Mapping multiprotein complexes by affinity purification and mass spectrometry

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The combination of affinity purification and tandem mass spectrometry (MS) has emerged as a powerful approach to delineate biological processes. In particular, the use of epitope tags has allowed this approach to become scaleable and has bypassed difficulties associated with generation of antibodies. Single epitope tags and tandem affinity purification (TAP) tags have been used to systematically map protein complexes generating protein interaction data at a near proteome-wide scale. Recent developments in the design of tags, optimisation of purification conditions, experimental design and data analysis have greatly improved the sensitivity and specificity of this approach. Concomitant developments in MS, including high accuracy and high-throughput instrumentation together with quantitative MS methods, have facilitated large-scale and comprehensive analysis of multiprotein complexes.

Introduction

The development of protein-tagging methods and protein identification by tandem mass spectrometry (MS) has revolutionised the way we look at cell biology. The combined use of protein tagging and MS has been used to systematically map protein interactions in many species including yeast [1, 2, 3••, 4••], Escherichia coli [5, 6] and human cell lines [7••]. In addition, this approach has enabled mapping of specific biological pathways such as the TNFα/NF-κβ [8] and WNT/β-catenin [9] pathways as well as specific interactomes such as the human transcription machinery [10] and the embryonic stem cell pluripotency network [11]. The major requirements for this type of protein interaction mapping are specific, efficient and scaleable protein complex purification methods, sensitive and accurate MS acquisition platforms for reproducible and high-throughput protein identification and data analysis pipelines that exploit qualitative and quantitative features of MS data that effectively identify false protein identifications with minimum loss of sensitivity.

In this review we focus on technological developments over the past two years, in particular methods for generation and analysis of tagged-protein complexes. This encompasses developments in the design of tandem affinity purification (TAP) tags, large-scale protein-interaction mapping, novel strategies for the identification of transient protein interactions and structural analysis of protein complexes.

Section snippets

Novel TAP-tagging strategies

The original TAP tag developed for yeast consisted of a Protein A tag and a calmodulin-binding peptide (CBP) tag separated by a tobacco etch virus (TEV) protease cleavage site (Figure 1A) [12]. Although it has been used to purify complexes from mammalian cells, yields from these purifications are low and consequently large numbers of cells are needed as starting material [13]. Burckstummer et al. [13] compared the efficiency of this yeast TAP tag with that of three other TAP tags containing

Systematic analysis of protein complexes

In the past two years a number of large-scale studies of protein complexes have been reported; more comprehensive analyses of yeast and E. coli interactomes as well as the first analysis of a human interactome. In 2006, two comprehensive studies of protein complexes in yeast were published [3••, 4••]. Both of these studies employed systematic TAP tagging by homologous recombination and increased proteome coverage significantly (up to 72% [4••]) compared to initial large-scale TAP and

Mapping transient protein interactions

Maintenance and identification of transiently interacting proteins during affinity purification remains a challenge. These dynamic protein interactions are likely to be important in the context of signalling pathways where modulation of signalling can occur rapidly. It is thought that such interactions can be lost during the longer purification times necessary for tandem purifications, while single-step purifications may retain these interactions to some degree. However, decreased purity of

Architecture of multiprotein complexes

Hernández et al. [26] combined TAP and specialised MS analysis of intact non-covalent complexes to investigate subunit architecture of selected yeast complexes. Highly selective TAP experiments were performed to yield pure complexes that are necessary for top-down MS analysis. The TAP protocol was modified to remove detergents before the last purification step and buffer exchange and concentration steps were necessary to make purified complexes compatible for intact MS analysis. Additionally,

Conclusions

Although the analysis of protein complexes is still not routine, significant improvements in tagging, purification and analysis of protein complexes have been made. In particular, the ability to perform large-scale protein interaction mapping in mammalian cells should produce biological insights that are not achievable using other methods. We envisage that quantitative MS-based approaches and stable isotope labelling will be increasingly used for exploring dynamics of protein complexes involved

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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