R.Bakhtiar1 and F.L.S.Tse MS) in 1984, the field of bioanalytical chemistry has seenexplosive growth (Fenn et al., 1989; Loo et al., 1999a;Department of Drug Metabolism and Pharmacokinetics, Novartis InstituteMcLafferty et al., 1999; Kelleher, 2000; Thomas et al., 2000).for Biomedical Research, East Hanover, NJ 07936, USAThe compatibility of ESI with separation techniques such asBiological polymers undergo numerous significant andcapillary electrophoresis (CE) and high performance liquidfascinating interactions, such as post-translational modi-chromatography (HPLC) allows characterization of a largefications, non-covalent associations and conformationalarray of components, such as small organic molecules, peptides,changes. A valuable parameter for the characterization ofproteins, DNA fragments, inorganic/organometallic complexesa biopolymer is molecular weight. Modern methods ofand synthetic polymers. In addition, matrix-assisted lasermass spectrometry, including electrospray ionization anddesorption ionization (MALDI) mass spectrometry, a comple-matrix-assisted laser desorption ionization mass spectro-mentary approach to ESI which was introduced by Hillenkampmetry, are ideally suited for the accurate determination ofand Karas (Hillenkamp et al., 1991), is now widely utilizedthe molecular weight of a biopolymer of interest. Molecularfor protein/peptide analysis and in characterization of combin-weight measurements are now routinely utilized in theatorial chemistry libraries, protein mapping and DNA sequen-qualitative and quantitative analysis of macromolecules. Incing (Murray, 1996; Hop and Bakhtiar, 1997; Brewer andmany cases small sample quantities (i.e. a few micrograms)Henion, 1998; de Jong, 1998; Altman et al., 1999; Costellolimit the utility of nuclear magnetic resonance spectroscopy1999; Deng and Smith, 1999; Ding and Vouros, 1999; Fengand X-ray crystallography in obtaining structural informa-et al., 1999; Gygi et al., 1999a; Harvey, 1999; Kiselar andtion. Thus, mass spectrometry offers an attractive alternat-Downard, 1999; Kuster and Mann, 1999; McCloskey et al.,ive to the more traditional bioanalytical methods for rapid1999; Pramanik et al., 1999; van Baar, 2000). These newand sensitive measurements. The ultimate goal of theseapproaches promise a stunning breath of perspective, driven byexperiments is to obtain sufficient information in order toa continued pursuit of macromolecular structural information inmap the complex molecular circuitry which operates withinproteomics and genomics research (Lamond and Mann, 1997;the cell. In the analysis of complex mixtures mass spectro-Lottspeich, 1999).metry is even more powerful when utilized in conjunction
At the outset it should be noted that both techniques arewith separation methods. Herein we present some of thesensitive and allow observation of intact biopolymers with aaspects of modern biological mass spectrometry for themol. wt of 100 000 Da or higher. Since the ESI and MALDIinvestigation of large molecules. For more advanced orspectra of modified biopolymers show little or no fragmenta-detailed technical descriptions we refer the reader to ation, these techniques can be useful in obtaining accuratenumber of recently published reports.identification of the specimen and both are referred to as‘soft’ ionization techniques. In this article we present a briefdescription of the ionization processes involved in ESI and
Introduction MALDI as well as a number of examples, which will demon-strate some of the capabilities of the ESI-MS and MALDI-In 1898 Wien succeeded in deflecting charged rays using a
combination of electric and magnetic fields. Thomson demon- MS techniques.strated the presence of two neon isotopes in 1912. Moreadvanced mass analyzers were designed and constructed by
MALDI-MS and ESI-MSDempster and Aston in 1918 and 1919, respectively (Biemann,1962; Matsuo and Seyama, 2000). Although early mass spectro- In a typical MS experiment the sample of interest is ionized
in the ionization source and guided via a series of electric and/metry (MS) provided important information about stable iso-topes and radionuclides, it was limited to lower mol. wt or magnetic lenses to the detector. The three main events
during MS analysis are ion production, ion transmission andcompounds that could be readily volatilized. The problems ofinvolatility and high mass (�1000 Da) limited the scope of ion detection. In order to control the motion of the ions during
their transmission to the detector it is necessary to control theMS applications. Larger species simply could not be transferredto the gas phase without substantial degradation and/or frag- influences of pressure and temperature on ion mobility (kinetic
energy). Therefore, a vacuum system with a pressure rangingmentation.Recent advances in ionization methods (vide infra) have from ~10–5 to 10–8 Torr is utilized in all mass spectrometers.
