Macromolecular complexes are involved in a broad spectrum of cellular processes including protein biosynthesis, protein secretion and degradation, metabolism, DNA replication and repair, and signal transduction along with other important biological processes. The analysis of protein complexes in health and disease is important to gain insights into cellular physiology and pathophysiology. In the last few decades, research has focused on the identification and the dynamics of macromolecular complexes. Several techniques have been developed to isolate native protein complexes from cells and tissues to allow further characterization by microscopic and proteomic analysis. In the present paper, we provide a brief overview of proteomic methods that can be used to identify protein–protein interactions, focusing on recent developments to study the entire complexome of a biological sample.

Introduction

Most proteins require additional biomolecules, i.e. other proteins, lipids or nucleic acids, to exert their biological function and form large stable or dynamic macromolecular complexes. The isolation of large protein complexes, and the identification of their components and their dynamic interactions are difficult tasks requiring advanced proteomic strategies. In particular, proteins involved in cellular signalling pathways form transient complexes and their dynamics are often regulated by post-translational modifications. Transient signalling protein complexes are of low abundance in the cell and require sensitive methods for their biochemical and proteomics analysis. For the analysis of membrane protein complexes the situation is even more complicated and requires mild solubilization protocols to identify labile and transient interaction partners, for example adapter molecules on cell-surface receptors or assembly factors of the OXPHOS (oxidative phosphorylation system) in the mitochondrial inner membrane. In general, there are two basic strategies to identify and to characterize macromolecular complexes. (i) Targeted approaches that use AP (affinity purification) to co-purify interacting molecules of a protein of interest [1] or expression of a tagged bait protein in heterologous cellular systems [2]. (ii) Techniques that are not focused on a specific protein complex such as BNE (blue native electrophoresis) or sucrose density centrifugation that can be combined with quantitative MS. The latter approaches have been extended to proteomic strategies called PCP (protein correlation profiling) [3] and complexome profiling [4] that can provide a comprehensive overview of all macromolecular complexes of a biological sample. In the present paper, we review current methods to analyse protein–protein interactions and demonstrate the power of non-targeted profiling approaches as tools to study known and unknown protein complexes.

Targeted identification of protein interaction partners

Many different in vivo and in vitro strategies have been developed to identify and characterize macromolecular complexes in cells and tissues (Table 1). Yeast two-hybrid screens identify interactions between bait and prey proteins in vivo [2]. Although yeast two-hybrid analysis is very sensitive and is able to identify labile and transient protein–protein interactions, the method is applicable only to a subset of proteins entering the nucleus of yeast cells. Limited compatibility of the heterologous system for specific post-translational modification could prevent protein complex formation leading to false negative results. Other in vivo strategies such as the split-ubiquitin system and FRET are suitable for membrane protein complexes and enable elucidation of protein complex dynamics under different physiological conditions [5,6].

