Decoding Signalling Networks by Mass Spectrometry-based Proteomics

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F O C U S O N S I G N A L I N T E G E V I E WN R R AT I O S Decoding signalling networks by mass spectrometry-based proteomics Chunaram Choudhary* and Matthias Mann*‡ Abstract | Signalling networks regulate essentially all of the biology of cells and organisms in normal and disease states. Signalling is often studied using antibody-based techniques such as western blots. Large-scale ‘precision proteomics’ based on mass spectrometry now enables the system-wide characterization of signalling eve
  Cells are information processing devices that integratemyriad extracellular and intracellular signals to producean optimal output. Many diseases, for example cancer,can be thought of as pathological alterations in signal-ling networks. Consequently, studying the nature andmechanisms of signalling events is a large and crucialpart of biological and medical research. Extracellularligands can activate plasma membrane-bound receptors,which transmit the signal through a phosphorylationcascade into the cell nucleus, ultimately leading tochanges in the expression of specific genes. Signallingevents also involve intricate networks, which encom-pass feedback loops, crosstalk with other signals andthe integration of information relating to the internalstate of the cell. FIGURE 1 illustrates the principles of signalling net-works using receptor Tyr kinases (RTKs) and tumournecrosis factor- α (TNF α ) signalling as examples. In thecase of RTKs, ligand binding typically induces dimer-ization of the receptors, leading to trans -phosphorylationof their intracellular region and activation of their kinasedomains. These phosphorylation events can create dock-ing sites for the recruitment of downstream signallingeffectors, which contain specific modification inter-action domains, such as SH2 domains and PTB domains ,which recognize phosphorylated Tyr residues in specificsequence contexts 1 . Following these early events, the sig-nal propagates through kinase cascades and adaptor pro-tein interactions to various cellular compartments. Thetranslocation of some activated kinases to the nucleus,and the subsequent phosphorylation of transcriptionfactors, leads to changes in gene transcription. Regulationby many other signals follows the same general princi-ples but there are also important differences. For exam-ple, in the case of the inflammatory cytokine TNF α , theinitial signalling events involve activ ation of ubiquitinligases and the attachment of ubiquitin to signal media-tors. Downstream events involve both phosphorylationand ubiquitylation, as in the case of RTKs. Note thatdifferent signalling activators initiate multiple signal-ling pathways and share some of the same components,which is known as signalling crosstalk  2 .Signal propagation involves protein changes on threedifferent levels: regulated protein post-translationalmodifications (PTMs), including but not limited to phos-phorylation, acetylation, ubiquitylation, methylationand gylcosylation; protein–protein interactions, oftenowing to PTMs; and signal-induced protein expressionchanges (FIG. 1) . All three levels are synchronized in ahighly dynamic and often spatially segregated mannerand may themselves lead to changes in protein activity,localization and association with small molecules suchas phospholipids. A primary task of signalling research istherefore the measurement of PTMs, protein interactionsand proteome dynamics. The principles of cell signal-ling have been worked out over decades using ingeniousanalytical approaches. One of these is the recognition of proteins and their modifications by specific antibodies:protein–protein interactions are often studied by co-immunoprecipitation followed by western blotting,whereas changes in PTMs are detected by modification-specific antibodies and protein expression changes are *The Novo NordiskFoundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3,2200 Copenhagen, Denmark. ‡ Department of Proteomicsand Signal Transduction,Max Planck Institute for Biochemistry, Martinsried,; doi:10.1038/nrm2900Published online 12 May 2010 SH2 domain (SRC homology 2 domain).An ~100 amino acid domainthat recognizes phosphoTyrresidues in a specific sequencecontext. PTB domain (PhosphoTyr-binding domain).Like the SH2 domain, the PTBdomain binds to phosphoTyr,but usually binding specificityis determined by the sequenceN-terminal to thephosphorylation site. Decoding signalling networks bymass spectrometry-based proteomics Chunaram Choudhary* and Matthias Mann* ‡ Abstract | Signalling networks regulate essentially all of the biology of cells and organisms innormal and disease states. Signalling is often studied using antibody-based techniques suchas western blots. Large-scale ‘precision proteomics’ based on mass spectrometry now enablesthe system-wide characterization of signalling events at the levels of