Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS ...

17 Pages·2011·1.02 MB·English

AnalBioanalChem(2011)401:1109–1125
DOI10.1007/s00216-011-5207-9
REVIEW
–
Studying protein protein affinity and immobilized
–
ligand protein affinity interactions using MS-based methods
Jeroen Kool&Niels Jonker&Hubertus Irth&
Wilfried M. A. Niessen
Received:7March2011/Revised:12June2011/Accepted:24June2011/Publishedonline:14July2011
#TheAuthor(s)2011.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract This review discusses the most important current Abbreviations
methodsemployingmassspectrometry(MS)analysisforthe ADAP adhesion and degranulation promoting adapter
study of protein affinity interactions. The methods are protein
discussed in depth with particular reference to MS-based CggR central glycolytic genes repressor
approaches for analyzing protein–protein and protein– ESI electrospray ionization
immobilized ligand interactions, analyzed either directly or FBP fructose 1,6-bisphosphate
indirectly. First, we introduce MS methods for the study of GPCR G-protein-coupled receptor
intact protein complexes in the gas phase. Next, pull-down His histidine
methods for affinity-based analysis of protein–protein and ICAT isotope-coded affinity tags
protein–immobilized ligand interactions are discussed. IMAC immobilized metal affinity chromatography
Presently, this field of research is often called interactomics IMS ion mobility spectrometry
or interaction proteomics. A slightly different approach that iTRAQ isobarictagsforrelativeandabsolutequantitation
willbediscussed,chemicalproteomics,allowsonetoanalyze LC liquid chromatography
selectivityprofilesofligandsformultipledrugtargetsandoff- HSA human serum albumin
targets.Additionally,ofparticularinterestistheuseofsurface MALDI matrix-assisted laser desorption ionization
plasmon resonance technologies coupled with MS for the MS mass spectrometry
study of protein interactions. The review addresses the Nampt nicotinamide phosphoribosyltransferase
principle of each of the methods with a focus on recent NMR nuclear magnetic resonance
developmentsandtheapplicabilitytoleadcompoundgenera- PEBP phosphatidylethanolamine-binding protein
tion in drug discovery as well as the elucidation of protein RXR retinoid X receptor
interactionsinvolvedincellularprocesses.Thereviewfocuses SILAC stable isotope labeling by amino acids in cell
ontheanalysisofbioaffinityinteractionsofproteinswithother culture
proteinsandwithligands,wheretheproteinsareconsideredas SPR surface plasmon resonance
thebioactivesanalyzedbyMS. TAP tandem affinity purification
Keywords Mass spectrometry.Protein–protein
interactions.Chemical proteomics.affinity.Native mass Introduction
spectrometry and interaction proteomics
Thisreviewaddressesmassspectrometry(MS)methodsfor
the study of (intact) protein complexes and so-called
J.Kool(*):N.Jonker:H.Irth:W.M.A.Niessen protein–protein-interaction-based pull-down strategies to
BioMolecularAnalysis,DepartmentofChemistryand elucidate these bioactive interactions. In addition, it
PharmaceuticalSciences,FacultyofSciences, discusses automated digestion steps after affinity purifica-
VUUniversityAmsterdam,
tions and surface plasmon resonance technologies coupled
DeBoelelaan1083,
with MS. The methods described are used mainly in two
1081HVAmsterdam,TheNetherlands
e-mail:[email protected] research areas. Firstly, they are used in fundamental studies
1110 J.Kooletal.
of protein interactions with other proteins and with small substrate, DNA duplex and quadruplex species, intact
molecules [1]. This is an important research area aimed at multimeric proteins, host–guest, oligonucleotide–ligand
gaining a better understanding of biological processes in and protein-ligand complexes, and protein–protein com-
general and more specifically their cellular processes. plexes [5–14]. Even intact virus assemblies are currently
Looking more elaborately at cellular processes, signaling analyzed with native MS approaches [15].
cascades involve many coordinated multiprotein binding The advantage of native MS for structural-biology-
events, production and metabolism of signaling molecules, oriented studies compared with other approaches, such as
modifications of proteins, and binding of small signaling crystallography, protein nuclear magnetic resonance (NMR)
molecules to proteins [2]. These processes facilitate the andisothermaltitrationcalorimetry,isthepossibilitytolook
cellularmachineryneededforhomeostasisand,forexample, directlyatprotein–proteinandprotein–ligandinteractionsin
allow coordinated tissue growth. Furthermore, other solution. This permits scientists to rapidly effectuate
processes such as cellular localization of proteins and changes (e.g., add ligand or protein) to the in vitro system
cellular morphology processes are mediated by protein and thereby study directly the effects on the protein
binding events and are pivotal for cells and the functioning complexes dynamically under real-time conditions. Also,
of the body. Secondly, the study or screening of small MS allows the study of extremely large protein complexes
moleculesthatbindtoproteinscanalsobeusedtoscreenfor and even virus assemblies, which is out of scope for, e.g.,
hits or lead compounds in drug discovery. With the crystallography and protein NMR. Furthermore, for MS
emergence of protein–protein interactions and biopharma- approaches in the native MS area, only low amounts of
ceuticals in drug discovery and development, screening and proteins are needed as they are commonly introduced via a
studying these interactions are becoming increasingly nano-ESI source. With sufficient analytical resolution, the
important in this research area as well [3]. successful analysis of picomolar amounts of large heterog-
The different distinct MS approaches which are enous protein complexes becomes reality. In addition, it
discussed in this review are as follows. First, the study of allows real-time addition of cofactors, substrates, and
noncovalentcomplexesdirectlyfromsolutionbyMS(native ligands while monitoring the resulting changes to the
MS). The second topic is the study of cellular protein complexes. This contrasts with the limited possibilities of
complexes involved in signaling events by pull-down other methods used for analyzing the stoichiometry of
(“fishing”)-based interaction proteomics. Third, chemical protein complexes, such as crystallography and protein
proteomicsislooked at; thisallows one toscreenselectivity NMR. To obtain sufficient MS signals, however, high
profiles of ligands for multiple drug targets and off-targets. protein concentrations (e.g., generally low micromolar
Finally, surface plasmon resonance (SPR) coupled with MS concentration range, while only consuming low volumes
forproteinaffinityanalysis,quantification,andidentification introduced via nano-ESI capillaries) are required because
is discussed. Many reviews have been published about the the overall MS signal is divided over isotopic patterns and
fourdistinctapproachesthatarediscussedhere.Thisreview, differently charged protein complexes.
