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insightreview articles

198

NATURE

VOL 422

13 MARCH 2003

www.nature.com/nature

roteomics in general deals with the large-scaledetermination of gene and cellular functiondirectly at the protein level. But as theaccompanying articles in this issue describe,the field is a collection of various technicaldisciplines, all of which contribute to proteomics. Theseinclude cell imaging by light and electron microscopy,array and chip experiments, and genetic readoutexperiments, as exemplified by the yeast two-hybrid assay.Another powerful proteomic approach focuses on the

denovo

analysis of proteins or protein populations isolatedfrom cells or tissues. Such studies typically pose challengesowing to the high degree of complexity of cellularproteomes and the low abundance of many of theproteins, which necessitates highly sensitive analyticaltechniques. Mass spectrometry (MS) has increasinglybecome the method of choice for analysis of complexprotein samples. MS-based proteomics is a disciplinemade possible by the availability of gene and genomesequence databases and technical and conceptual advancesin many areas, most notably the discovery anddevelopment of protein ionization methods, as recognizedby the 2002 Nobel prize in chemistry.Here we survey the state of the field, particularly as it hasevolved over the three years since the last review in thesepages

. Already, many of the dreams of the discipline have atleast been partly realized. MS-based proteomics hasestablished itself as an indispensable technology to interpretthe information encoded in genomes. So far, proteinanalysis (primary sequence, post-translational modifica-tions (PTMs) or protein–protein interactions) by MS hasbeen most successful when applied to small sets of proteinsisolated in specific functional contexts. The systematicanalysis of the much larger number of proteins expressed ina cell, an explicit goal of proteomics, is now also rapidlyadvancing, due mainly to the development of new experi-mental approaches.Today, proteomics still remains a multifaceted, rapidlydeveloping and open-ended endeavour. Although it hasenjoyed tremendous recent success, proteomics still facessignificant technical challenges. Each breakthrough thateither allows a new type of measurement or improves thequality of data made by traditional types of measurementsexpands the range of potential applications of MS to molec-ular and cellular biology. Indeed, this field is already tooexpansive for a comprehensive, single review; thus we apol-ogize in advance for the many omissions. However, we dohope that this article captures the excitement of recentachievements in MS-based proteomics, and points the waytowards the direction future developments will likely take.

Principles and instrumentation

Mass spectrometric measurements are carried out in the gasphase on ionized analytes. By definition, a mass spectrome-ter consists of an ion source, a mass analyser that measuresthe mass-to-charge ratio (

) of the ionized analytes, and adetector that registers the number of ions at each

value.Electrospray ionization (ESI) and matrix-assisted laserdesorption/ionization (MALDI) are the two techniquesmost commonly used to volatize and ionize the proteins orpeptides for mass spectrometric analysis

2,3

. ESI ionizes theanalytes out of a solution and is therefore readily coupled toliquid-based (for example, chromatographic andelectrophoretic) separation tools (Fig. 1). MALDI subli-mates and ionizes the samples out of a dry, crystalline matrixvia laser pulses. MALDI-MS is normally used to analyserelatively simple peptide mixtures, whereas integratedliquid-chromatography ESI-MS systems (LC-MS) arepreferred for the analysis of complex samples.The mass analyser is, literally and figuratively, central tothe technology. In the context of proteomics, its key parame-ters are sensitivity, resolution, mass accuracy and the abilityto generate information-rich ion mass spectra from peptidefragments (tandem mass or MS/MS spectra) (see Fig. 1 andrefs 1,4,5). There are four basic types of mass analyser cur-rently used in proteomics research. These are the ion trap,time-of-flight (TOF), quadrupole and Fourier transformion cyclotron (FT-MS) analysers. They are very different indesign and performance, each with its own strength andweakness. These analysers can be stand alone or, in somecases, put together in tandem to take advantage of thestrengths of each (Fig. 2).In ion-trap analysers, the ions are first captured or‘trapped’ for a certain time interval and are then subjected toMS or MS/MS analysis. Ion traps are robust, sensitive andrelatively inexpensive, and so have produced much of theproteomics data reported in the literature. A disadvantage of ion traps is their relatively low mass accuracy, due in part tothe limited number of ions that can be accumulated at theirpoint-like centre before space-charging distorts theirdistribution and thus the accuracy of the mass measure-ment. The ‘linear’ or ‘two-dimensional ion trap’