The vacuum minimizes interference from collision of thecircumvented the limitations of traditional MS and it is nowpossible to analyze high mol. wt compounds of all types. Since analyte ions with the background neutral gaseous molecules
and facilitates their transmission to the detector.Fenn and co-workers introduced electrospray ionization (ESI-
1To whom correspondence should be addressed at: Building 405, Room 229, 59 Route 10, Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936,USA. Tel: �1 973 781 3562; Fax: �1 973 781 6076; Email: [email protected]
Fig. 1. In a MALDI experiment, the sample is mixed or dissolved with an excess amount (e.g. 1 part sample to 10 000 parts matrix) of a matrix component(having an absorption wavelength which matches closely with the laser wavelength). Upon laser irradiation, a plume of neutral molecules and ions isdesorbed. The ions are then guided to the mass analyzer and the detector by electrostatic lenses. In contrast to ESI, MALDI generally does not yield multiplycharged ions, does not require mass spectral deconvolution and is more suitable for analysis of complex mixtures.
A majority of commercial mass spectrometers utilize an MALDI the sample preparation procedure can be extremelycrucial because the ion population depends upon the type ofelectron multiplier detector, which provides an internally
amplified electrical current subsequent to exposure to charged matrix and the presence of impurities (Chapman, 1996; Stimsonet al., 1997; Jespersen et al., 1998; Breaux et. al., 2000;ions. The ion current output corresponding to each specific
analyte is then processed by the instrument electronics and Garner, 2000; Landry et al., 2000). Depending on the special-ist’s experience and instrumentation, it is now possible totranslated to a mol. wt. In the resulting mass spectrum the
ordinate indicates the relative intensity or abundance while the acquire mol. wt information on a biopolymer using picomolequantities of samples. However, in some cases additionalabscissa shows the observed ratio of mass to the number of
charges on the ions. The latter is referred to as the mass-to- sample quantities may be required for detailed analyses, suchas peptide or oligonucleotide sequencing.charge ratio or m/z. Regardless of the ionization source, it is
the m/z that is measured by the mass spectrometer. Figure 3 shows a representative MALDI spectrum obtainedfrom an 8mer oligonucleotide, d(GGAGGCCT), containingMALDI (Figure 1) uses pulses of laser light (e.g. a nitrogen
laser at 337 nm) to desorb the analyte from a solid phase the codon 249 sequence (AGG) of the p53 gene (Jones et al.,1999). In this experiment, the sample (200 fmol) was mixedsurface (the analyte co-crystallized with a light-absorbing
matrix; Figure 2) and yield gaseous ions. A laser is a device with 1% α-cyano-4-hydroxycinnamic acid in 1:1 acetonitrile/deionized water, loaded onto the MALDI sample holder andthat can deliver coherent and high density energy (photons) to
a small space. Pulsed laser radiation tuned to the absorption evaporated to dryness. A nitrogen laser using 4 ns pulses at337 nm was used to desorb and ionize the sample, yielding amaximum of the matrix is used to initiate the desorption/
ionization event and to simultaneously generate a packet of signal corresponding to a [M–H]– species. Since MALDI isconsidered a mild ionization process, the sample experiencesions of different m/z values. The laser may be tuned to UV,
visible or infrared wavelengths (Zenobi and Knochenmuss, little or no fragmentation during analysis (Muddiman et al.,1997). Therefore, the mixture can be analyzed in a single1998). The matrix is typically a small organic molecule which
has an absorption band that closely coincides with the energy experiment, because each component generally produces onlyone predominant signal ([M�H]� or [M–H]–).of the laser radiation. Figure 2 depicts the molecular structures
of some of the commonly used matrices (Siuzdak, 1996). The A complementary technique to MALDI is ESI (Figure 4),which produces single or multiply charged gaseous ions directlymatrix is generally co-crystallized in large excess over the
analyte and facilitates ionization of the analyte as well as from solution by generating a fine spray of highly chargeddroplets in the presence of a strong electric field. There areminimizes sample degradation due to the laser radiation. For
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Fig. 4. A simplified schematic diagram of an ESI source. A spray of finedroplets which contain the analyte and solvent molecules is generated uponapplication of a high electrical tension through a needle. In some instrumentsa heated capillary is placed following the electrospray needle to facilitatesolvent evaporation (courtesy of the Finnigan Corp., San Jose, CA).