however, provides an overview of the different MS-based The study of noncovalent complexes directly by MS
approaches with recent examplesand focuses on the protein relies on extensive optimization to obtain sufficiently
binding aspects and methods behind them in relation to stabile protein–protein or protein–ligand complexes in
biological binding events and less on the MS technologies. solution and in the gas phase. Furthermore, for the study
Foreverymethod,recentapplicationsandspecificexamples ofprotein–ligandinteractionsitismandatorytodistinguish
ofstudiesperformedarebrieflydiscussed. the protein–ligand complex from the unbound protein
without a separation step. Presently, progress in the
Electrospray ionization (ESI) and matrix-assisted laser development of MS and its application to protein analysis
desorption ionization (MALDI) are the most suitable in the gas-phase have led to major improvements in this
methods for generating gas-phase ions of large biomole- field.Also,thesemethodsprovidethemostdirectevidence
cules.In thecase ofESI, many different intact noncovalent of protein–ligand and protein–protein complexation and
protein complexes can be studied in the gas phase under can be used as a model for in vivo complexation, with
certain conditions. These studies are typically called native the caveat that gas-phase complexation in the mass
MS studies. They also allow one to study protein–ligand spectrometer is a good representation of in vivo binding,
complexes(receptors aswellasenzymes).Ganemetal.[4] and is not an analytical artifact caused by the analysis
andKattaandChait[5]wereamongthefirsttouseESI-MS [16]. Contrary to ESI, MALDI techniques are less suited
to study noncovalent receptor–ligand complexes and for the study of noncovalent protein–protein and protein–
biological myoglobin interactions. For the analysis of ligandinteractionsastheseinteractionsareoftendisrupted
noncovalentcomplexesbyMS,agreatvarietyofbiological under the conditions needed and the procedures followed
interactions have been studied: receptor–ligand, enzyme– to produce the MALDI matrix.
StudyingproteinaffinitywithMS 1111
A distinctly different way of analyzing protein–protein become much more difficult to interpret, which is not an
interactionsisperformedindirectlyafterso-calledpull-down issue with native MS. Currently, among the largest protein
assays. For this, the protein (or ligand) of interest is structures resolved by protein NMR are the 300-kDa
immobilizedtocreateanaffinitycolumninamannersimilar aspartate transcarbamoylase [24] and the 670-kDa protea-
to that for affinity chromatography approaches. After all some [23] obtained by the group of Kay. With native MS,
boundproteinshavebeentrappedandsubsequentlyreleased, among the currently largest protein complexes (over 10
MSanalysisisoftenperformedaftera1Dgelelectrophoresis MDa) studied is the Norwalk virus assembly [15].
separation and a (tryptic) digestion of the separated proteins All technologies used to study protein complexes have
[17]. Instead of a normal affinity-chromatography-based their own intrinsic advantages and disadvantages. In this
protein complex purification, immunoprecipitation [18] or regard, MS allows the dynamic real-life study of protein
tandem affinity purification [19] can be used. The protein- complexes, the study of very large complexes, and the
“fishing”-based MS approaches discussed in this review are stoichiometric determination of the complexes analyzed,
not suited for the dynamic study of protein binding events, and importantly only requires small amounts of protein. In
but rather allow the identification of large multiprotein contrast, high-resolution 3D protein structures cannot be
complexes involving many different proteins. determined by MS techniques.
Finally, SPR has been coupled with MS to study protein AdvancesinMS,however,doprovidenewopportunities
binding events on an SPR chip directly followed by MS regardingtheanalysisofthesecomplexes.Onecanthinkof
identification of the bound proteins. This approach allows advanced nano-ESI sources, new ionization techniques
protein quantification combined with structural character- such as ambient temperature ionization, and implementa-
ization/identification of the proteins. Consequently, MS tion of ion mobility spectrometry (IMS) technologies, but
complements the SPR detection and may reveal structural also new and adapted MS configurations and hardware
modifications not detected by SPR [20]. [25]. Still, the buffers used for biochemical studies that
mimic physiological conditions often contain phosphates
The study of noncovalent complexes by native MS and other nonvolatile salts [e.g., phosphate-buffered saline
or tris(hydroxymethyl)aminomethane buffer with NaCl]
Analysis of noncovalent complexes by MS, also known as and cannot be used in combination with ESI-MS because
native MS, requires ESI-compatible buffer solutions. This theyarenonvolatile. Also, theuseofalowpHfor efficient
implies that in a number of cases maximum sensitivity is ESI-MS in positive ionization mode is not an option when
notachievedand/ornonphysiologicalconditionshavetobe studying biological noncovalent complexes, nor is the
used. Although noncovalent complexes observed by MS commonly used high percentage of organic modifier.