Table 1
Strategies to identify and characterize protein–protein interactions
 Advantages Limitations Reference(s) 
In vivo strategies    
 Yeast two-hybrid screen Protein–protein interaction in vivo, suitable for high-throughput screening Limited compatibility of yeast cells for specific post-translational modification of mammalian proteins, applicable only to a subset of proteins interacting in the yeast nucleus [2
Split-ubiquitin system Protein–protein interaction in vivo, suitable for membrane proteins, dynamics of protein interactions and protein conformations under different physiological conditions Expression of fusion proteins could interfere with protein binding and function [5
 FRET Identification of protein–protein interactions in vivo and in vitro, dynamics of protein interactions and protein conformations under different physiological conditions, suitable for membrane proteins Expression of fusion proteins with GFP derivatives, fluorescent tag could interfere with protein binding and function [6
Targeted in vitro strategies    
 Co-IP Applicable to any kind of sample including cells and tissues from animal models and patients, antibody generation for virtually any protein possible, many antibodies commercially available High rate of non-specific binders, protocol adaptation required for every new target and sample [7
 AP Expression of tagged proteins and purification of protein complexes with standardized proteins, high-quality kits for AP available Introduction of a tag can impair function of target proteins, overexpression of protein could lead to unusual appearance in subcellular compartments and may give false positive results, many non-specific binders [1,8
 TAP (tandem AP) Very low rate of non-specific binders due to tandem purification Introduction of a tandem-tag can impair function of target proteins, loss of labile and transient binders due to tandem purification [9,10
 QUICK (quantitative immunoprecipitation combined with knockdown) Assesses endogenous protein complexes, antibody generation for virtually any protein possible, quantitative MS allows discrimination of specific and non-specific binders Knockdown experiments required, applicable only for cell culture or few SILAC-labelled animal models [11,12
 QUBIC [quantitative BAC (bacterial artificial chromosome) interactomics] Low rate of non-specific binders, quantitative MS allows discrimination of specific and non-specific binders, high-quality purification, expression of a tagged protein under endogenous promoter, GFP as tag allows proteomic and imaging studies with the same sample Introduction of a tag can impair function of target proteins [13,14
 LILBID-MS (laser-induced liquid bead desorption MS) Top-down method to study composition and subunit stoichiometry of an isolated protein complex, applicable to large membrane protein complexes, tolerance to various buffers and detergents Equipment not commercially available [44,45
 EtEP (equimolarity through equalizer peptides) Method for absolute quantification and stoichiometry determination using internal standard peptides Applicable only as targeted approach [46
Non-targeted in vitro strategies    
 Cross-linking Stabilizes labile and transient protein–protein interactions in vivo and in vitro, identifies interfaces between interacting proteins Protocol optimization is always required to restrict cross-linking to two interacting proteins and to avoid extensive random cross-linking, complicated MS data analysis [47
 BNE and LP-BNE High resolution of native protein complexes up to 50 MDa, multidimensional native and denaturing electrophoresis allow intensive studies on supercomplexes and labile binders, microscale method for scarce samples, superior for membrane protein complex isolation Limited availability of pre-cast gels, gel casting of special native gradient gels required, large-pore gels are very soft and need careful handling [15,16
 Density gradient centrifugation Fractionation of large complexes or entire organelles from any kind of sample Very low resolution, sample dilution, long centrifugation steps required, large amount of sample required [48
 Size-exclusion chromatography Fractionation of large complexes up to 5 MDa from any kind of sample Low resolution, sample dilution [49
 PCP Comparison of separation profiles from known marker proteins and potential interaction partners, applicable to any protein separation method and sample, high sensitivity of MS enables identification of low-abundant protein complexes Elaborate MS analysis [3,34,35
 Complexome profiling Unbiased bottom-up approach to identify known and unknown protein complexes in entire biological samples, high resolution, native gel separation cover mass range from 10 kDa to 50 MDa, at least 60 fractions of native gels support complex identification by hierarchical clustering, suitable as microscale method to study dynamics of protein complexes under different physiological condition in health and disease Elaborate MS analysis [4,41
 Advantages Limitations Reference(s) 
In vivo strategies    
 Yeast two-hybrid screen Protein–protein interaction in vivo, suitable for high-throughput screening Limited compatibility of yeast cells for specific post-translational modification of mammalian proteins, applicable only to a subset of proteins interacting in the yeast nucleus [2
Split-ubiquitin system Protein–protein interaction in vivo, suitable for membrane proteins, dynamics of protein interactions and protein conformations under different physiological conditions Expression of fusion proteins could interfere with protein binding and function [5
 FRET Identification of protein–protein interactions in vivo and in vitro, dynamics of protein interactions and protein conformations under different physiological conditions, suitable for membrane proteins Expression of fusion proteins with GFP derivatives, fluorescent tag could interfere with protein binding and function [6
Targeted in vitro strategies    
 Co-IP Applicable to any kind of sample including cells and tissues from animal models and patients, antibody generation for virtually any protein possible, many antibodies commercially available High rate of non-specific binders, protocol adaptation required for every new target and sample [7
 AP Expression of tagged proteins and purification of protein complexes with standardized proteins, high-quality kits for AP available Introduction of a tag can impair function of target proteins, overexpression of protein could lead to unusual appearance in subcellular compartments and may give false positive results, many non-specific binders [1,8
 TAP (tandem AP) Very low rate of non-specific binders due to tandem purification Introduction of a tandem-tag can impair function of target proteins, loss of labile and transient binders due to tandem purification [9,10
 QUICK (quantitative immunoprecipitation combined with knockdown) Assesses endogenous protein complexes, antibody generation for virtually any protein possible, quantitative MS allows discrimination of specific and non-specific binders Knockdown experiments required, applicable only for cell culture or few SILAC-labelled animal models [11,12
 QUBIC [quantitative BAC (bacterial artificial chromosome) interactomics] Low rate of non-specific binders, quantitative MS allows discrimination of specific and non-specific binders, high-quality purification, expression of a tagged protein under endogenous promoter, GFP as tag allows proteomic and imaging studies with the same sample Introduction of a tag can impair function of target proteins [13,14
 LILBID-MS (laser-induced liquid bead desorption MS) Top-down method to study composition and subunit stoichiometry of an isolated protein complex, applicable to large membrane protein complexes, tolerance to various buffers and detergents Equipment not commercially available [44,45
 EtEP (equimolarity through equalizer peptides) Method for absolute quantification and stoichiometry determination using internal standard peptides Applicable only as targeted approach [46
Non-targeted in vitro strategies    
 Cross-linking Stabilizes labile and transient protein–protein interactions in vivo and in vitro, identifies interfaces between interacting proteins Protocol optimization is always required to restrict cross-linking to two interacting proteins and to avoid extensive random cross-linking, complicated MS data analysis [47
 BNE and LP-BNE High resolution of native protein complexes up to 50 MDa, multidimensional native and denaturing electrophoresis allow intensive studies on supercomplexes and labile binders, microscale method for scarce samples, superior for membrane protein complex isolation Limited availability of pre-cast gels, gel casting of special native gradient gels required, large-pore gels are very soft and need careful handling [15,16
 Density gradient centrifugation Fractionation of large complexes or entire organelles from any kind of sample Very low resolution, sample dilution, long centrifugation steps required, large amount of sample required [48
 Size-exclusion chromatography Fractionation of large complexes up to 5 MDa from any kind of sample Low resolution, sample dilution [49
 PCP Comparison of separation profiles from known marker proteins and potential interaction partners, applicable to any protein separation method and sample, high sensitivity of MS enables identification of low-abundant protein complexes Elaborate MS analysis [3,34,35
 Complexome profiling Unbiased bottom-up approach to identify known and unknown protein complexes in entire biological samples, high resolution, native gel separation cover mass range from 10 kDa to 50 MDa, at least 60 fractions of native gels support complex identification by hierarchical clustering, suitable as microscale method to study dynamics of protein complexes under different physiological condition in health and disease Elaborate MS analysis [4,41