two widely proposed theories for ion formation in ESI (Gaskell,1997; Bruins, 1998; Constantopoulos et al., 2000; Fernandezde la Mora, 2000; Gamero-Castano and Fernandez de la Mora,2000; Kebarle and Peschke, 2000). One theory suggests thationized sample molecules are expelled from the droplets.Alternatively, it has been proposed that individual ionizedsample molecules remain after solvent evaporation and dropletfragmentation.
As shown in Figure 4, a solution of the analyte(s) and thesolvent are introduced into a sampling metal capillary (~100
Fig. 2. Chemical structures of some of the commonly utilized matrices in µm in internal diameter) which is charged by application ofMALDI. an electrical voltage (4–5 kV). The voltage polarity of the
metal capillary is positive or negative for positive or negativeion generation, respectively. At some point mutual repulsionbetween the ions at the surface becomes greater than thesurface tension of the liquid, which gives rise to formation ofthe so-called Taylor cone (Wilm and Mann, 1994). If theelectrical field is sufficiently strong, spraying commences andsmall charged droplets form. The ions generated by ESI carrymultiple charges, provided the sample molecules have a mol.wt of more than ~1000 Da. The characteristic feature of ESIthat distinguishes it from other ionization techniques is that itgenerally imparts multiple charges to larger analyte moleculesand the extent of multiple charging increases in near proportionto mol. wt. The resulting highly charged molecular ions arethus within the m/z range in which most conventional massspectrometers function quite well (Bakhtiar et al., 1996;Bakhtiar and Nelson, 2000). It is the multiple charging phenom-enon that allows assay of high mass ions by mass analyzerswith only a modest m/z range.
For the sake of clarification, let us dissect a hypotheticalpositive ion ESI-MS spectrum (Figure 5) of a biopolymer witha mol. wt of 5 kDa. Unevenly spaced signals correspondingto charge states 1� to 5� are evident. In comparison with aMALDI spectrum, the ESI spectrum shown in Figure 5 clearlyappears rather complex and convoluted. Thus, ESI spectrarequire deconvolution algorithms, which are commonly utilizedon all commercial MS instruments. Assuming that a positiveion series represents different protonation states, then the m/zof two successive peaks can be denoted P1 and P2, correspond-ing to (m/z)1 and (m/z)2, respectively (Figure 6). The objectiveFig. 3. A representative negative ion MALDI-MS spectrum of an 8-mer
oligonucleotide with a sequence motif 5�-GGAGGCCT-3�. About 500 fmol is to extract the charge state of each individual signal in orderof sample was utilized for the analysis. to deconvolute the spectra and obtain the mol. wt of the
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Fig. 5. A hypothetical positive ion ESI-MS spectrum for a biopolymer with a mol. wt of 5 kDa. Signals corresponding to multiple charge states �1 to �5 areevident. In contrast to MALDI, ESI spectra require a deconvolution algorithm for mol. wt determination. Multiple charging enables the conventional massspectrometers to measure mol. wts in excess of their dynamic range for singly charged molecule.
panels represent the originally acquired convoluted and sub-sequently processed deconvoluted spectra, respectively.