6,7

is anexciting recent development where ions are stored in acylindrical volume that is considerably larger than that of

Mass spectrometry-based proteomics

Ruedi Aebersold

& Matthias Mann

†*

Institute for Systems Biology, 1441 North 34th Street, Seattle, Washington 98103-8904, USA (e-mail: raebersold@systemsbiology.org)

†

Center for Experimental BioInformatics(CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark,Campusvej 55, DK-5230 Odense M, Denmark (e-mail: mann@bmb.sdu.dk)

Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool formolecular and cellular biology and for the emerging field of systems biology. These include the study ofprotein–protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping ofnumerous organelles, the concurrent description of the malaria parasite genome and proteome, and thegeneration of quantitative protein profiles from diverse species. The ability of mass spectrometry to identifyand, increasingly, to precisely quantify thousands of proteins from complex samples can be expected toimpact broadly on biology and medicine.

2003NaturePublishingGroup

the traditional, three-dimensional ion traps, allowing increasedsensitivity, resolution and mass accuracy. The FT-MS instrument isalso a trapping mass spectrometer, although it captures the ionsunder high vacuum in a high magnetic field. Its strengths are highsensitivity, mass accuracy, resolution and dynamic range

8–11

. But inspite of the enormous potential, the expense, operational complexityand low peptide-fragmentation efficiency of FT-MS instruments haslimited their routine use in proteomics research.MALDI is usually coupled to TOF analysers that measure the massof intact peptides, whereas ESI has mostly been coupled to ion trapsand triple quadrupole instruments and used to generate fragment ionspectra (collision-induced (CID) spectra) of selected precursor ions

.More recently, new configurations of ion sources and mass analysershave found wide application for protein analysis. To allow thefragmentation of MALDI-generated precursor ions, MALDI ionsources have recently been coupled to quadrupole ion-trap mass spec-trometers

and to two types of TOF instruments. In the first, two TOFsections are separated by a collision cell (‘TOF-TOF instrument’)

,whereas in the second, the hybrid quadrupole TOF instrument, thecollision cell is placed between a quadrupole mass filter and a TOFanalyser

. Ions of a particular

are selected in a first mass analyser(TOF or quadrupole), fragmented in a collision cell and the fragmention masses are ‘read out’ by a TOF analyser. These instruments havehigh sensitivity, resolution and mass accuracy, and the quadrupoleTOF instrument can be used interchangeably with an ESI ionizationsource. The resulting fragment ion spectra are often more extensiveand informative than those generated in trapping instruments.Although TOF, ion-trap and hybrid TOF instruments dominateproteomics today, other configurations including linear ion traps andFT-MS instruments could become widespread in the near future.As a result of its simplicity, excellent mass accuracy, highresolution and sensitivity, MALDI-TOF is still much used to identifyproteins by what is known as peptide mapping, also referred to aspeptide-mass mapping or peptide-mass fingerprinting. In thismethod, proteins are identified by matching a list of experimentalpeptide masses with the calculated list of all peptide masses of eachentry in a database (for example, a comprehensive protein database).Because mass mapping requires an essentially purified targetprotein, the technique is commonly used in conjunction with priorprotein fractionation using either one- or two-dimensional gelelectrophoresis (1DE and 2DE, respectively). The addition of sequencing capability to the MALDI method should make proteinidentifications by MALDI-MS/MS more specific than those obtainedby simple peptide-mass mapping (see below). It should also extendthe use of MALDI to the analysis of more complex samples, therebyuncoupling MALDI-MS from 2DE. However, if MALDI-MS/MS isto be used with peptide chromatography, the effluent of a liquidchromatography run must be deposited on a sample plate and mixedwith the MALDI matrix, a process that has thus far proven difficult toautomate. In general, it can be expected that the trend towards thecombination of liquid chromatography with ESI- or MALDI-MS/MS (Fig. 1) will continue.Protein identifications using peptide CID spectra are more clear-cut than those achieved by mass mapping because, in addition to thepeptide mass, the peak pattern in the CID spectrum also providesinformation about peptide sequence. This information is not readilyconvertible into a full, unambiguous peptide sequence, that is, the ‘

denovo

’ sequencing problem via MS is still not generally solved. Instead,the CID spectra are scanned against comprehensive protein sequencedatabases using one of a number of different algorithms, each with itsstrengths and weaknesses. The ‘peptide sequence tag’ approachextracts a short, unambiguous amino acid sequence from the peak pattern that, when combined with the mass information, is aspecific probe to determine the origin of the peptide