Co-IP (co-immunoprecipitation) co-purifies interaction partners of a specific protein that can be subsequently detected by immunoblotting or identified by MS [7]. Co-IP can be applied to any kind of biological sample including tissues from model organisms and human specimens. The approach relies on the quality of antibodies binding to peripheral native epitopes of a protein complex, and optimization of the protocol is required for every new target and specimen. For tagged proteins (e.g. FLAG-tag, Strep-tag, His-tag), standardized purification protocols can be used to purify protein complexes [1,8]. Isolated protein complexes are digested with trypsin and their components are identified by MS. The result is often a long list of identified proteins, and it is difficult to distinguish between specific interaction partners and contaminants. To reduce the amount of false positives, several strategies have been applied. The TAP (tandem AP) technology uses a dual tag for sequential mild purification steps to reduce contaminants [9,10]. Other strategies include quantitative MS into the workflow. Protein interaction screening by QUICK (quantitative immunoprecipitation combined with knockdown) assesses interactions of endogenous proteins. The method uses SILAC (stable isotope labelling by amino acids in cell culture) to identify interacting proteins in co-IPs comparing wild-type and knockdown cells [11,12]. QUBIC [quantitative BAC (bacterial artificial chromosome) interactomics] is based on expression of tagged proteins under physiological conditions and utilizes AP in combination with quantitative MS [13,14]. The introduction of the GFP as an affinity tag allows a direct combination of quantitative proteomics data with fluorescent microscopy to gain insights into protein function at the molecular and cellular levels [14].