ESI does have limitations in that it is not very tolerant ofthe presence of salts, detergents and inorganic buffers (MALDIhas proven to be more amenable in such cases). Thus, in orderto minimize signal suppression effects, ESI is often exploitedas an interface between CE or HPLC and a mass spectrometer(Niessen, 1999). Currently, HPLC-MS is an attractive tool inthe analysis of complex mixtures in biochemical research andmedical/diagnostic analysis. The up-front chromatographicseparation aids in sample purification/enrichment from mostcommon laboratory buffers and endogenous salts and providesadditional useful parameters, such as retention time (Cole,1997). For example, Figure 8 illustrates the influence of anorganic buffer, HEPES, which is commonly used between pH6.8 and 8.2, on the positive ion ESI-MS spectrum of horse
Fig. 6. A simplified procedure adapted to the deconvolution of ESI-MS skeletal muscle myoglobin (Mb). The typical working concen-spectra. Let the unknown mass of the biopolymer discussed in Figure 5 be tration range for HEPES is between 20 and 100 mM. WhileMr and let the number of unknown charges be z. Normally a range of values a solution of Mb in 0.1% formic acid yielded a satisfactoryis found for z, with each signal having one more charge (i.e. proton) than
spectrum (Figure 8a), the same solution containing 10 mMthe preceding one. Thus, two successive signals yield two equations andtwo unknowns, which can be solved to reveal the molecular weight of the HEPES exhibited significant ion suppression (Figure 8b).biopolymer (with an accuracy of �0.05–0.01%). Similar ESI-MS analyte signal suppression has also been
observed with several commonly used detergents and surfact-ants in protein chemistry (Ogorzalek Loo, 1996). The abovebiopolymer. This can be achieved easily by setting up twoexample (Figure 8) and reports from other laboratories (videequations and two unknowns using at least two adjacent signalssupra) serve to exemplify the need for proper on- or off-linein the spectrum (Figure 5). For example, solving for z1 andsample clean-up and preparation prior to MS analysis.mol. wt yields values of 5 Da and 5000 Da, respectively.
As an alternative to HPLC purification, several laboratoriesAnalogous outcomes could be obtained using any two sets ofhave explored the utility of on-line protein (DeGnore et al.,signals in the spectrum (Siuzdak, 1996). Of course, additional1998; Xu et al., 1998) or oligonucleotide (Liu et al., 1996;mathematical procedures, such as smoothing, background sub-Huber and Buchmeiser, 1998) dialysis prior to mass spectraltraction, noise filtering and automated algorithms, have beenanalysis. In our laboratory we have adapted a similar strategyintroduced as options on most modern MS computer worksta-to that reported by Liu et al. (1996) to perform on-line sampletions (Bruenner et al., 1994; Bonner and Shushan, 1995; Horndialysis and clean-up for several protein biomarkers. We choseet al., 2000). Figure 7 shows a ‘real life’ example of a positivehemoglobin (Hb) as our test model. The Hb experiment wasESI-MS spectrum acquired for a sample of bovine serum
albumin in 0.1% formic acid solution. The top and bottom performed with two objectives in mind. First, the performance
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Fig. 7. The top panel shows a positive ion mode ESI-MS spectrum of a sample of bovine serum albumin dissolved in 0.1% formic acid. The statisticallydifferent (bell-shaped distribution) charge states from �28 to about �62 represent the same molecule. The bottom panel illustrates the deconvoluted spectrumusing the equations shown in Figure 6. Note that the abscissa refers to the molecular weight of bovine serum albumin and not the m/z.
