Tandem affinity purification combined with mass spectrometry to identify components of protein complexes

Abstract

Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry-based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes.

Fig. 21.1

Tandem affinity purification using the protA/CBP tag: (A) N-TAP for N-terminal tagging and C-TAP for C-terminal tagging of proteins. The IgG-interacting domain of protein A (protA) and the calmodulin binding peptide (CBP) are separated by a TEV protease recognition site. Note the different tag configurations of N-TAP and C-TAP. (B) Protein complexes assembled on the tagged bait protein are first purified on IgG sepharose, eluted by site-specific proteolysis with TEV, and further purified on calmodulin beads. The purified protein complex is eluted with EGTA and processed for mass spectrometric analysis (MS). MS analysis (1D-LC-MS/MS) of individual protein bands can be performed after separation by SDS-PAGE and “in-gel” tryptic digest. Alternatively, the entire protein mixture can be analyzed directly by “in-solution” digest followed by 2D-LC-MS/MS (MudPIT)

Fig. 21.2

HBH-tag based tandem affinity purification combined with in-vivo cross-linking: (A) The HBH-tag consists of a bacterially derived in-vivo biotinylation signaling peptide (Bio), flanked by hexahistidine motifs (6xHis). The biotinylation signal peptide induces attachment of biotin in vivo and allows purification of HBH-tagged proteins on streptavidin resins. (B) Individual components of protein complexes can be covalently linked to each other by in-vivo cross-linking to preserve their composition during the purification procedure. Complexes are sequentially purified on Ni²⁺ sepharose and streptavidin sepharose. Both purification steps are performed under fully denaturing conditions to minimize background binding. Because of the irreversible nature of the streptavidin-biotin interaction, “on-bead” tryptic digest is used to elute peptides of the bound protein complex from the streptavidin sepharose for mass spectrometric identification