All of these proteomic approaches use pull-down strategies to co-purify interaction partners of the protein of interest and therefore give hardly any information on shape, stoichiometry, dynamics or molecular mass of a macromolecular complex. In addition, a protein of interest could be at the same time part of different protein complexes with specific biological functions, or included in subcomplexes or assembly intermediates that cannot be discriminated by a pull-down strategy.

For a more comprehensive analysis, non-targeted fractionation of native complexes by mild techniques, e.g. density gradient centrifugation, size-exclusion chromatography and native electrophoresis, is required before quantitative MS and bioinformatic analysis.

Non-targeted separation of native complexes

Fractions from density gradient centrifugation have been widely used to study the distribution of proteins in comparison with marker proteins from known protein complexes or cellular organelles by immunoblotting. The isolation of macromolecular complexes by density gradient centrifugation requires a relatively large quantity of the respective material and suffers from low resolution. Size-exclusion chromatography possesses a better resolution, but available media allow only a separation of complexes in the low-megadalton range and substantially dilute the sample. In order to compare many samples with a limited amount of material, e.g. patient biopsies, micro-scale and high-resolution fractionation methods such as BNE or CNE (clear native electrophoresis) are required to generate large comparative datasets. Since its development in 1991 [15], BNE has become a very popular method to analyse the composition and assembly of soluble and membrane protein complexes in eukaryotic cellular compartments and prokaryotic organisms [1618]. As a robust and reproducible method, BNE has been applied to medical proteomics to study complex assembly and stability defects in patients with mitochondrial disorders [19]. Standard blue native gels separate structurally and enzymatically intact protein complexes over a range from 10 kDa to 5 MDa. The recently introduced LP-BNE (large-pore BNE) extends the separation capacity up to 50 MDa [20] enabling isolation of megacomplexes such as oligomeric respirasomes (Figure 1A). The native mass of an isolated soluble or membrane protein complex can be easily estimated using suitable native mass calibration ladders [21]. CNE omitting the anionic Coomassie Blue dye has advantages for the isolation of detergent-labile protein–protein interactions [22]. The composition of isolated native protein complexes can be studied in more detail by application of SDS/PAGE in the second dimension [23]. Characteristic patterns of subunits in a Coomassie Blue- or silver-stained gel [16] and complete 2D gel maps including identified protein spots [24] provide information of known protein complexes from a sample immediately. Subcomplexes of lower abundance and assembly intermediates have been frequently analysed by 2D BNE–SDS/PAGE followed by immunoblotting [2527]. Multidimensional native and denaturing electrophoresis have been used to study the interface of supramolecular assemblies without [28] and with [29,30] application of chemical cross-linking to stabilize protein complexes.

Mass range of BNE and LP-BNE and workflow of complexome profiling

Figure 1
Mass range of BNE and LP-BNE and workflow of complexome profiling

(A) The gradients of total percentage of acrylamide with the percentage fraction of the cross-linker bisacrylamide in subscript and the corresponding separation range for native protein complexes is indicated for the two gel types. The position or size range of representative mitochondrial protein complexes is shown. I–V, complexes I–V of the OXPHOS; S, supercomplexes of respiratory chain containing complex I and III and copies of complex IV [43]; O, oxoglutarate dehydrogenase complex; P, pyruvate dehydrogenase complex; MINOS, mitochondrial inner membrane organizing system. (B) Protein complexes are separated by BNE, fixed and stained with Coomassie Blue. Gel lanes are cut into even slices and subjected to trypsin digestion, and peptides are analysed by label-free quantitative nano-LC–MS/MS. Protein abundance profiles are analysed by hierarchical clustering. The resulting heat map shows protein groups with similar migration profiles that correspond to known and newly discovered macromolecular complexes.

