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High-throughput: Affinity purification mass spectrometry

Another high-throughput method that is significantly contributing to the growth in published protein-protein interaction data is affinity purification mass spectometry (AP-MS, Figure 4).

AP-MS is an affinity-based assay. Its specificity and sensitivity depend greatly on the strength and stability of the interaction between the proteins involved and, crucially, on the absolute and relative abundance of the prey proteins.8

In AP-MS, a single protein or molecule of interest is immobilised in a matrix as a bait. Then a protein mixture is passed through the matrix and interacting partners (prey) are captured by the bait protein. Any form of technique relying on mass spectrometry (MALDI, LC-MS/MS, etc.) is then used to identify the captured proteins.

Information The affinity purification step and type of mass-spectrometry-based identification varies. For example, you can perform an immunoprecipitation and then identify the captured proteins using LC-MS/MS or you can perform a pull-down of epitope-tagged molecules and then identify the proteins using MALDI-MS.9,10
Figure 4 Affinity purification and mass spectometry (AP-MS). The bait protein (yellow) is immobilised on a matrix [1]. A protein mixture is passed through and only the interacting partners (prey) are retained [2]. In the following step the prey proteins are removed, digested with a protease and the resulting peptides are analysed by MS [3].3

Advantages:  

  • The technique detects complexes of several proteins interacting together
  • Potentially, depending on the sensitivity of your MS-approach and the affinity of the interacting partners, this method has the ability to examine interactions among multiple proteins at subpicomole concentrations
  • The prey proteins are present in their native state (so long as they are not affected by the sample lysis process) and concentration

Disadvantages:

  • The technique detects complexes of several proteins interacting together, but not binary relationships
  • Prey proteins without a peptide signature recognisable by MS (owing to obscure post-translational modifications) or present in very low amounts will not be identified
  • Relevant transient and/or weak interactions may be missed
  • Mixing of compartments during cell lysis/purification is a potential source of false positives. For example, interactions between proteins that would not normally be in the same cellular compartment may confound your results

There is an overwhelming variety of techniques that can be used to detect  protein-protein interactions using low- or medium-throughput setups. Next we summarise some methods that are often used to improve the confidence of an interaction detected by high-throughput methods or on their own merits in small-scale experiments.