of our fabricated microdialysis device could be evaluated. ProteomicsSecond, we could demonstrate that a relatively simple ESI-
The human body is estimated to contain ~70 000–100 000 genesMS experiment is amenable to differentiation of a single amino(the entire human genome is composed of ~3 000 000 000 bp)acid substitution.potentially encoding 100 000 different proteins (Rowen et al.,There are more than 700 Hb abnormalities known to be the1997). Furthermore, post-translational modification, mutation,result of a single amino acid variation (due to mutations indegradation and other cellular processes increase the numberthe coding sequence). In recent years a number of Hb variantsof proteins. This extremely high degree of complexity warrantshave been successfully characterized by MS (Shackleton andthe need for a conglomerate of sensitive and rapid analyticalWitkowska, 1996; Kaneko et al., 1999; Gatlin et al., 2000).techniques to yield qualitative and quantitative informationIn this regard, genetically transmitted sickle cell anemia iswith high efficiency. The science of proteomics involves thecharacterized by thin and elongated red blood cells. Sickle celldetection and identification of proteins, and complementsanemia is generally accompanied by cardiac enlargement,genomics. The proteome is a highly dynamic system which canswelling of lymph nodes, jaundice and anemia. The topologicalbe influenced by environmental variations, such as quantitativealteration in individuals with sickle cell anemia is due to achanges in protein expression as a result of drug administration.single amino acid mutation in the β-chain of their Hb. In aFor example, a drug may elicit inhibition or overexpressionnormal adult, position six of the β-chain is occupied by Gluof a specific enzyme (hepatic cytochrome P450), which maywhile in a patient with sickle cell anemia this amino acid isyield alterations in the therapeutic outcome. Enzyme inductionsubstituted by Val. This mutation dramatically reduces thecan decrease drug levels or increase the formation of toxicsolubility of the deoxygenated form of Hb. Sickle cell Hb ismetabolites (Guengerich, 1999; Whitlock, 1999). The informa-referred to as Hb S to distinguish it from the normal adult Hbtion obtained from proteome analysis can aid in identification(Hb A). Figure 9 shows the deconvoluted positive ion ESI-of therapeutic targets or surrogate markers in understanding theMS spectra for Hb S (top) and Hb A (bottom) subsequent toinitiation and progression of a disease state. Thus, proteomicson-line dialysis. The result clearly indicates the mol. wtresearch can be a valuable tool in drug discovery and for thedifference of 29 Da between the two β-chains correspondingfirst time offers the scientist an integration of genomics, mRNAto the substitution β6Glu→Val. There are no amino acidanalysis and protein expression (Blackstock and Weir, 1999).sequence differences in the α-subunit of Hb S and Hb A and,
One of the most commonly utilized techniques for proteintherefore, the observed mol. wt values (mass accuracy 0.01%separation has been based on gel separation. Simple proteinor �1.5 Da) for both samples are similar. In other experimentsmixtures (�100 components) are normally separated using 1-high resolution instruments have identified a mass differencedimensional (1D) SDS–PAGE. On the other hand, for complexof 1 Da in proteins having mol. wts of ~12 kDa (Marshall
et al., 1997). protein mixtures (i.e. cell or tissue extracts) the resolving
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Fig. 8. (a) Positive ion ESI-MS spectrum of horse skeletal muscle Mb obtained from a solution containing 0.1% formic acid. Mb denatures under acidicconditions resulting in loss of the non-covalently bound heme moiety (m/z 616.1). (b) The same solution was exposed to 10 mM HEPES buffer and infuseddirectly into the mass spectrometer at a flow rate of 2 µl/min. Significant signal suppression is observed due to the presence of HEPES. The mass spectrum isdominated by signals corresponding to HEPES aggregates.