Figure 1
Mass range of BNE and LP-BNE and workflow of complexome profiling

(A) The gradients of total percentage of acrylamide with the percentage fraction of the cross-linker bisacrylamide in subscript and the corresponding separation range for native protein complexes is indicated for the two gel types. The position or size range of representative mitochondrial protein complexes is shown. I–V, complexes I–V of the OXPHOS; S, supercomplexes of respiratory chain containing complex I and III and copies of complex IV [43]; O, oxoglutarate dehydrogenase complex; P, pyruvate dehydrogenase complex; MINOS, mitochondrial inner membrane organizing system. (B) Protein complexes are separated by BNE, fixed and stained with Coomassie Blue. Gel lanes are cut into even slices and subjected to trypsin digestion, and peptides are analysed by label-free quantitative nano-LC–MS/MS. Protein abundance profiles are analysed by hierarchical clustering. The resulting heat map shows protein groups with similar migration profiles that correspond to known and newly discovered macromolecular complexes.

Identification of proteins in complexes isolated by BNE and CNE is achieved by in-gel digestion and LC–MS/MS analysis [31,32]. The combination of native gel electrophoresis and MS analysis allowed the characterization of labile assembly intermediates of the mitochondrial ATP synthase [32] and revised the subunit composition of mitochondrial complexes I and IV [33].

Co-migration of proteins under native conditions in the same fractions on BNE, size-exclusion chromatography or density gradient centrifugation can provide valuable hints for possible protein–protein interactions, but, without quantitative information about protein distribution in neighbouring fractions, this could result in false positive detection of interaction candidates. To overcome this limitation, two strategies have been introduced: (i) PCP [3] to identify new components of known protein assemblies, and (ii) complexome profiling [4] to obtain more information about known protein–protein interactions and to discover new protein complexes.

PCP and complexome profiling

PCP analyses fractions of mild protein complex separation techniques such as density gradient centrifugation or BNE with quantitative MS to identify co-migrating proteins based on reference profiles of known proteins and complexes. Andersen et al. [3] introduced PCP to study new components of human centrosomes in fractions from sucrose density gradient centrifugation. Label-free quantitative MS was used to generate abundance distribution profiles of proteins in comparison with centrosomal markers [3]. PCP was further applied to generate a mammalian organelle map of more than 1400 proteins to ten subcellular compartments [34]. Wessels et al. applied PCP to separated human mitochondrial complexes by BNE [35]. The whole native gel was divided into 24 equal pieces and analysed by label-free quantitative MS to obtain protein migration profiles across the entire lane. Average profiles from well-characterized mitochondrial complexes were used to identify low-abundant assembly intermediates and assembly factors. This study impressively demonstrated that this approach is very powerful to gain information even on low-abundant and more dynamic protein complexes in a very complex sample [35]. Similar approaches including hierarchical clustering were used to analyse mitochondrial complexes from the yeast Saccharomyces cerevisiae [36] and soluble protein complexes from Nicotiana tabacum cv. Bright Yellow-2 cells [37]. Among complexes that were validated are the MITRAC (mitochondrial translation regulation assembly intermediate of cytochrome c oxidase) complex [25] and the 20S proteasome from Plasmodium falciparum [38]. Recently, the comprehensive profiling study from Arabidopsis thylakoids and whole cells of the cyanobacterium Synechocystis sp. have been used to generate a protein co-migration database [39]. An integrative co-fractionation strategy by application of non-denaturing multibed IEX-HPLC (ion-exchange HPLC), sucrose density gradient centrifugation and IEF (isoelectric focusing) generated a global proteomic profile of human soluble protein complexes [40].