. In thecross-correlation method, peptide sequences in the database areused to construct theoretical mass spectra and the overlap or ‘cross-correlation’ of these predicted spectra with the measured massspectra determines the best match

. In the third main approach,‘probability based matching’, the calculated fragments from peptidesequences in the database are compared with observed peaks. Fromthis comparison a score is calculated which reflects the statisticalsignificance of the match between the spectrum and the sequencescontained in a database

.In each of these methods the identified peptides are compiledinto a protein ‘hit list’, which is the output of a typical proteomicexperiment. Because protein identifications rely on matches withsequence databases, high-throughput proteomics is currentlyrestricted largely to those species for which comprehensive sequencedatabases are available.

Protein identification and quantification

No method or instrument exists that is capable of identifying andquantifying the components of a complex protein sample in a simple,single-step operation. Rather, individual components for separating,

insightreview articles

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VOL 422

13 MARCH 2003

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199

Excisedproteins(2) TrypsindigestionPeptidemixture

LLEAAAQSTK

(5) MS/MS

516.27 (2+)

y9y8y7

LEAAQ

y5y4y3

y60100200

200 600 1000

0200400

400 600 800

(4) MS

516.27 (2+)

(3) Peptidechromatographyand ESI

a2b2

(1) SamplefractionationSDS– PAGE

I n t e n s i t y ( a r b i t r a r y u n i t s )

m/z m/z

Figure 1

Generic mass spectrometry (MS)-based proteomics experiment. The typicalproteomics experiment consists of five stages. In stage 1, the proteins to be analysedare isolated from cell lysate or tissues by biochemical fractionation or affinity selection.This often includes a final step of one-dimensional gel electrophoresis, and defines the‘sub-proteome’ to be analysed. MS of whole proteins is less sensitive than peptide MSand the mass of the intact protein by itself is insufficient for identification. Therefore,proteins are degraded enzymatically to peptides in stage 2, usually by trypsin, leadingto peptides with C-terminally protonated amino acids, providing an advantage insubsequent peptide sequencing. In stage 3, the peptides are separated by one or moresteps of high-pressure liquid chromatography in very fine capillaries and eluted into anelectrospray ion source where they are nebulized in small, highly charged droplets.After evaporation, multiply protonated peptides enter the mass spectrometer and, instage 4, a mass spectrum of the peptides eluting at this time point is taken (MS

spectrum, or ‘normal mass spectrum’). The computer generates a prioritized list ofthese peptides for fragmentation and a series of tandem mass spectrometric or‘MS/MS’ experiments ensues (stage 5). These consist of isolation of a given peptideion, fragmentation by energetic collision with gas, and recording of the tandem orMS/MS spectrum. The MS and MS/MS spectra are typically acquired for about onesecond each and stored for matching against protein sequence databases. Theoutcome of the experiment is the identity of the peptides and therefore the proteinsmaking up the purified protein population.

2003NaturePublishingGroup

identifying and quantifying the polypeptides as well as tools forintegrating and analysing all the data must be used in concert. Out of abewildering multitude of techniques and instruments, two main trackscan be identified. The first, and most commonly used, is a combinationof 2DE and MS. The second track combines limited proteinpurification with the more recently developed techniques of automat-ed peptide MS/MS and, if accurate quantification is desired, stable-isotope tagging of proteins or peptides. In either track a suitable dataprocessing, storage and visualization infrastructure needs to bedeveloped, if the platform is intended for high-throughput operation.In the first track, the proteins in a sample are separated by 2DE,stained, and each observed protein spot is quantified by its stainingintensity. Selected spots are excised, digested and analysed by MS.Sophisticated pattern-matching algorithms as well as interpretationby skilled researchers are required to relate the 2DE patterns to eachother in order to detect characteristic patterns and differences amongsamples. 2DE has been a mature technique for more than 25 years andwas the first technique capable of supporting the concurrent quanti-tative analysis of large numbers of gene products. In fact, many of theprinciples now commonly used for global, quantitative analysis of messenger RNA expression patterns, such as clustering algorithmsand multivariate statistics, were developed in the context of 2DE