power of 2-dimensional (2D) SDS–PAGE is required. In this regulation in a disease state. The resulting information couldbe utilized to identify biomarkers in clinical and toxicolo-approach proteins are separated by isoelectric point in the first
dimension and subsequently by their mol. wt in the second gical studies.Conceptually, a similar and complementary approach butdimension. Visualization of the gel is easily achieved by
Coomassie blue staining, silver staining, fluorescent tagging with higher accuracy (i.e. better than 10 p.p.m.), sensitivityand speed can be implemented with the aid of MS-basedor radioactive labeling, with some approaches having a detec-
tion limit of ~10 ng of protein (Rabilloud, 2000). However, techniques. The gel ‘spots’ can be excised, washed, subjectedto proteolytic digestion and characterized by MALDI-MS orvisualization does not provide unambiguous protein identifica-
tion and, therefore, scientists typically use western blotting or ESI-MS (Bantscheff et al., 1999; Jungblut and Thiede, 1997;Keough et al., 1999; Loo et al., 1999b; Neubauer and Mann,classical Edman sequencing for this purpose. Fortunately,
integration of the inherent benefits of MS (i.e. sensitivity, 1999; Schrotz-King et al., 1999). Sometimes, affinity chroma-tography techniques are necessary to enrich a specific class ofselectivity and speed) with those conferred by protein and
expressed sequence tag (EST) databases has contributed to proteins prior to additional sample manipulations (Link et al.,1999; Gruninger-Leitch et al., 2000). Commonly, on-line CE-significant advances (Blackstock and Weir, 1999; Yates, 2000).
Figure 10 depicts a simplified strategy which is being MS or HPLC-MS analysis can be employed to further separatecomplex protein or peptide mixtures (Cao and Stults, 1999;widely utilized in high throughput polypeptide characterization.
Typically, samples obtained from different cellular fractions Jensen,P.K. et al., 1999; Tong et al., 1999). A number ofsoftware packages are currently available to query largeare processed by 2D SDS isoelectric focusing gel electrophor-
esis. Each cell or tissue type may require a specific visualization databases and enhance the speed of the MS protein identifica-tion process (Jaffe et al., 1998; Clauser et al., 1999; Demirevapproach, such as Coomassie blue or silver staining or fluores-
cence tagging, for protein detection (Hancock et al., 1999; et al., 1999; Green et al., 1999). Non-redundant proteindatabases with ~350 000 entries and human EST databasesLottspeich, 1999; Wang and Hewick, 1999; Williams,K.L.,
1999). Subsequently, gel images are electronically retrieved with ~1 200 000 entries can yield more sophisticated andaccurate identification output compared with 2D gel analysisby high resolution scanners and analyzed (spot finding) using
pattern recognition techniques against 2D gel database queries. (Mann, 1996; Jensen,O.N. et al., 1999). In addition, 2D gelsample components below a mol. wt of 10 000 Da or aboveSophisticated computer software packages can be employed
to enhance contrast, subtract background, align images, remove 100 000 Da that are not easily characterized can be readilyobserved by MS (vide infra). The following examples willartifacts and perform gel comparisons. Proteome maps are
compared against databases for identification of up- or down- clarify the above discussions.
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Fig. 9. (a) The deconvoluted spectrum transformed from the positive ion ESI-MS spectrum of a sample of Hb S dissolved in 0.1% formic acid. (b) Thedeconvoluted spectrum transformed from the positive ion ESI-MS spectrum of a sample of Hb A dissolved in 10 mM ammonium acetate containing 0.1%formic acid. A Glu6→Val mutation in the β-chain results in the pathological disorder known as sickle cell anemia. MS clearly shows the corresponding massshift (29 Da) associated with this mutation.