post-translationalmodifications, protein–protein interactions and changes in protein expression. Thistechnology delivers accurate and unbiased information about the quantitative changes of thousands of proteins and their modifications in response to any perturbation. Current studiesfocus on phosphorylation, but acetylation, methylation, glycosylation and ubiquitylation arealso becoming amenable to investigation. Large-scale proteomics-based signalling researchwill fundamentally change our understanding of signalling networks. REVIEWS NATURE REVIEWS |   MOLECULAR CELL BIOLOGY  VOLUME 11 | JUNE 2010 |    427   FOCUS ON SIGNAL INTEGRATION © 20 Macmillan Publishers Limited. All rights reserved10    Protein mixtureDigestion intopeptidesEnrichment for PTM-bearingpeptides (for PTM analysis)Cells or tissue1D PAGE abcd Elution time (min)         I      n       t      e      n      s        i       t      y Electrospray ionizationChromatographicpeptide separation Collision cellMass analyserMass analyser         I      n       t      e      n      s        i       t      y m/zMS and MS/MS spectraDatabase searchList of proteins and PTMsBioinformatic dataanalysis TVGTWR EVGTWKGCGTYR DSGTWR KCGTWR SVGTAKGVMSWR ECGTPKSVLTVR AVGTWR QVGTNK DEFYGR  A B C MSMS/MS SIGNALINGNETWORK-SREGULATETVGTWRESSEN-TIALLYAECGTPKLLOFTHE-BIOOOFCELLSANDEVGTWKORGANISMSINNORMALANDSVLTVRDISEASESTATESSIGNALINGISTRADITIONAL- an additional peptide separation step — usually basedon ion exchange chromatography — before on-lineLC MS/MS (see below).Three pieces of information are needed for MS analy-sis of each peptide: its mass, its ion intensity and a listof its fragments. The mass and fragments identify thepeptide, whereas the intensity is used for quantification.To obtain these data the mass spectrometer is used intwo different modes. In the MS mode, a spectrum of all peptides eluting from the column at any given timeis acquired, yielding the mass and intensity. The massspectrometer then isolates each peptide species andfragments them by imparting enough energy to break chemical bonds. The mass spectrum of these peptidefragments is called the tandem or MS/MS spectrum. Inmodern mass spectrometers, ions can be moved in milli-seconds between different parts of the instrument. MSand MS/MS can be carried out in the same mass analyser  or, in hybrid mass spectrometers, in different massanalysers in the same instrument.Among many different types of mass spectrometers 6 ,two configurations are often used in proteomics: quad-rupole time-of-flight (TOF) instruments and hybrid lin-ear ion trap–orbitrap instruments. In TOF instruments,peptide ions are separated in time by their arrival at thedetector. In the orbitrap mass analyser, the frequency of peptide ions oscillating in the trap is measured andthe mass spectrum is obtained by  Fourier transforma-tion . MS resolution is an important parameter, regard-less of which instrument is being used, because at any given time during the gradient the many peptides co-eluting from the chromatographic column need to bedistinguished in the mass spectrum. TOF instrumentsnow have resolution in excess of 10,000 and the orbitrapis routinely used at a resolution of 60,000 (MS resolution isa unit-less quantity). This is a big advance over the ubiq-uitous ion traps with typical resolution below 1,000,which are therefore generally restricted to the analysisof simple mixtures. Mass accuracy can be in the lowparts-per-million range for TOF instruments and evenlower for the orbitrap, greatly improving the percentageof peptides that can be identified 7   (BOX 1) .There are also different ways to fragment peptideions. Most commonly they are collided with a lowpressure of an inert gas (collision-induced dissocia-tion (CID); see REF. 8 for an introduction to MS-basedpeptide sequencing). In ion trap mass spectrometers,peptide ions are resonantly excited by an electric field,leading to an increase in internal energy and fragmen-tation primarily through cleavage at the peptide bonds.Ion trap fragmentation spectra can be uninformativeif the lowest energy pathway only involves the loss of a small chemical group, such as a water molecule or alabile PTM. For this reason, ion traps are usually oper-ated in more complicated modes, with additional frag-mentation of these uninformative ions 9 . In the ion trap,peptide fragments are always measured at low resolution.In quadrupole TOF instruments, the quadrupole servesas a mass filter that only passes the ions of a particularmass-to-charge (m/z) ratio. These ions are fragmentedin a collision chamber and a TOF spectrum of the frag-ments is subsequently obtained. These instrumentscan produce more informative MS/MS spectra becauseuninformative fragment ions frequently fragment Figure 2 | Typical work flow for proteome and PTM analysis using shotgun proteomics.a | Proteins extracted from organs, tissues or cells are separated by one-dimensionalpolyacrylamide gel electrophoresis (1D PAGE) and ‘in-gel digested’ into peptidesusing proteases such as trypsin. The peptides containing specific post-translationalmodifications (PTMs) can be enriched using different approaches (see TABLE 1 ).Non-modified peptides are used to identify and quantify total cellular proteins. b | Purifiedpeptides are separated on a miniaturized reverse phase chromatography column with anorganic solvent gradient. Peptides eluting from the column are ionized by electrosprayat the tip of the column, directly in front of the mass spectrometer (known as on-linecoupling). c | The electrosprayed ions are transferred into the vacuum of the massspectrometer. In the mass spectrometry (MS) mode, all ions are moved to theorbitrap mass analyser, where they are measured at high resolution (top mass spectrum).The first mass analyser then selects a particular peptide ion and fragments it in a collisioncell. The inset in the MS panel indicates the stable isotope labelling by amino acids in cellculture (SILAC) ratio of one of the peptides. The MS/MS spectrum can be obtained in theion trap mass analyser at low resolution or in the orbitrap at high resolution. For modifiedpeptides, the peptide mass will be shifted by the mass of the modification, as will allfragments containing the modification, allowing the unambiguous placement of the PTMon the sequence. d | The mass and list of fragment masses for each peptide are scannedagainst protein sequence databases, resulting in a list of identified peptides and proteins.These lists of proteins and their quantitative changes are the basis for biological discovery. REVIEWS NATURE REVIEWS |   MOLECULAR CELL BIOLOGY  VOLUME 11 | JUNE 2010 |    429   FOCUS ON SIGNAL INTEGRATION © 20 Macmillan Publishers Limited. All rights reserved10  Electrospray ionization An ionization methoddeveloped by J. Fenn, forwhich he shared the 2002Nobel Prize in chemistry.A liquid is passed through acharged needle, producingelectrosprayed dropletsthat contain the peptides.On evaporation of the solvent,intact and protonated peptides(or other analyte molecules)are left in the gas phase. Mass analyser A part of a mass spectrometerthat measures mass to charge(m/z) ratios of ions (forexample, ionized peptides).Multiplying the m/z value bythe charge and subtracting theweight of the charging entity(typically two protons) yieldsthe mass of the peptide.A mass spectrometer cancontain several mass analysersof the same or different types,and ions can be movedbetween these analysers atwill. Fourier transformation A mathematical operationthat transforms onecomplex-valued function of a real variable (typically afrequency spectrum) intoanother domain. In Fouriertransformation MS, thefrequencies associated withions moving in a trap are massdependent and this signal istransformed by Fouriertransformation into a massspectrum. MS resolution This value is defined as thewidth of the peak at half height divided by the massof the peak and is thereforea dimensionless number.High resolution distinguishesco-eluting peptides withsimilar mass, a prerquisite forunambiguous identificationand quantification of peptides. Chemical derivatization A chemical method usedto transform one chemicalcompound into a derivative.In proteomics, side chains of amino acids can be chemicallymodified, which can be usedfor enriching these peptidesfrom complex mixtures or forquantification of the modifiedpeptide in MS. further to yield informative ones. The recent introduc-tion of higher energy collisional dissociation (HCD)has made a similar fragmentation mode to that in quad-rupole TOF available to linear ion trap–orbitrap instru-ments, without diminishing sensitivity  10,11 . In HCD, theMS/MS spectrum is analysed with high mass accuracy in the orbitrap analyser.Two further fragmentation techniques rely on acompletely different physical mechanism. In electroncapture dissociation (ECD) and electron transfer dis-sociation (ETD), peptides obtain excess energy froman electron, which neutralizes one of the peptide’s posi-tive charges 12,13 . Fragmentation is much faster and moredirect than in CID, and often cleaves the backbone of the peptide without cleaving the most labile bonds first.Thus, ECD and ETD are attractive for analysing peptideswith labile PTMs, such as the glycosylation of proteins by   -linked β -  -acetylglucosamine (  -GlcNac).  