methods to analyse composition and dynamics of macromolecular complexes

Bioenergetics in Mitochondria, Bacteria and Chloroplasts 1235

Methods to analyse composition and dynamics ofmacromolecular complexesHeinrich Heide* and Ilka Wittig*1

*Functional Proteomics, SFB815 Core Unit, Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany

AbstractMacromolecular complexes are involved in a broad spectrum of cellular processes including proteinbiosynthesis, protein secretion and degradation, metabolism, DNA replication and repair, and signaltransduction along with other important biological processes. The analysis of protein complexes in healthand disease is important to gain insights into cellular physiology and pathophysiology. In the last fewdecades, 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 toallow further characterization by microscopic and proteomic analysis. In the present paper, we provide abrief overview of proteomic methods that can be used to identify protein–protein interactions, focusing onrecent developments to study the entire complexome of a biological sample.

IntroductionMost proteins require additional biomolecules, i.e. otherproteins, lipids or nucleic acids, to exert their biologicalfunction and form large stable or dynamic macromolecularcomplexes. The isolation of large protein complexes, andthe identification of their components and their dynamicinteractions are difficult tasks requiring advanced proteomicstrategies. In particular, proteins involved in cellular signallingpathways form transient complexes and their dynamics areoften regulated by post-translational modifications. Transientsignalling protein complexes are of low abundance in thecell and require sensitive methods for their biochemical andproteomics analysis. For the analysis of membrane proteincomplexes the situation is even more complicated andrequires mild solubilization protocols to identify labile andtransient interaction partners, for example adapter moleculeson cell-surface receptors or assembly factors of the OXPHOS(oxidative phosphorylation system) in the mitochondrialinner membrane. In general, there are two basic strategiesto identify and to characterize macromolecular complexes.(i) Targeted approaches that use AP (affinity purification) toco-purify interacting molecules of a protein of interest [1] orexpression of a tagged bait protein in heterologous cellularsystems [2]. (ii) Techniques that are not focused on a specificprotein complex such as BNE (blue native electrophoresis)or sucrose density centrifugation that can be combined withquantitative MS. The latter approaches have been extendedto proteomic strategies called PCP (protein correlation

Key words: blue native electrophoresis, complexome profiling, membrane protein complexes,

protein correlation profiling.

Abbreviations used: 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.1To whom correspondence should be addressed (email [email protected]).

profiling) [3] and complexome profiling [4] that can providea comprehensive overview of all macromolecular complexesof a biological sample. In the present paper, we reviewcurrent methods to analyse protein–protein interactions anddemonstrate the power of non-targeted profiling approachesas tools to study known and unknown protein complexes.

Targeted identification of proteininteraction partnersMany different in vivo and in vitro strategies have beendeveloped to identify and characterize macromolecularcomplexes in cells and tissues (Table 1). Yeast two-hybridscreens identify interactions between bait and prey proteinsin vivo [2]. Although yeast two-hybrid analysis is verysensitive and is able to identify labile and transient protein–protein interactions, the method is applicable only to asubset of proteins entering the nucleus of yeast cells. Limitedcompatibility of the heterologous system for specific post-translational modification could prevent protein complexformation leading to false negative results. Other in vivostrategies such as the split-ubiquitin system and FRETare suitable for membrane protein complexes and enableelucidation of protein complex dynamics under differentphysiological conditions [5,6].

Co-IP (co-immunoprecipitation) co-purifies interactionpartners of a specific protein that can be subsequentlydetected by immunoblotting or identified by MS [7]. Co-IP can be applied to any kind of biological sample includingtissues from model organisms and human specimens. Theapproach relies on the quality of antibodies binding toperipheral native epitopes of a protein complex, andoptimization of the protocol is required for every newtarget and specimen. For tagged proteins (e.g. FLAG-tag,Strep-tag, His-tag), standardized purification protocols can

Biochem. Soc. Trans. (2013) 41, 1235–1241; doi:10.1042/BST20130153 C©The Authors Journal compilation C©2013 Biochemical SocietyBio

chem

ical

So

ciet

y T

ran

sact

ion

s

ww

w.b

ioch

emso

ctra

ns.

org

1236 Biochemical Society Transactions (2013) Volume 41, part 5

Table1 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]