are not analyzed directly from real cellular systems, most Instead, physiological buffer conditions have to be substi-
often the stoichiometry of complexes determined by native tuted with MS-compatible buffers, such as ammonium
MSmatchesthatdeterminedinotherways,suchaselectron formate, acetate and bicarbonate. Furthermore, the percent-
microscopy, X-ray crystallography, and NMR. There are, age of methanol, acetonitrile, or 2-propanol has to be low
however, a few recognized exceptions [21]. (usually lower than 5%) to prevent dissociation or
In general, native MS is a very powerful technique for denaturation of the noncovalent complexes to be studied.
the study of protein complexes, complementary to more Finally, nonvolatile additives such as detergents and block-
traditional approaches. Other approaches such as crystal- ing reagents may cause ionization suppression, and their
lography and NMR have different analytical capabilities. use should be avoided or they should only be used in very
Whereas crystallography allows a detailed 3D image of a low concentrations. Another factor to take into account is
protein–ligand complex to be obtained, the analysis of that the complexes that are formed and studied in native
multiprotein complexes, large protein complexes, and MS depend on both protein–protein and protein–ligand
several types of protein classes in general is difficult or affinities and their concentrations. For most proteins (and
the protein complexes may be impossible to crystallize. ligands), much higher concentrations have to be used than
Furthermore, crystallography only permits the analysis of a those present under physiological conditions. Therefore,
static crystal and real-life dynamic analysis in vitro is one must be aware of the physiological relevance of the
therefore not possible. Protein NMR, on the other hand, is complexes studied as in the body the concentrations of
anemergingtechnologybutthestudyoflargeandcomplex proteins studied are much lower. This implies that low-
protein structures, such as whole virus assemblies, is still affinity protein complexes may be seen under the artificial
not feasible. For protein NMR, protein sizes in the 0.1–1- conditions with high protein concentrations in the mass
MDa range have beenstudied[22,23]. With NMR studies, spectrometer, but might have less relevance in the body
homomericcomplexesareanalyzedmoreconvenientlythan (or only under specific conditions) when they are not
heteromeric protein complexes as the NMR spectra then formed or are only formed at very low percentages.
1112 J.Kooletal.
A way to study these possible effects might be by
analyzing the protein complexes in different ratios and
concentrations and by omitting specific binding partners.
Although analysis of the protein complexes in lower
concentrations will give a worse signal-to-noise ratio,
observedchangesintheratiosofthecomplexesseenmight
give indications of the affinities of the different binding
partnersinthesecomplexes.Also,chemicalcross-linkingat
lower protein concentrations followed by analysis of the
complexes under denaturing conditions can by utilized to
verify if the complexes are relevant at low concentration.
In-solution dissociation experiments can also give valuable
additional information about the binding interactions of the
interaction partners. Nonspecific oligomerization, for
example, can be distinguished from specific interactions
by looking at the distribution of the molecules, which is
related to the initial concentration and droplet sizes in the
ESIsource[26].Alltheseconsiderationsdictatethebalance
required between efficient and representative analyses. Fig.1 Interactionofthenuclearhormonereceptordimerretinoicacid
ESI-MS can be seen as a complementary tool to receptor(RAR)−retinoidXreceptor(RXR)bindingtoashortstrand
established biochemical methods for investigating protein of DNA (induced by retinoic acid binding). All RXR heterodimers
preferentially bind DNA at two sites of a direct repeat (DR)
structure and conformation under nondenaturating condi-
configuration, separated by one to five nucleic acids, called DR1,
tions.TypesofinformationthatcanbeobtainedbyESI-MS DR2, DR3, DR4, and DR5. The binding to a DR5 configuration is
include protein conformation properties and molecular shown, resembling binding to hormone response elements (HREs).
interactions, protein–protein interactions, protein–ligand UponbindingtoactualHREs,genetranscriptioncanoccur.(Reprinted
fromBichetal.[31].Withpermission)
interactions,andprotein–cofactorinteractions.Sometypical
examples are now briefly discussed.
Noncovalentinteractionsbetweenlowmolecularweight theanalysisofcomplexesinvolvedinthechaperonin-assisted
antiamyloid agents and amyloid β peptides were studied refoldingofthemajorcapsidprotein(gp23)ofbacteriophage
byMartineauetal.[27]torankbindersthatmaybeableto T4. Intermediate complexes that are involved in chaperonin
modulate/inhibit the amyloid β aggregation process. (GroEL-GroES) folding were studied as such. It was found
Jecklin et al. [28] compared different approaches, ESI- thatchaperonincomplexescanbinduptotwounfoldedgp23
MS, SPR, and isothermal titration calorimetry, for label- proteins.Whenincomplexwiththecochaperoningp31,only
free quantitative assessment of binding strengths of the one gp23 can bind. Figure 2 shows typical results obtained
protein human carbonic anhydrase I with small ligands. forthisstudy.Ionswithdifferentchargestatescorresponding
Real-time monitoring of enzymatic conversions and inhi- to the 801-kDa complex (GroEL; blue dots), the 857-kDa
bition and formation of complexes is also possible with complex(onegp23moleculeboundtoGroEL;yellowdots),
MS [29]. Hydrogen–deuterium exchange experiments can and the 913-kDa complex (two gp23 molecules bound to
be used to study proteins by MS and allow one to monitor GroEL; red dots) are seen in Fig. 2a. Figure 2b shows the
protein dynamics and binding interactions over time [30]. deconvoluted spectrum of the three different complexes.