Recently we introduced complexome profiling as a bottom-up approach to identify the interactome of entire cells or subcellular compartments [4]. Whereas PCP uses a reference protein or average profile of a known complex to identify putative interaction partners, our approach implies global hierarchical clustering of migration profiles and visualization as heat maps to allow an unbiased fast verification of known complexes, selection of new candidates participating in well-characterized complexes and identification of new less stable or transient complexes. The workflow (Figure 1B) of complexome profiling includes large high-resolution BNE and LP-BNE gels to cover a mass range from 10 kDa to 50 MDa. Lanes of approximately 14 cm are divided into 60 even slices and subjected to trypsin digestion for subsequent label-free quantitative nano-ESI–LC–MS/MS. The resulting set of protein abundance profiles is hierarchically clustered and visualized as interaction profiles in a heat map. The native mass corresponding to each slice is calibrated using known complexes as internal markers or a parallel lane as external mass ladder. To exploit the resolution of long BNE and LP-BNE lanes for better clustering a subdivision in a larger number of at least 60 slices is advisable. Using complexome profiling we identified in intact rat heart mitochondria an association of a protein with so far unknown function, i.e. TMEM126B (transmembrane protein 126B), together with assembly factors of the mitochondrial complex I, namely CIA30 (complex I intermediate-associated protein 30), Ecsit (evolutionarily conserved intermediate in Toll pathways) and ACAD9 (acyl-CoA dehydrogenase family member 9). A knockdown of TMEM126B in human cells revealed that this transmembrane protein is essential for complex I assembly [4]. Application of complexome profiling to bovine heart mitochondrial membranes identified ApoO (apolipoprotein O) and ApoOL (ApoO-like) protein as components of the MINOS (mitochondrial inner membrane organizing system) [41]. In this study, overexpression and knockdown of ApoOL caused altered mitochondrial cristae morphology.

Perspectives

Global views on the dynamic behaviour of protein complexes in cell division, cell cycle, apoptosis, environmental adaptation, stress response and differentiation is an ambitious aim in cell biology. Reliable and comprehensive proteomics tools capable of analysing the complexome of an entire cell or organelle are needed to address these topics. PCP and complexome profiling are well suited to study efficiently multiple physiological conditions and dynamic processes. ‘Profiles within profiles’ using pulsed SILAC [42] to identify newly translated proteins and their assembly into protein complexes, identification of post-translational modifications in macromolecular complexes, analysis of samples from patients or animal models are additional options to gain deep insights into cellular physiology.

Bioenergetics in Mitochondria, Bacteria and Chloroplasts: Third Joint German/UK Bioenergetics Conference, a Biochemical Society Focused Meeting held at Schloss Rauischholzhausen, Ebsdorfergrund, Germany, 10–13 April 2013. Organized and Edited by Fraser MacMillan (University of East Anglia, Norwich, U.K.) and Thomas Meier (Max Planck Institute of Biophysics, Frankfurt am Main, Germany).

Abbreviations

     
  • AP

    affinity purification

  •  
  • ApoO

    apolipoprotein O

  •  
  • ApoOL

    apolipoprotein O-like

  •  
  • BNE

    blue native electrophoresis

  •  
  • CNE

    clear native electrophoresis

  •  
  • Co-IP

    co-immunoprecipitation

  •  
  • LP-BNE

    large-pore BNE

  •  
  • OXPHOS

    oxidative phosphorylation system

  •  
  • PCP

    protein correlation profiling

  •  
  • SILAC

    stable isotope labelling by amino acids in cell culture

  •  
  • TMEM126B

    transmembrane protein 126B

  •  
  • Y2H

    yeast two-hybrid

We thank Stefan Dröse and Ulrich Brandt for helpful discussions and a critical reading of the paper before submission.

Funding

This work was supported by the Cluster of Excellence ‘Macromolecular Complexes’ at the Goethe University Frankfurt [grant number EXC 115] and the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 815 project Z1-Redox-Proteomics and by Bundesministerium für Bildung und Forschung [grant number 01GM1113B] mitoNET-Deutsches Netzwerk für mitochondriale Erkrankungen (to I.W.).

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