C©The Authors Journal compilation C©2013 Biochemical Society


Table1 Continued

Advantages Limitations Reference(s)

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]

be used to purify protein complexes [1,8]. Isolated proteincomplexes are digested with trypsin and their componentsare identified by MS. The result is often a long listof identified proteins, and it is difficult to distinguishbetween specific interaction partners and contaminants. Toreduce the amount of false positives, several strategies havebeen applied. The TAP (tandem AP) technology uses adual tag for sequential mild purification steps to reducecontaminants [9,10]. Other strategies include quantitativeMS into the workflow. Protein interaction screening byQUICK (quantitative immunoprecipitation combined withknockdown) assesses interactions of endogenous proteins.The method uses SILAC (stable isotope labelling by amino

acids in cell culture) to identify interacting proteins inco-IPs comparing wild-type and knockdown cells [11,12].QUBIC [quantitative BAC (bacterial artificial chromosome)interactomics] is based on expression of tagged proteins underphysiological conditions and utilizes AP in combination withquantitative MS [13,14]. The introduction of the GFP asan affinity tag allows a direct combination of quantitativeproteomics data with fluorescent microscopy to gain insightsinto protein function at the molecular and cellular levels [14].

All of these proteomic approaches use pull-down strategiesto co-purify interaction partners of the protein of interestand therefore give hardly any information on shape, stoi-chiometry, dynamics or molecular mass of a macromolecular



complex. In addition, a protein of interest could be atthe same time part of different protein complexes withspecific biological functions, or included in subcomplexesor assembly intermediates that cannot be discriminated by apull-down strategy.

For a more comprehensive analysis, non-targeted fraction-ation of native complexes by mild techniques, e.g. densitygradient centrifugation, size-exclusion chromatography andnative electrophoresis, is required before quantitative MS andbioinformatic analysis.

Non-targeted separation of nativecomplexesFractions from density gradient centrifugation have beenwidely used to study the distribution of proteins incomparison with marker proteins from known proteincomplexes or cellular organelles by immunoblotting. Theisolation of macromolecular complexes by density gradientcentrifugation requires a relatively large quantity of therespective material and suffers from low resolution. Size-exclusion chromatography possesses a better resolution,but available media allow only a separation of complexesin the low-megadalton range and substantially dilute thesample. In order to compare many samples with a limitedamount of material, e.g. patient biopsies, micro-scale andhigh-resolution fractionation methods such as BNE orCNE (clear native electrophoresis) are required to generatelarge comparative datasets. Since its development in 1991[15], BNE has become a very popular method to analysethe composition and assembly of soluble and membraneprotein complexes in eukaryotic cellular compartmentsand prokaryotic organisms [16–18]. As a robust andreproducible method, BNE has been applied to medicalproteomics to study complex assembly and stability defectsin patients with mitochondrial disorders [19]. Standardblue native gels separate structurally and enzymaticallyintact protein complexes over a range from 10 kDa to5 MDa. The recently introduced LP-BNE (large-pore BNE)extends the separation capacity up to 50 MDa [20] enablingisolation of megacomplexes such as oligomeric respirasomes(Figure 1A). The native mass of an isolated soluble ormembrane protein complex can be easily estimated usingsuitable native mass calibration ladders [21]. CNE omittingthe anionic Coomassie Blue dye has advantages for theisolation of detergent-labile protein–protein interactions [22].The composition of isolated native protein complexes canbe studied in more detail by application of SDS/PAGEin the second dimension [23]. Characteristic patterns ofsubunits in a Coomassie Blue- or silver-stained gel [16] andcomplete 2D gel maps including identified protein spots [24]provide information of known protein complexes from asample immediately. Subcomplexes of lower abundance andassembly intermediates have been frequently analysed by2D BNE–SDS/PAGE followed by immunoblotting [25–27].Multidimensional native and denaturing electrophoresis have

been used to study the interface of supramolecular assemblieswithout [28] and with [29,30] application of chemical cross-linking to stabilize protein complexes.

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

Co-migration of proteins under native conditions in thesame fractions on BNE, size-exclusion chromatographyor density gradient centrifugation can provide valuablehints for possible protein–protein interactions, but, withoutquantitative information about protein distribution inneighbouring fractions, this could result in false positivedetection of interaction candidates. To overcome thislimitation, two strategies have been introduced: (i) PCP [3]to identify new components of known protein assemblies,and (ii) complexome profiling [4] to obtain more informationabout known protein–protein interactions and to discovernew protein complexes.