Bich et al. [31] applied MS to study the retinoic acid These results nicely illustrate the capabilities of native MS
induced heterodimerization of the nuclear retinoid X for the study of protein–protein interactions.
receptor (RXR), resulting in formation of an activated Following the advent of native MS, very large protein
dimer that binds to DNA hormone response elements, complexes such as ribosomes and even whole viruses can
mimickedbyDNA-baseddirectrepeatconfigurations.This now be studied in the gas phase [32–34]. With the recent
is exemplified in Fig. 1. The dots in the MS spectrum addition of ion mobility to MS analysis, new doors have
represent the m/z values of the differentially charged ions been opened for the study of such large complexes [35]. In
corresponding to the complex of the RXR–retinoic acid IMS, biomolecules and noncovalent complexes are sepa-
receptor dimer bound to the double-helix DNA fragment rated inthegasphase according totheirdifferencesinsize,
DR5.The dotson top ofthepeaksrepresentthesame ions shape, and charge prior to actual MS analysis. For IMS,
withdifferentchargestates.Aftercross-linking,theauthors new possibilities lie, for example, in the analysis of
also successfully studied these complexes with high-mass heterogeneous protein complexes, providing information
MALDI-MS.VanDuijnetal.[12]haveusednativeMSfor on the topology, stoichiometry, and cross section. This new
StudyingproteinaffinitywithMS 1113
Fig.2 Typicalnativemassspectrometry(MS)resultsfromvanDuijn (912 kDa; magenta circles) are seen. b The corresponding deconvo-
etal.[12]aNano-electrosprayionizationmassspectrumofamixture luted spectrum, which reveals the three chaperonin complexes with
ofGroELandunfoldedpolypeptidegp23(1:4).Charge-stateseriesof their binding stoichiometries. (Reprinted from van Duijn et al. [12].
GroEL (800 kDa; blue circles), one gp23 molecule bound to GroEL Withpermission)
(856 kDa; yellow circles) and two gp23 molecules bound to GroEL
addition to the available MS tools does, however, require quaternary structures of protein complexes [43]. The central
additionalandextensivedatahandlingtohavefeasibledata glycolyticgenesrepressor(CggR)playsaroleinglycolysisin
interpretation [36]. Research has already shown that results Bacillussubtilis.Theeffectorsugarfructose1,6-bisphosphate
obtained by IMS-MS for noncovalent complexes show (FBP) abolishes binding cooperativity of CggR and DNA.
good correlation with results obtained by traditionally Atmanene et al. [44] used native MS to investigate FBP-
applied methods, such as cryoelectron microscopy and dependent CggR–DNA interactions using automated chip-
X-raycrystallography[37].However,thereisalsoevidence based nano-ESI MS and traveling wave IMS-MS. Among
that proteins and protein complexes may become more others findings, it was revealed that tetrameric CggR
compactorcollapseinthegasphaseintheabsenceofwater dissociates into dimers upon FBP binding. In a more
[38]. Besides the technologies mentioned, electron micros- recent study, the assembly states of the nucleosome
copy is an alternative method to MS to look at protein assembly protein 1 were studied by sedimentation velocity
complexes, and also protein complexes consisting of many andnativeMS[45].Fromthisitwasconcludedthatthebasic
different proteins, provided that the complexes are very assembly was a dimer from which even-numbered higher-
large [39]. With crystallography approaches, often ligand assembly states formed. Phosphatidylethanolamine-binding
binding to a receptor or enzyme is studied. protein (PEBP) can be associated with morphine and
A selected set of very recent typical examples of studies morphine glucoronides. In a native MS study, Atmanene
involvingnativeMSarenowdiscussed.BycombiningIMS- et al. [46] characterized these interactions and finally
MS with tandem MS, one can characterize non-covalently suggested that PEBP might protect morphine 6-glucuronide
bound macromolecular complexes (mass, cross-sectional following its secretion into blood, which leads to a longer
area, and stability) with only one experiment, which was half-life. The ribosomal stalk complex has a role in the
demonstrated by Knapman et al. [40] by determining the delivery of translation factors to the ribosome. The stoichi-
topology of virus assembly intermediates. Boeri-Erba et al. ometryofthesecomplexesisimportanttofurtherunderstand
[41]usedIMS-MStostudytheinfluenceofsubunitpacking their functioning, which was investigated by Gordiyenko
andthechargeonthedissociationofmultiproteincomplexes et al. [47].
