Bioinformatics Mass Spectrometry Molecular Biology


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Affinity purification is the capture of biological material through specific enrichment with a ligand coupled to a solid support. Many types of ligands can be used in affinity purification, including DNA and RNA molecules, chemicals, lipids, peptides, or proteins, with some of the most widely used ligands for affinity purification being antibodies.
In AP-MS, a single protein or molecule of interest is affinity captured in a matrix as bait. A protein mixture (often a lysate from the cell or tissue of interest) is passed through the matrix, and interacting partners (prey) are retained by interaction with the bait. Proteins that do not interact pass through the matrix and are discarded. There are multiple variations in the affinity purification step, including immunoprecipitation and pull-down of epitope tagged molecules. Once purified, proteins can be processed for direct analysis by MS or, fractionated to reduce sample complexity.

The mapping of protein–protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein’s interactome. Affinity purification combined with mass spectrometry (AP-MS) has emerged as a particularly attractive method for PPI mapping. A major advantage is that this method allows unbiased detection of PPIs under physiological conditions. Importantly, AP-MS can assess PPIs in relevant biological contexts such as mammalian cell lines or even tissues. Moreover, AP-MS experiments have the advantage that they can provide quantitative information (q-AP-MS). This greatly increases the confidence in interaction partners that are identified and can also be used to study the impact of perturbations on PPIs.

Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. 

Bait selection

The first step to design an AP-MS study is to select the proteins of interest, or baits, that will be used to characterize a network of protein-protein interactions. As the proteins in this set of baits are scored against each other, they should be selected to maximize the likelihood of identifying interactions that are unique to the bait compared with other proteins in the set. 


Within each bait set, include a positive control and a negative control. Positive-control bait may be a protein that previously underwent AP-MS analysis and that has a high-confidence set of interacting proteins identified by reciprocal binding assays. 

Cell lines

Ideally, bait proteins would be expressed in the background of the interactome targeted for interrogation. However, an important consideration is that the input material needed for AP and MS analysis is reasonably large (<25 million cells yield microgram-scale quantities of expressed bait protein). Thus, the experimental design must balance optimizing bait expression with maintaining relevance to the biology or tropism of the proteins of interest. 

Affinity tags

A number of synthetic or naturally occurring affinity tags have been implemented in AP-MS studies. Common epitope tags include FLAG, Strep, Myc, hemagglutinin, protein A, His6-tag, calmodulin-binding protein, GFP and maltose-binding protein. In some cases, multiple tags are fused together, such as 2×Strap 3×FLAG, which is widely used by the authors of this protocol and which contains five tandem affinity tags of two types. 

Affinity purification

Early AP-MS approaches used tandem affinity tags to perform sequential APs that yielded highly pure protein complexes that could be characterized by MS. At that time, analyzing a complex protein mixture with MS platforms was limited, which caused nonspecifically interacting proteins in the purification to obscure the identification of true members of a protein complex. 

MS analysis

Sample preparation methods are fairly standardized and generally include denaturation of proteins with chaotropes such as urea or guanidine hydrochloride, reduction of disulfide bonds with a reducing agent, alkylation of free cysteine residues and proteolytic digestion with trypsin or an alternate protease such as LysC. After digestion, samples are typically desalted with C18 cartridges or tips to remove salts and other digestion impurities.

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