PCP and complexome profilingPCP analyses fractions of mild protein complex separationtechniques such as density gradient centrifugation or BNEwith quantitative MS to identify co-migrating proteins basedon reference profiles of known proteins and complexes.Andersen et al. [3] introduced PCP to study new componentsof human centrosomes in fractions from sucrose densitygradient centrifugation. Label-free quantitative MS was usedto generate abundance distribution profiles of proteins incomparison with centrosomal markers [3]. PCP was furtherapplied to generate a mammalian organelle map of more than1400 proteins to ten subcellular compartments [34]. Wesselset al. applied PCP to separated human mitochondrial com-plexes by BNE [35]. The whole native gel was divided into 24equal pieces and analysed by label-free quantitative MS to ob-tain protein migration profiles across the entire lane. Averageprofiles from well-characterized mitochondrial complexeswere used to identify low-abundant assembly intermediatesand assembly factors. This study impressively demonstratedthat this approach is very powerful to gain information evenon low-abundant and more dynamic protein complexes ina very complex sample [35]. Similar approaches includinghierarchical clustering were used to analyse mitochondrialcomplexes from the yeast Saccharomyces cerevisiae [36] andsoluble protein complexes from Nicotiana tabacum cv. BrightYellow-2 cells [37]. Among complexes that were validatedare the MITRAC (mitochondrial translation regulationassembly intermediate of cytochrome c oxidase) complex[25] and the 20S proteasome from Plasmodium falciparum[38]. Recently, the comprehensive profiling study fromArabidopsis thylakoids and whole cells of the cyanobacteriumSynechocystis sp. have been used to generate a proteinco-migration database [39]. An integrative co-fractionation



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.

strategy by application of non-denaturing multibed IEX-HPLC (ion-exchange HPLC), sucrose density gradient cent-rifugation and IEF (isoelectric focusing) generated a globalproteomic profile of human soluble protein complexes [40].

Recently we introduced complexome profiling as abottom-up approach to identify the interactome of entirecells or subcellular compartments [4]. Whereas PCP uses a

reference protein or average profile of a known complex toidentify putative interaction partners, our approach impliesglobal hierarchical clustering of migration profiles and visu-alization as heat maps to allow an unbiased fast verification ofknown complexes, selection of new candidates participatingin well-characterized complexes and identification of new lessstable or transient complexes. The workflow (Figure 1B) of



complexome profiling includes large high-resolution BNEand LP-BNE gels to cover a mass range from 10 kDa to50 MDa. Lanes of approximately 14 cm are divided into 60even slices and subjected to trypsin digestion for subsequentlabel-free quantitative nano-ESI–LC–MS/MS. The resultingset of protein abundance profiles is hierarchically clusteredand visualized as interaction profiles in a heat map. The nativemass corresponding to each slice is calibrated using knowncomplexes as internal markers or a parallel lane as externalmass ladder. To exploit the resolution of long BNE andLP-BNE lanes for better clustering a subdivision in a largernumber of at least 60 slices is advisable. Using complexomeprofiling we identified in intact rat heart mitochondria anassociation of a protein with so far unknown function, i.e.TMEM126B (transmembrane protein 126B), together withassembly factors of the mitochondrial complex I, namelyCIA30 (complex I intermediate-associated protein 30), Ecsit(evolutionarily conserved intermediate in Toll pathways) andACAD9 (acyl-CoA dehydrogenase family member 9). Aknockdown of TMEM126B in human cells revealed that thistransmembrane protein is essential for complex I assembly[4]. Application of complexome profiling to bovine heartmitochondrial membranes identified ApoO (apolipoproteinO) and ApoOL (ApoO-like) protein as components of theMINOS (mitochondrial inner membrane organizing system)[41]. In this study, overexpression and knockdown of ApoOLcaused altered mitochondrial cristae morphology.