ofheatshock protein 16.9and stable protein 1.Also, native
MS can be used to identify protein aggregates after (size- Thestudyofcellularproteincomplexesinvolvedinsignaling
exclusion) chromatography, which was demonstrated for eventswithpull-down-basedinteractionproteomics
humanmonoclonalantibodyaggregates[42].Recentadvances
in native MS, including specifically applied surface-induced For many drug target systems, genomics approaches, e.g.,
dissociation approaches, allow one to get a closer look at by RNA array analysis of gene expression, have revealed
1114 J.Kooletal.
that receptor stimulation results in numerous pathway (spin) affinity column or (magnetic) beads. When the key
regulations. More recentadvances inproteomicsalsoallow interactor is incubated with the lysate, complexes are
not only the unraveling of the complex protein regulations formed with the key interactor under the conditions used
mediated by ligand signaling, but moreover give insights for the study, thereby selectively extracting or “fishing” the
into protein phosphorylation processes that precede this. interacting proteins from the complex mixture. Figure 3
These processes are efficiently studied with, e.g., phospho- gives an overview of a typical interaction proteomics
proteomics approaches based on phosphopeptide purifica- workflow. The exemplary procedure depicted starts with
tion with affinity chromatography [with, e.g., immobilized an affinity purification step (2a and 2b) of the sample (1a)
metal affinity chromatography (IMAC) or TiO ] performed and the control (1b). After the affinity purification, the
2
after proteolytic digestion of all proteins. The affinity- proteins bound areseparated ona gel (3aand 3b). The two
purified phosphopeptides are then separated by liquid experiments can be performed in one experiment if, e.g., a
chromatography (LC) and analyzed by MS [48]. stable isotope labeling with amino acids in cell culture
We are starting to understand that ligand-mediated (SILAC) is used. Subsequently, protein bands are excised
signaling is not a one-directional linear process, but rather anddigestedin-gel.Then,MS-basedanalysisoccurs.Inthe
a parallel process with different dependent, independent, exampleshown,nano-LC-MSisusedforpeptideseparation
cross-linking, correlating, and influencing pathways and and analysis. This approach is essentially based on the
eventual effects [49–51]. Protein kinases play pivotal roles reversed principle of affinity-selection MS approaches
in transmitting ligand-mediated signals through many discussed by Jonker et al. [58]: Rather than immobilizing
different pathways. Before eventual gene expression in the target proteins to retain ligands, one immobilizes the
cells takes place, phosphorylation and dephosphorylation ligands to retain the target proteins. Subsequent washing
steps of subsequent protein kinases and other proteins away of all nonbinding entities (e.g., proteins, membranes,
occur, thereby activating and/or deactivating them, in order and small molecules) followed by release of all binding
to pass on signaling events. Also, protein localization proteins (by, e.g., heat shock, pH shock, chaotropic agents,
processes, e.g., based on phosphorylation state, are impor- organic modifiers, ligand-based displacement, or tryptic
tant mediators in (localized) cellular processes. Finally, key digestion when a bottom-up approach is used) allows the
proteins in different pathways are upregulated or down- proteomicanalysisoftheinteractome. Here itismandatory
regulated and dictate the final cellular (desired or unde- that the cell lysate used is carefully prepared in order to
sired) effects. Through all these signaling cascades, protein maintainthecellularconditionsintermsoftheinteractome/
complexes play crucial roles. In other words, the way that noncovalent complexes under study. Cellular conditions or
proteins interact with each other, form noncovalent com- cell-mimicking conditions are easily disturbed chemically
plexes,andlocalize,internalize,andrecruitotherproteinsis bybufferconditions/lysisconditions,aswellasbiologically
fundamentaltocellularsignaling[49,52,53].Sometypical andarecell-mediatedduringtheinitiationof,e.g.,celllysis.
examplesincludeG-protein-(in)dependentand/orβ-arrestin Furthermore, often cellular conditions are unknown (and
signaling for G-protein-coupled receptors (GPCRs), coac- cannot be specified for different cell compartments) and
tivator and/or repressor protein recruitment for the nuclear thus it is difficult to mimic them. The use of homogenized
receptors, and localization of certain proteins (e.g., protein tissues or even whole organs can be problematic. First, to
kinasesandGPCRs)bybinding/complexationto/with,e.g., investigate cellular pathways it is important to know the
anchoring proteins [50, 51, 54–57]. These important correspondingtypeofcelltowhichtheproteinsbelong.An
processes in signaling are difficult to study with traditional association of different cell types should only be used to
biological/biochemical approaches as they comprise a give the first hints. Furthermore, if different cell types are
complex interplay of many different events. One relatively used in one batch, uncontrolled reactions may be initiated.
new way of studying these complexes in a more compre- It is difficult to control changes occurring when only one
hensive manner is by interactome proteomics. type of cell is homogenized because cell compartments are
In interactome proteomics, an affinity purification of the destroyed.Forexample,disruptingthevacuolemightcause
protein complexes to be studied precedes the actual significant artefacts in the study of plant cells.
