accordingly impact the activity of a whole subset of proteins. Instead of focusing on individual genes or loci implicated in human disease, PPI-based analyses study the parts of pathway connections that are most changed by the disease
state, thus offering an alternative to identify a mutation’s impact on cellular function. The MSF has experience with multiple methods for studying PPIs. In Figure 1 we show a representative workflow for three of the most common workflows.
Affinity purification coupled with MS (AP–MS), proximity-dependent biotinylation identification (BioID), and crosslinking MS (XL-MS) methods have made substantial contributions to interaction proteomics studies. Whereas AP−MS results in the identification
of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, and XLMS identifies direct binders of proteins, resulting in overlapping yet distinct protein identifications. For AP-MS, it is
preferred to have added an epitope tag to the protein of interest. The resulting bait protein functions as an affinity capture probe for interacting proteins and allows for easy purification of the protein and interactors. Interacting proteins can
be visualized using a network-based approach, with nodes representing the “bait” proteins of interest of a PPI study. Here we show the PPI network from a study on histone interacting proteins. Proximity labeling is performed by expressing a bait protein
of interest fused to a promiscuous labeling enzyme in cells (Figure 1B). The addition of a small molecule substrate, such as biotin, allows the covalent tagging of endogenous proteins within a 10–20 nm range, capturing the protein’s
surrounding environment, including potential interactors. After cell lysis, proteins are denatured and solubilized, followed by selective enrichment of biotinylated proteins through streptavidin binding. Due to the strong binding affinity between
biotin and streptavidin, proximity labeling permits more. efficient protein extraction, lysis methods and harsher washing conditions than AP-MS, allowing the identification of weak or transient interactions that might be missed in AP-MS or similar
experiments.
Crosslinking can be performed on a variety of sample types, from purified proteins to cell or tissues, and requires no additional molecular biology, unlike AP-MS or BioID. Crosslinking helps to provide not only the identity of the proteins involved in
interactions, but also information as to the regions involved, and can be combined with informatic tools to provide structural information. During crosslinking, covalent bonds are generated between proximal residues using a crosslinker. These covalent
linkages occur in a residue specific manner, allowing for the use of crosslinkers of various reactivity to be employed. Much like general proteomics, each peptide identified in a crosslinked pair is then used to identify the protein from which they
came, thus elucidating the PPI. If performed on cells or tissues, global interaction PPI networks can be determined (Figure 1C bottom panel). However, if investigators are interested in a particular protein, there are no guarantees
it will show up in the data set, as crosslinks are generally low in abundance, requiring enrichment, and reaction conditions would need to be optimized.. All PPI analyses can be displayed in interaction plots, where nodes are connected by edges to
the interacting proteins identified by AP-MS, BioID proximity labeling, XL-MS, or similar experiments (Tan et al, Sci Adv, 2021; Huang et al, Mol Cell, 2020, Richards et al, MSB, 2021). As with any experiment, the selection of appropriate controls
is essential to these experiments and we recommend discussing design of the experiments in advance.