PerspectivesGlobal views on the dynamic behaviour of protein com-plexes in cell division, cell cycle, apoptosis, environmentaladaptation, stress response and differentiation is an ambitiousaim in cell biology. Reliable and comprehensive proteomicstools capable of analysing the complexome of an entire cellor organelle are needed to address these topics. PCP andcomplexome profiling are well suited to study efficientlymultiple physiological conditions and dynamic processes.‘Profiles within profiles’ using pulsed SILAC [42] to identifynewly translated proteins and their assembly into proteincomplexes, identification of post-translational modificationsin macromolecular complexes, analysis of samples frompatients or animal models are additional options to gain deepinsights into cellular physiology.

Acknowledgments

We thank Stefan Drose 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 ‘Macro-

molecular Complexes’ at the Goethe University Frankfurt [grant

number EXC 115] and the Deutsche Forschungsgemeinschaft

Sonderforschungsbereich 815 project Z1-Redox-Proteomics and by

Bundesministerium fur Bildung und Forschung [grant number

01GM1113B] mitoNET-Deutsches Netzwerk fur mitochondriale

Erkrankungen (to I.W.).

References1 Oeffinger, M. (2012) Two steps forward–one step back: advances in

affinity purification mass spectrometry of macromolecular complexes.Proteomics 12, 1591–608

2 Fields, S. and Song, O. (1989) A novel genetic system to detectprotein–protein interactions. Nature 340, 245–246

3 Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A. andMann, M. (2003) Proteomic characterization of the human centrosomeby protein correlation profiling. Nature 426, 570–574

4 Heide, H., Bleier, L., Steger, M., Ackermann, J., Drose, S., Schwamb, B.,Zornig, M., Reichert, A.S., Koch, I., Wittig, I. and Brandt, U. (2012)Complexome profiling identifies TMEM126B as a component of themitochondrial complex I assembly complex. Cell Metab. 16,538–549

5 Johnsson, N. and Varshavsky, A. (1994) Split ubiquitin as a sensor ofprotein interactions in vivo. Proc. Natl. Acad. Sci. U.S.A. 91,10340–10344

6 Sun, Y., Rombola, C., Jyothikumar, V. and Periasamy, A. (2013) Forsterresonance energy transfer microscopy and spectroscopy for localizingprotein-protein interactions in living cells. Cytometry A 83, 780–793

7 Gygi, S.P., Han, D.K., Gingras, A.C., Sonenberg, N. and Aebersold, R.(1999) Protein analysis by mass spectrometry and sequence databasesearching: tools for cancer research in the post-genomic era.Electrophoresis 20, 310–319

8 Paul, F.E., Hosp, F. and Selbach, M. (2011) Analyzing protein–proteininteractions by quantitative mass spectrometry. Methods 54, 387–395

9 Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Seraphin, B.(1999) A generic protein purification method for protein complexcharacterization and proteome exploration. Nat. Biotechnol. 17,1030–1032

10 Volkel, P., Le Faou, P. and Angrand, P.O. (2010) Interaction proteomics:characterization of protein complexes using tandem affinitypurification–mass spectrometry. Biochem. Soc. Trans. 38, 883–887

11 Selbach, M. and Mann, M. (2006) Protein interaction screening byquantitative immunoprecipitation combined with knockdown (QUICK).Nat. Methods 3, 981–983

12 Heide, H., Nordhues, A., Drepper, F., Nick, S., Schulz-Raffelt, M., Haehnel,W. and Schroda, M. (2009) Application of quantitativeimmunoprecipitation combined with knockdown and cross-linking toChlamydomonas reveals the presence of vesicle-inducing protein inplastids 1 in a common complex with chloroplast HSP90C. Proteomics 9,3079–3089

13 Hubner, N.C. and Mann, M. (2011) Extracting gene function fromprotein–protein interactions using Quantitative BAC InteraCtomics(QUBIC). Methods 53, 453–459

14 Hubner, N.C., Bird, A.W., Cox, J., Splettstoesser, B., Bandilla, P., Poser, I.,Hyman, A. and Mann, M. (2010) Quantitative proteomics combined withBAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol.189, 739–754

15 Schagger, H. and von Jagow, G. (1991) Blue native electrophoresis forisolation of membrane protein complexes in enzymatically active form.Anal. Biochem. 199, 223–231

16 Wittig, I., Braun, H.P. and Schagger, H. (2006) Blue native PAGE. Nat.Protoc. 1, 418–428

17 Wittig, I. and Schagger, H. (2009) Native electrophoretic techniques toidentify protein–protein interactions. Proteomics 9, 5214–5223