Recently, a high throughput protein identification (double proteins subsequent to cadmium-mediated stress. The abund-ance of several intact and isotopically distinctive proteinsparallel digestion, DPD) method was reported by Sanchez and
co-workers (Bienvenut et al., 1999). In the DPD approach was qualitatively monitored for up to several hours usingthis method.partially digested proteins were obtained using an immobilized
trypsin membrane and transblotted. The resulting peptides Another related area in proteome analysis is protein expres-sion mapping, which is defined as the quantitative measurementwere trapped on a polyvinylidene difluoride membrane and
scrutinized by MS. The DPD approach was successfully of protein dynamics in a specimen (i.e. a cell, tissue, or bodyfluid) of interest. In this approach proteome analysis is typicallyapplied to a mini-2D gel electrophoresis of Escherichia coli
extract. Several critical issues, however, need to be further performed in a subtractive fashion whereby alterations inindividual proteins for two or more states are compared. Theseimproved in order to realize the full potential of 2D gel
analysis in conjunction with MS analysis. These include a so-called ‘cell states’ could refer to a cell prior to andsubsequent to treatment with a xenobiotic or cells obtainedreduction in background chemical noise (i.e. due to keratins),
which can mask the detection of lower abundance or ‘low from normal and pathological states. Recently an elegantquantitative microcapillary-LC-ESI-MS strategy for the ana-copy number’ proteins. Keratin interference can even originate
from inadequately purified trypsin, which is widely used for lysis of protein mixtures in Saccharomyces cerevisiae wasreported by Aebersold and co-workers (Gygi et al., 1999b).peptide mapping in MS experiments (Zhang,Y. et al., 1998).
Thus, a clean sample preparation environment, automation, An isotope-containing affinity tag (ICAT), which consisted ofan affinity tag (biotin), a linker containing a stable isotope andminimum sample handling procedures and higher quality gel
materials compatible with MS analysis could alleviate the a reactive moiety with a propensity to react with free sulfhydrylgroups (i.e. cysteines), was utilized (Figure 11). Two sets ofproblem of possible contamination. In addition, post-transla-
tional modifications, oxidation of protein during sample pre- cell states (or tissue extracts) were independently treated withisotopically light and heavy (8 Da higher in mol. wt due toparation and poor recovery of large proteins from the gel have
the potential to extend the duration of the unambiguous incorporation of 2H) ICAT reagents. The cells were combinedand subjected to proteolytic cleavage. The ICAT-labeled pep-analyte(s) identification process.
Smith and co-workers (Pasa-Tolic et al., 1999) demonstrated tides were isolated using the biotin tag and analyzed bymicrocapillary-LC-ESI-MS. Peptide sequence information wasthe utility of ultra-high resolution MS measurements in con-
junction with the resolving power of capillary isoelectric obtained by tandem mass spectrometry experiments and identi-fied by computer searches against protein data banks. Quantita-focusing for characterization of the cadmium stress response
in E.coli K-12 strain MG1655 cells. The cells were cultured tion of proteins was performed using the ratios of the respectivelight and heavy ICAT-labeled peptides, which were generatedin normal as well as rare isotope (i.e. 13C, 15N and 2H) depleted
media in order to provide internal calibrants for all detected by enzymatic digestion. The stable isotope labeling procedure
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Fig. 10. A simplified proteomics scheme outlining steps involved in the characterization of polypeptides (see text for details).