analysis. The affinity purification uses the key interactor Alternatively, immunoprecipitation can be performed
to be studied for its interactome in a relevant biological instead of interactome fishing with immobilized key
surrounding. Here, the key interactor can be a ligand, an interactors. An advantage of immunoprecipitation is the
inhibitor, a protein, DNA, RNA, or another biomolecule, possibilityofin-solutionincubation,whichcanavoidsteric
whereas the biological surroundings are often lysates of hindranceof(co-)binderstothecomplexesformedwiththe
(cultured) cells, but also tissues, subcellular compartments, key interactor when it is not immobilized (the indirect
organs, and insects have been studied this way. The key approach). A limitation is that the method depends on the
interactor is immobilized onto a solid support, such as a specificityoftheantibodies used,thechoiceoftheantigen,
StudyingproteinaffinitywithMS 1115
Fig. 3 Pull-down proteomics,
interactomics,orinteraction
proteomics.Thetypical
workflowisillustrated.The
sampletobeanalyzed(1a)and
oftenacontrolsample(1b)are
subjectedtoaffinitychromatog-
raphy(2aand2b).Alternatively,
immunoprecipitationcanalsobe
used(notshown).After
trapping,allboundproteinsare
eluted(by,e.g.,disruptionor
sequentialelutionwithaffinity
displacers)and(often)subjected
togelelectrophoresis(3aand
3b).Sampleandcontrolproteins
arecomparedandall(oronly
interesting)proteinsareexcised
fromthegelslab,andarein-gel
digested(ingelbands)(4).The
resultingpeptidescanthenbe
separatedby(nano)-liquid
chromatography(LC)(6)and
detectedbyMS(7).Alterna-
tively,matrix-assistedlaser
desorptionionizationMSmay
alsobeused.Theresultingtotal
ioncurrentofachromatogram
(8),theMSspectrumofa
peptide(9),theMS/MS
spectrumofthefragmented
peptide(10),andadatabase
search(11)areshown
and the accessibility of the antibodies for binding the and 13C to achieve isobars) [61], isotope-coded affinity
antigens when the complexes are formed in solution. tags (ICAT) [62], labeling with 18O [63], and [2H ]
6
Withinteractomeproteomics,acontrolexperimentisoften dimethyl labeling [64]. These labeling approaches have to
comparedwiththeactualexperimentinwhichthebiological be performed after the interactome fishing process, that is,
matrix under study is stimulated by a certain chemical, after or during a digestion step of the isolated proteins, in
biological, or physical stimulus. For this approach, both contrast to SILAC. The actual proteomics experiment can
biologicalmatrixescanbeisotopicallylabeled.Althoughthis be performed in a bottom-up approach in which first all
isanelegantwayofincorporatingthecontrolexperimentand proteins are digested, followed by LC-MS analysis and
stimulated experiment in one analysis with a labeling database searching [48]. The common approach involves
approach, controls can be performed without isotopic protein separation by 1D sodium dodecyl sulfate–
labeling.The advantage of labeling is that both the control polyacrylamide gel electrophoresis. Here, gel bands are
andthestimulatedexperimentscanbepooledandanalyzed subsequently excised, in-gel digested, and the peptides
in one simultaneous proteomics experiment where the formed are then analyzed by LC-MS (or potentially
peptide ratios used to calculate protein ratios can be MALDI-MS). An advantage of this approach is the
distinguished in MS owing to the differences in molecular possibility for additional Western blotting for confirmation
masses between the differently labeled peptides. Labeling of the identity of specific proteins. Also the molecular
can be achieved in situ during cell growth via SILAC for mass of the excised proteins in denaturating gels can be
proteomic comparison of the stimulus’ effect later on [59, estimated, thus yielding additional confirmation. A disad-
60]. Other applicable labeling approaches can be per- vantage is that this method is quite labor-intensive.
formed, such as isobaric tags for relative and absolute An example of a typical pull-down proteomics study
quantitation (iTRAQ; based on a combination of 18O, 15N, involvestheuseofanimmobilizedinhibitorforphosphodi-
1116 J.Kooletal.
esterase 5 to study its interactome [65]. To gain specificity, TAP tag can be broken enzymatically for further process-
selective precleaning and elution protocols were developed ing and eventual MS analysis. One manner of efficient
forefficientdiscriminationbetweenspecificandnonspecific quantificationofproteinsafteraffinitytrappingprocedures
or less-specific binding proteins. A similar approach was is the recently developed quantitative bacterial artificial
used with cyclic AMP affinity column materials to study a chromosomes interactomics [72]. In this approach, tagged
specific protein kinase anchoring protein for type I cyclic full-length baits are employed which are expressed under
AMPdependency[66].Thisproteinplaysimportantrolesin endogenous control. Different cell lines with tagged
localization processes for specific kinases during complex proteins are available for this approach. Actual quantifi-
interplays of signaling events. For studying protein–protein cationoccursbySILAC,butitcanalsobeperformedbya
complexes, a method was developed in which the “bait label-free approach.
protein” was constructed as a glutathione S-transferase The α nicotinic receptor, which is an important
7
fusion protein for interactome pull-down chromatography potentialdrugtargetagainstseveralbrain-residingdiseases,
withglutathionebeads.Thesepulldownscanbeenvisioned has also been studied indirectly with an interactomics
as the protein-based version of yeast two-hybrid screens approach. Bungarotoxin, which has a high affinity for this
[67]. Prior to eventual MS analysis and data handling for nicotinic receptor, was used as a key binding partner. For
protein identification, different sample preparations of this, isolated carbachol-sensitive α-bungarotoxin-binding
interactingproteins(e.g.,specializedgelstainingtechniques complexes from total mouse brain tissue were selectively
and in-gel tryptic digestions) were evaluated and used. An eluted and analyzed [73]. By comparison of results
example of immunoprecipitation pull downs is given for obtained from wild-type mice and from α nicotinic
7
GTPcyclohydrolaseI,whichisanimportantenzymeinthe receptor knockout mice, binding proteins were identified
biosynthesisoftetrahydrobiopterin,anessentialcofactorfor from the brain tissues used.
aromatic amino acid hydroxylase and nitric oxide synthase The importance of interactomics studies in life science
[68].Itwasfoundthat29proteinsfromdifferentsubcellular today is reflected by the many different studies performed
components interacted with GTP cyclohydrolase I. In an to investigate the binding partners of specifically selected
example where affinity protein columns were manufactured interaction proteins. Other recent examples include an
for the pull down, proteins targeted by the thioredoxin interactomics study towards the most widely expressed
superfamily in Plasmodium falciparum were identified, isoforms of p63, a transcription factor for the p53 tumor
yielding 21 potential target proteins [69]. Another example suppression protein [74]. Relevant binding partners of
targeted the phosphatidylinositol 3,4,5-trisphosphate inter- helicases, which are important in the unwinding of the
actome [70], important in regulations of cell physiological strands of DNA double helixes, have been studied by
processes,e.g., viaGPCR-mediated signaling. In this study, Jessulat et al. [75] by a TAP approach in an in vivo study.