18 Wittig, I. and Schagger, H. (2008) Features and applications ofblue-native and clear-native electrophoresis. Proteomics 8,3974–3990

19 Calvaruso, M.A., Smeitink, J. and Nijtmans, L. (2008) Electrophoresistechniques to investigate defects in oxidative phosphorylation. Methods46, 281–287

20 Strecker, V., Wumaier, Z., Wittig, I. and Schagger, H. (2010) Large poregels to separate mega protein complexes larger than 10 MDa by bluenative electrophoresis: isolation of putative respiratory strings orpatches. Proteomics 10, 3379–3387

21 Wittig, I., Beckhaus, T., Wumaier, Z., Karas, M. and Schagger, H. (2010)Mass estimation of native proteins by blue native electrophoresis:principles and practical hints. Mol. Cell. Proteomics 9, 2149–2161



22 Wittig, I. and Schagger, H. (2005) Advantages and limitations ofclear-native PAGE. Proteomics 5, 4338–4346

23 Schagger, H. (2006) Tricine-SDS-PAGE. Nat. Protoc. 1, 16–2224 Klodmann, J., Senkler, M., Rode, C. and Braun, H.P. (2011) Defining the

protein complex proteome of plant mitochondria. Plant Physiol. 157,587–598

25 Mick, D.U., Dennerlein, S., Wiese, H., Reinhold, R., Pacheu-Grau, D.,Lorenzi, I., Sasarman, F., Weraarpachai, W., Shoubridge, E.A., Warscheid,B. and Rehling, P. (2012) MITRAC links mitochondrial proteintranslocation to respiratory-chain assembly and translational regulation.Cell 151, 1528–1541

26 Fernandez-Vizarra, E., Tiranti, V. and Zeviani, M. (2008) Assembly of theoxidative phosphorylation system in humans: what we have learned bystudying its defects. Biochim. Biophys. Acta 1793, 200–211

27 Vogel, R.O., Dieteren, C.E., van den Heuvel, L.P., Willems, P.H., Smeitink,J.A., Koopman, W.J. and Nijtmans, L.G. (2007) Identification ofmitochondrial complex I assembly intermediates by tracing taggedNDUFS3 demonstrates the entry point of mitochondrial subunits J. Biol.Chem. 282, 7582–7590

28 Wittig, I., Velours, J., Stuart, R. and Schagger, H. (2008) Characterizationof domain interfaces in monomeric and dimeric ATP synthase. Mol. Cell.Proteomics 7, 995–1004

29 Fronzes, R., Weimann, T., Vaillier, J., Velours, J. and Brethes, D. (2006)The peripheral stalk participates in the yeast ATP synthase dimerizationindependently of e and g subunits. Biochemistry 45, 6715–6723

30 Saddar, S. and Stuart, R.A. (2005) The yeast F1Fo-ATP synthase: analysisof the molecular organization of subunit g and the importance of aconserved GXXXG motif. J. Biol. Chem. 280, 24435–24442

31 Fandino, A.S., Rais, I., Vollmer, M., Elgass, H., Schagger, H. and Karas, M.(2005) LC–nanospray–MS/MS analysis of hydrophobic proteins frommembrane protein complexes isolated by blue-native electrophoresis. J.Mass Spectrom. 40, 1223–1231

32 Wittig, I., Meyer, B., Heide, H., Steger, M., Bleier, L., Wumaier, Z., Karas,M. and Schagger, H. (2010) Assembly and oligomerization of human ATPsynthase lacking mitochondrial subunits a and A6L. Biochim. Biophys.Acta 1797, 1004–1011

33 Balsa, E., Marco, R., Perales-Clemente, E., Szklarczyk, R., Calvo, E.,Landazuri, M.O. and Enrıquez, J.A. (2012) NDUFA4 is a subunit of complexIV of the mammalian electron transport chain. Cell Metab. 16, 378–386

34 Foster, L.J., de Hoog, C.L., Zhang, Y., Zhang, Y., Xie, X., Mootha, V.K. andMann, M. (2006) A mammalian organelle map by protein correlationprofiling. Cell 125, 187–199