Making MS quantitative. One of the attractions of MSover classical methods in signalling research is its poten-tial for highly accurate quantitative results for thousandsof proteins and PTMs. To achieve highest accuracy, thetwo proteomes to be compared are differentially iso-topically labelled. In metabolic labelling, cells incorporatethe isotopic label as part of normal bio synthesis 14 . Stableisotope labelling by amino acids in cell culture (SILAC)is one example of metabolic labelling and generally usesArg and Lys labelled with the stable 13 C isotope and/or 15 N isotope — after tryptic digestion peptides then con-tain a labelled amino acid at their carboxy terminus.The known molecular weight difference between the‘light’ (normal) and ‘heavy’ (labelled) amino acid thatis used during the growth of the two cell populationsallows the proteomes to be distinguished. After mixingthe light and heavy cells, they can be fractionated, orotherwise manipulated, without introducing quantita-tive errors between them 15,16 . Tryptic peptides appear asheavy and light pairs separated by a defined mass off-set (for example, heavy  13 C 6 - and 15 N 4 -labelled Arg hasa mass that is 10.00827 daltons higher than light Arg,and heavy  13 C 6 - and 15 N 2 -labelled Lys has a mass thatis 8.0142 daltons higher than light Lys; note that theseisotopes are stable and not radioactive). Triple labellingof Lys and Arg is also straightforward using 13 C 6 -labelledArg and D 4 -labelled Lys as additional labels that do notintroduce overlap between the natural isotope distribu-tions of the SILAC forms. Triple labelling allows thedirect comparison of three proteomes, which is usefulfor three state comparisons and in time-series measure-ments. Because of its simplicity and accuracy, SILAChas become a method of choice in MS-based signallingresearch and its use has recently been extended beyondcell culture systems to small mammals 17 and even tohuman tumour tissues 18 .The most popular chemical labelling technique isisobaric tag for relative and absolute quantification(iTRAQ). Like many other chemical labelling tech-niques, iTRAQ attaches a chemical group to primary amino groups (the N-terminus and Lys side chains) 19 ;however, in iTRAQ, the differential labelling is only apparent in the fragmentation spectra (that is, afterthe peptides have been fragmented during MS/MS).When working with iTRAQ it is important to exclude‘co-fragmented’ peptides that have similar mass andelute at the same time from quantitative analysis, as thiswould skew observed ratios 20,21 . Many other chemicallabelling techniques have also been described. For exam-ple, labelling by heavy or light dimethyl groups 22 is very economical and therefore also allows chemical labellingin cases where large amounts of starting material arerequired 23 . Note that some level of side reaction is una- voidable in chemical derivatization and this may interferewith unbiased PTM analysis 24 .The attraction of label-free quantification, in whichpeptide signals between different experimental condi-tions are compared directly, is that no experimentalmanipulation of the sample is necessary. However, ithas been very difficult to control for overall differencesin the signals between runs, which, unlike in the caseof isotopic labelling, are not automatically taken intoaccount. Several successful applications of label-free Box 1 | Precision proteomics A key advance in mass spectrometry (MS)-based proteomics in the past few years has been a huge increase in the qualityof the data. The three decade-old technology of two-dimensional gel electrophoresis typically resolved only theproducts of a few hundred genes at best, was low throughput and had a low dynamic range and, thus, was never a seriousfoundation for proteomics. Even the analysis of complex peptide mixtures, the current mainstay technology in MS-basedproteomics, was previously associated with ion trap mass spectrometers that had a mass spectrometric resolution of onlya few hundred and, consequently, could not distinguish co-eluting peptides of similar mass in complex peptide mixtures.This low quality of data resulted in low identification rates (often only a few percent 119 ) and precluded accuratequantification. The situation has changed dramatically. First, modern quadrupole time-of-flight instruments achievedmedium resolution and mass accuracy in MS and MS/MS spectra. Next, the hybrid linear ion trap–orbitrap instrumentsmade high resolution and high accuracy mass measurements routine without sacrificing robustness, speed orsensitivity 13,120–122 . However, the peptide fragmentation spectra on these instruments were still measured at relativelylow resolution in the ion trap part of the instrument. This is called a ‘high-low’ strategy because MS spectra are taken athigh resolution and MS/MS spectra at low resolution. Recently, the introduction of ion sources with much higher iontransmission from the atmosphere to the vacuum of the mass spectrometer, combined with the higher energy coalitionaldissociation (HCD) fragmentation method, has made it possible to routinely obtain extremely high mass accuracy in boththe survey spectrum and the fragmentation spectrum 10 . This finally allows a routine ‘high-high’ strategy in shotgunproteomics 123 without loss of analysis depth, and even ensures near certain identification of, for example, peptidesdefining new genes and unexpected modifications. REVIEWS  430 | JUNE 2010 | VOLUME 11   REVIEWS © 20 Macmillan Publishers Limited. All rights reserved10
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