282 proteins were found to directly or indirectly interact Another TAP approach, by Guo et al. [76], centered on the
with phosphatidylinositol 3,4,5-trisphosphate. identification of human tuberous sclerosis protein 1 com-
The family of 14-3-3 proteins are regulatory proteins plexes. In the case of binding partners of the estrogen
conserved across species with the ability to bind many receptor as a key mediator in certain breast cancer cells,
differentproteinsinvolvedinsignaling,suchaskinasesand knowledge of binding partners of the ligand-activated
membrane receptors. The importance of these proteins in receptor is important for a better understanding of trans-
signaling processes renders them excellent candidates for duction of the hormonal signal that allows the cancer cells
interactomics studies helping to unravel their exact binding to grow. These binding partners were revealed in an
partners. This is true not only for mammalian 14-3-3 interaction proteomics study using TAP by Tarallo et al.
proteins, but also for plant proteins. Paul et al. [71] studied [77].Integrinsaretransmembraneproteinsthatareinvolved
Arabidopsis 14-3-3 complexes, which revealed highly in regulation of cellular mobility, shape, and cell cycle
conserved interactions between humansand plants. Among processes. To look closely at associated proteins that might
other important proteins, also in plants, are the protein be involved in these processes, Raab et al. [78] looked at
kinases. One study focused on transgenic rice plants to theinteractomeoftheplateletintegrinα regulatorymotif.
IIb
identifybinderstorice-leaf-expressedproteinkinasesfused The use of tethered RNAs to detect RNA–protein inter-
to tandem affinity purification (TAP) tags [19]. TAPs are actions was described by Lioka et al. [79], revealing
two-step affinity purification protocols which allow isola- specificproteinbinders.Thenuclearlaminaisamongother
tion of protein complexes under close-to-physiological factors an important regulator of the structural integrity of
conditions with the help of fusion proteins. These fusion the nucleus. It is involved in nuclear processes, including
proteins can have a “bait” part, an enzymatic cleavage DNA replication. Unraveling of binding partners of the
part, and a trapping part, for example, protein Awhich lamina can allow scientists to further understand the
binds to immobilized IgG. After initial purification, the processes behind the regulations involved. For this, protein
StudyingproteinaffinitywithMS 1117
interactors with lamin A and progerin were studied by 1b, 1c, and 1d), each incubated with a different stimulus
Kubben et al. [80]. The immune adapter protein adhesion (differentligandconcentrationsinthis case).Aftercelllysis
and degranulation promoting adapter protein (ADAP) is and sample preparation, the lysed cells are incubated with
involved in integrin-dependent migration and adhesion affinity beads. The proteins bound for each incubation
processes after T-cell stimulation. To investigate and (2a-d) are subsequently isolated by washing the beads (3 to
differentiate between phosphorylation-specific and nonspe- 4) followed by elution with help of, e.g., a disruption step
cificproteininteractions,Langeetal.[81]usedSILACand (5a to 5b). Different approaches for protein separation can
enzymatic 18O-labeling to identify ADAP interaction be used prior to analysis (10a to 10b, or potentially 11a to
partners. Jäger et al. [82] described an affinity purification 11b). iTRAQ labeling is performed, allowing the different
method to characterize HIV protein complexes. The experiments (1a, 1b, 1c, and 1d) to be combined after
interaction partners of dysferlin, an important protein digestion and labeling. With this approach, the samples can
involved in muscle membrane repair, were recently also thenbepooledpriortoLC-MSanalysis(9).Thetechnology
studied [83]. It was shown that dysferlin is not only usesaffinitybeadswithimmobilizedligandtofishouttarget
involved in membrane repair, but that it is also important proteins for MS-based analysis. By addition of solution-
for maintenance and integrity of muscle membranes. For phase test ligands of pharmaceutical interest (which bind
proteins that interact with muscarinic receptor, Borroto- with different affinities to the target proteins), the target
Escuela et al. [84] revealed many protein interactions in proteins, when bound to the test ligands, do not bind
various signaling pathways that will allow a better anymore or bind in a lower percentage to the beads,
understanding of the muscarinic interactome. depending on their intrinsic test ligand affinities. For
An alternative, attractive approach uses protein trapping specific interactions, this results in a reduced amount of
with reactive chemical affinity tags which efficiently traps target protein extracted and consequently lower amounts
proteins for MS-based analysis. Fischer et al. [85] and Luo quantified per target protein by MS. Using this approach,
et al. [86] used this so-called capture compound MS proteins complexing with the target proteins bound are not
approach which involves binding of a small reactive looked at, but can theoretically be looked at if desired.