.Peptide-mass mapping by MALDI-TOF and peptide sequencingby ESI-MS/MS have become highly efficient at the identification of gel-separated proteins. In the many reports using this technology,largely the same proteins were identified repeatedly, irrespective of the system studied, which suggests limited dynamic range of 2DE-based proteomics. Systematic studies of the budding yeast

Saccharomyces cerevisiae

indeed revealed that typically only the mostabundant proteins can be observed by this method

. Incrementalimprovements in 2DE technology, including more sensitive stainingmethods

20,21

, large-format higher resolving gels

and sample frac-tionation prior to 2DE have alleviated, but not eliminated, these andother shortcomings of the 2DE/MS approach.Studying major histocompatibility complex class I-associatedpeptides, a natural and complex peptide library, Hunt and colleaguespioneered the use of LC-MS/MS for the analysis of complex peptidemixtures and it is this method that is today at the core of MS-basedproteomics

. However, before LC-MS/MS could be used both for theidentification of protein mixtures and for quantitative proteomicexperiments, a number of technical issues had to be addressed.First, single-dimension peptide chromatography does not providesufficient peak capacity to separate peptide mixtures as complex asthose generated by the proteolysis of protein mixtures of, forexample, total cell lysates. Second, in both MALDI- and ESI-MS, therelationship between the amount of analyte present and measuredsignal intensity is complex and incompletely understood. Massspectrometers are therefore inherently poor quantitative devices.

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NATURE

VOL 422

13 MARCH 2003

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Ion sourceMass analyserDetectorLiquidchromatographyElectrospray ionization(ESI)Triple quadrupoleor linear ion trap

fdabce

Ion trapNozzleSampling coneIonsQ

TOFReflectorTOF

TOF

Sample platePulsed laserQ

SprayneedlePulsed laserExtraction gridSampleplateIonsCollision cellTOFReflectorSuperconductingmagnetFourier transformion cyclotronresonance massspectrometer (FT-MS)Quadrupoletime-of-flightMatrix-assisted laserdesorption/ionization(MALDI)Reflectortime-of-flight (TOF)Time-of-flighttime-of-flight (TOF-TOF)

Figure 2

Mass spectrometers used in proteome research. The left and right upperpanels depict the ionization and sample introduction process in electrospray ionization(ESI) and matrix-assisted laser desorption/ionization (MALDI). The different instrumentalconfigurations (

–

) are shown with their typical ion source.

, In reflector time-of-flight(TOF) instruments, the ions are accelerated to high kinetic energy and are separatedalong a flight tube as a result of their different velocities. The ions are turned around in areflector, which compensates for slight differences in kinetic energy, and then impinge ona detector that amplifies and counts arriving ions.

, The TOF-TOF instrumentincorporates a collision cell between two TOF sections. Ions of one mass-to-charge (

)ratio are selected in the first TOF section, fragmented in the collision cell, and the massesof the fragments are separated in the second TOF section.

, Quadrupole massspectrometers select by time-varying electric fields between four rods, which permit astable trajectory only for ions of a particular desired

. Again, ions of a particular m/zare selected in a first section (Q

), fragmented in a collision cell (q

), and the fragmentsseparated in Q

. In the linear ion trap, ions are captured in a quadruple section, depictedby the red dot in Q

. They are then excited via resonant electric field and the fragmentsare scanned out, creating the tandem mass spectrum.

, The quadrupole TOFinstrument combines the front part of a triple quadruple instrument with a reflector TOFsection for measuring the mass of the ions.

, The (three-dimensional) ion trap capturesthe ions as in the case of the linear ion trap, fragments ions of a particular

, and thenscans out the fragments to generate the tandem mass spectrum.

, The FT-MSinstrument also traps the ions, but does so with the help of strong magnetic fields. Thefigure shows the combination of FT-MS with the linear ion trap for efficient isolation,fragmentation and fragment detection in the FT-MS section.

2003NaturePublishingGroup

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