Tandem Affinity Purification and Mass Spectrometry (TAP-MS) for the Analysis of Protein Complexes

Abstract

Affinity purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.

Keywords: TAP; affinity purification; epitope tags; mass spectrometry; protein complex; protein-protein interaction.

Figure 1:

Overview of the Basic Protocols described in this section.

Figure 2:

Workflow diagram of the subcellular fractionation protocol.

Figure 3:

Relative expression of several tandem-tagged human proteins and their endogenous counterparts. Cytoplasmic and nuclear extracts were prepared from HeLa S3 cells expressing N-terminal FLAG:HA-tagged PRMT5, N-terminal FLAG:SII-tagged PRMT5, N-terminal FLAG:HA-tagged WDR77, N-terminal FLAG:SII-tagged hnRNPH1, and C-terminal FLAG:SII-tagged PSF. 12.5 µg of each subcellular fraction was loaded on the gel. Western blot detection was performed using commercial antibodies raised against the respective endogenous proteins.

Figure 4:

Expression of several tandem-tagged human proteins relative to recombinant FLAG-tagged BAP. In addition to extracts described in Figure 1, cytoplasmic and nuclear extracts were prepared from HeLa-S3 cells expressing N-terminal FLAG:SII-tagged RIOK1. A serial dilution of FLAG-BAP was run alongside each fractionated cell extract (17.5 µg). Western blot detection was performed using an HRP-labeled anti-FLAG antibody.

Figure 5:

Quality control (quantitative). Nuclear extracts (4 mg) from HeLa S3 cells expressing ERCC8:FLAG:HA were subjected to FLAG-IP (2.5 hrs.) and HA-IP (O/N). Samples for quality controls were taken at indicated purification steps and analyzed by western blot. Western blot detection was performed using an HRP-labeled anti-FLAG antibody.

Figure 6:

Quality control (qualitative). Cytoplasmic extracts (4 mg) from control (empty pOZ-N vector) HeLa S3 cells and from HeLa S3 cells expressing FLAG:HA:PRMT5 were subjected to FLAG-IP (2.5 hrs.) and HA-IP (O/N). Samples from the FLAG-IP elutions (5%) and from the HA-IP elutions (15%) were analyzed by silver staining.

Figure 7:

Automated TAP on KingFisher. Nuclear extracts (2.4 mg) from control (empty pST-C vector) HeLa S3 cells and from HeLa S3 cells expressing PSF:FLAG:SII were subjected to automated FLAG-IP (3 hrs.) and SII-IP (3 hrs.). Samples from the FLAG-IP elutions (5%) and from the SII-IP elutions (15%) were analyzed by silver staining. The wells containing the control and PSF:FLAG:SII extracts are indicated in the plate schematic above the silver stain image.

Figure 8:

Manual and Automated TAP comparison. Nuclear extracts (6.6 mg) from control (empty pOZ-C vector) HeLa S3 cells and from HeLa S3 cells expressing ERCC8:FLAG:HA were subjected to FLAG-IP (3 hrs.) and HA-IP (O/N). Samples from the manual (MAN.) and automated (KING.) FLAG-IP elutions (5%) and from the HA-IP elutions (15%) were analyzed by silver staining.