molecule(e.g.,adruglikecompound)tointeractingproteins Awell-known example uses affinity beads (Kinobeads)
followed by covalent reaction (e.g., after photoactivation) that are able to trap most, if not all, protein kinases via
with the binding proteins. An incorporated biotin function their binding pocket(s) [92]. The approach uses immobi-
then allows selective purification for MS analysis. In a lized broad-selectivity kinase inhibitors that bind protein
typical example, the broad-range and high-affinity protein kinases (and related proteins) mainly at their ATP binding
kinase binder staurosporine was used to trap and study sites and related sites. In the presence of increasing
protein kinases in the hepatocyte cell line HepG2 [87]. To concentrations of a ligand, the ligand and the affinity
study chemical cross-linking of covalently connected material compete for a binding site on the protein kinases
bindingpartnersinordertoallowidentificationofinteracting present in cellular lysates. This means that at low ligand
proteinsdirectlyincells,Sinz[88]discusseddifferentinvivo concentrations, only the high-affinity-binding kinases are
cross-linking strategies that allow protein–protein interac- not trapped on the affinity material anymore as they are
tions to be looked at under physiological conditions. Other bound to the ligand, whereas at higher ligand concen-
capture compound MS approaches include the profiling of trations, also the lower-affinity kinases are unable to bind
methyltransferases and S-adenosyl-L-homocysteine-binding to the affinity material anymore. Experiments with different
proteins[89]andtheuseofageneticallyincorporatedphoto- ligandconcentrationsaredoneandafterpulldownsfollowed
cross-linkable amino acid to study protein complexes of by proteomics analysis, decreasing amounts of affinity-
protein 2 bound to mammalian growth factor receptor [90]. material-trapped kinases are detected with increasing ligand
concentrations. This is used to construct typical IC dose–
50
Chemical proteomics to screen selectivity profiles response curves for all kinases studied (up to hundreds at
of ligands for multiple drug targets and off-targets once). There are 518 human protein kinases, and all protein
kinases that bind can theoretically be detected as can other
Affinitybeadsorcolumnscanbeusedinamorepharmaceu- proteins that interact with the affinity material. Of course,
ticallyorientedfashionthaninteractomeproteomics.Here,an their individual cellular concentrations and activation states
additionalstepisincludedinvolvingtheadditionofdifferent inspecificcelltypesmightpreventbindingand/ordetection.
concentrations of a ligand (e.g., lead compound) to cell Forthisspecificprocess,medicallyinterestingtargetcellsare
lysates prior to processing. This technology is the so-called used where the endogenous protein kinases are the target
chemical proteomics approach [91]. Figure 4 gives an proteins.Afterlysis,theligandisadded,followedbyaffinity
overview of a typical chemical proteomics approach. In trapping of the protein kinases, washing steps, release and
the exemplary figure, Petri dishes with cells are shown (1a, digestionoftheproteinkinases,labeling,andfinallyLC-MS.
1118 J.Kooletal.
Fig.4 Chemicalproteomics.
Thetypicalworkflowisshown.
Thesamplewithdifferentcon-
centrationsofthetestligand
(fourconcentrations;1a,1b,1c,
and1d)aresubjectedtolysates
ofculturedcellsofinterestand
incubatedwithimmobilized
ligandaffinitybeads(2a-d).
Afterincubation(3),theaffinity
beadswithboundtargetproteins
areremovedfromtheincubation
(4),washed,resuspended(5a),
andfinallyadisruptionstep(5b)
releasestheboundtarget
proteinsfromthebeads.The
crudeproteinmixturesarethen
preparedfor1D(10a)orpoten-
tially2D(11a)gelelectrophore-
sisfollowedbyexcisionofthe
proteins(10b,11b).Theproteins
canthenbein-geldigestedprior
toanalysis.Alternatively,
proteinscanbedirectlyprepared
fordigestion(6),digested(7and
8)andsubsequentlylabeled
with,e.g.,isobarictagsfor
relativeandabsolutequantita-
tion(iTRAQ).AfteriTRAQ
labeling,samplesarecombined
forstraightforwardeventual
relativeproteinquantification
fromMS/MSspectra(see
Fig.5)obtainedbyLC-MS/MS
analysis(9)
An iTRAQ labeling reagent is commonly used for this [92, Togiveoneexample,aquantitativechemicalproteomics
93]. The method also allows for measurement of ligand- approach was used to study the effects of small molecule
induced changes in phosphorylation states of the isolated ABL kinase inhibitor drugs on hundreds of endogenously
proteinkinases.Themainadvantageofthismethodisthatit expressed protein kinases and purine-binding proteins [92].
is capable of analyzing inhibitory panel profiles of protein Furthermore, drug-induced changes in the captured pro-
targets instead of aiming at a single drug target. This teome’s phosphorylation state were also looked at. Typical
possibility allows drug discovery projects to start aiming at results obtained are shown in Fig. 5. Figure 5a shows
drugscapableofselectivelyinhibitingseveraldrugtargetsin cultured cells with different chemical stimuli (ligands; in
a panel fashion. One challenge in protein kinase affinity this case drugs) at the top, and below this the schematic
screening is that the most interesting selective inhibitors for processofbindingofproteinkinasestotheaffinitybeadsin
protein kinases are expected to bind allosterically (at non- the presence of different concentrations of ligand is
ATP binding sites) and consequently might not be detected. depicted.TheMS/MSspectrumatthebottomleftofFig.5a
These methods, however, are also starting to aim more shows the four characteristic iTRAQ reporter signals
specifically at multiple binding sites. indicative of the relative amount of protein kinase trapped