35 Wessels, H.J., Vogel, R.O., van den Heuvel, L., Smeitink, J.A., Rodenburg,R.J., Nijtmans, L.G. and Farhoud, M.H. (2009) LC–MS/MS as an alternativefor SDS-PAGE in blue native analysis of protein complexes. Proteomics 9,4221–4228

36 Helbig, A.O., de Groot, M.J., van Gestel, R.A., Mohammed, S., de Hulster,E.A., Luttik, M.A., Daran-Lapujade, P., Pronk, J.T., Heck, A.J. and Slijper, M.(2009) A three-way proteomics strategy allows differential analysis ofyeast mitochondrial membrane protein complexes under anaerobic andaerobic conditions. Proteomics 9, 4787–4798

37 Remmerie, N., De Vijlder, T., Valkenborg, D., Laukens, K., Smets, K.,Vreeken, J., Mertens, I., Carpentier, S.C., Panis, B., De Jaeger, G. et al.(2011) Unraveling tobacco BY-2 protein complexes with BNPAGE/LC–MS/MS and clustering methods. J. Proteomics 74, 1201–1217

38 Sessler, N., Krug, K., Nordheim, A., Mordmuller, B. and Macek, B. (2012)Analysis of the Plasmodium falciparum proteasome using Blue NativePAGE and label-free quantitative mass spectrometry. Amino Acids 43,1119–1129

39 Takabayashi, A., Kadoya, R., Kuwano, M., Kurihara, K., Ito, H., Tanaka, R.and Tanaka, A. (2013) Protein co-migration database (PCoM-DB) forArabidopsis thylakoids and Synechocystis cells. SpringerPlus 2,148

40 Havugimana, P.C., Hart, G.T., Nepusz, T., Yang, H., Turinsky, A.L., Li, Z.,Wang, P.I., Boutz, D.R., Fong, V., Phanse, S. et al. (2012) A census ofhuman soluble protein complexes. Cell 150, 1068–1081

41 Weber, T.A., Koob, S., Heide, H., Wittig, I., Head, B., van der Bliek, A.,Brandt, U., Mittelbronn, M. and Reichert, A.S. (2013) ApoOL is acardiolipin-binding constituent of the mitofilin/MINOS protein complexdetermining cristae morphology in mammalian mitochondria. PLoS ONE8, e63683

42 Schwanhausser, B., Gossen, M., Dittmar, G. and Selbach, M. (2009)Global analysis of cellular protein translation by pulsed SILAC. Proteomics9, 205–209

43 Schagger, H. and Pfeiffer, K. (2000) Supercomplexes in the respiratorychains of yeast and mammalian mitochondria. EMBO J. 19,1777–1783

44 Morgner, N., Kleinschroth, T., Barth, H.D., Ludwig, B. and Brutschy, B.(2007) A novel approach to analyze membrane proteins by laser massspectrometry: from protein subunits to the integral complex. J. Am. Soc.Mass Spectrom. 18, 1429–1438

45 Meier, T., Morgner, N., Matthies, D., Pogoryelov, D., Keis, S., Cook, G.M.,Dimroth, P. and Brutschy, B. (2007) A tridecameric c ring of theadenosine triphosphate (ATP) synthase from the thermoalkaliphilicBacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemicalproton potential. Mol. Microbiol. 65, 1181–1192

46 Holzmann, J., Pichler, P., Madalinski, M., Kurzbauer, R. and Mechtler, K.(2009) Stoichiometry determination of the MP1–p14 complex using anovel and cost-efficient method to produce an equimolar mixture ofstandard peptides. Anal. Chem. 81, 10254–10261

47 Leitner, A., Walzthoeni, T., Kahraman, A., Herzog, F., Rinner, O., Beck, M.and Aebersold, R. (2010) Probing native protein structures by chemicalcross-linking, mass spectrometry, and bioinformatics. Mol. Cell.Proteomics 9, 1634–1649

48 Castle, J.D. (2003) Purification of organelles from mammalian cells. Curr.Protoc. Immunol. 56, 8.1B.1–8.1B.57

49 O’Fagain, C., Cummins, P.M. and O’Connor, B.F. (2011) Gel-filtrationchromatography. Methods Mol. Biol. 681, 25–33

Received 15 July 2013doi:10.1042/BST20130153


methods to analyse composition and dynamics of macromolecular complexes

Documents