was a clever approach to assist in the identification of two essential to identify them prior to Phase III clinical studies(Gould Rothberg et al., 2000). Therefore, it is of interestpeptides with identical sequences and mol. wts from two
different cell states. Since all the physical characteristics of to elucidate the identity and pharmacogenomic traits (i.e.polymorphically expressed enzymes) of key cellular proteinsthe two identical protein samples from the two cell states
remain the same, the resulting peptide fragments obtained and to design optimum medication for individual patients. MStechnology offers a viable platform which can be utilized toby enzymatic cleavage yield identical mass spectra. Thus,
incorporation of specific stable isotopes in one cell state results assay differential protein expression following drug treatment.Considering the recent report by Sweedler and co-workersin mass shifts, which in turn serve as an internal standard for
all other cell states within the same experiment (Mann, 1999). (Li et al., 1999; Rubakhin et al., 2000), the application of MSmeasurements to the identification of proteins in individualSince the light and heavy ICAT-tagged peptides are chemically
identical, one can safely assume that they would yield ana- organelles will not be far in the future. In their findingsSweedler and colleagues devised an elegant direct approachlogous MS detection (ionization) responses and behave as
mutual internal standards for quantification purposes. to the identification of peptides in 1–2 µm diameter vesiclesfrom the exocrine atrial gland of Aplysia californica as aIn general, the above strategies for qualitative and quantitat-
ive identification of key cellular proteins could have great model system. MALDI-MS was used to analyze samplevolumes of as low as 300 al (300�10–18 l) and identified apotential in several areas of drug development, such as pharma-
cogenomics (Evans and Relling, 1999). In the science of wide range of bioactive polypeptides. This technology offersan exciting window of opportunity to study discrete smallpharmacogenomics genetic polymorphisms in transporters,
drug metabolizing enzymes (e.g. cytochrome P450s and uridine cells and processes such as synaptosomes, vesicular transportbetween the endoplasmic reticulum and the various Golgi5�-triphosphate glucuronosyltransferases), receptors and thera-
peutic target proteins have been postulated to be one of the compartments, hepatocytes and protein packaging pathways.underlying reasons for variable responses to drug treatment inpatients. Currently these investigations are rather tedious and
Non-covalent complexesempirical. In most cases the human genetic variations resultingin different drug responses are realized in large studies at the A growing body of literature has been devoted to the application
of MS to the detection of non-covalent complexes (Przybylskipost-marketing stages (i.e. subject sizes exceeding 100 000).Some of these idiosyncratic responses are toxic and thus it is and Glocker, 1996; Loo, 1997; Jespersen et al., 1998; Coyle
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Fig. 11. The principle of quantitative proteomics by incorporation of a stable isotope-labeled tag. Cysteine in proteins in two different cell states (i.e. normalversus abnormal due to stress induced by a drug or other environmental factor) are covalently modified by the tag. One tag contains 2H while the otherincorporates 1H. The protein extracts from the two cell states are mixed, digested with trypsin and separated by affinity chromatography. The resulting peptidemixtures are then subjected to LC-MS and tandem mass spectrometry. The ratios of labeled to unlabeled peptides (differing by 8 Da) are manifestations of theabundance of the gene product in the two cell states. Subsequent LC-MS/MS peptide sequencing of these peptides can identify the gene product which isbeing quantified. A computer search algorithm is typically utilized during the identification process using large protein data banks.
et al., 1999; Griffey et al., 1999; Last and Robinson, 1999; Van Berkel et al., 2000). Furthermore, mass spectrometricdetermination of 1H-2H exchange has provided complementaryNordhoff et al., 1999; Rostom and Robinson, 1999; Rostom
et al., 2000). Using inherently ‘gentle’ ionization, MALDI- information to existing nuclear magnetic resonance (NMR)data on water soluble peptide and protein tertiary structuresMS and ESI-MS have provided valuable information on
structurally specific biomolecular interactions, including direct and conformations (Gross, 1999; Weber-Ban et al., 1999;Bouchard et. al., 2000; Demmers et al., 2000; Jager andstoichiometry determinations for specific protein–drug, DNA–
drug, protein–DNA, RNA–drug and protein–protein complexes Pluckthun, 2000).For example, interaction of the HIV protein gp120, on the(Bakhtiar and Stearns, 1995; Bakhtiar and Bednarek, 1996;
Hop and Bakhtiar, 1997; Pramanik et al., 1998; Loo, 2000; exterior of the HIV virus, with CD4 glycoprotein, on the
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surface of T helper cells, is of interest in understanding thetissue specificity of HIV virus infection. In humans the maincells infected by the HIV virus are CD4 T helper lymphocytesand macrophages via CD4 surface receptor interactions. CD4is a surface glycoprotein which is involved in the T helper cellreaction with other cells (cell–cell adhesion). As glycoproteins,gp120 and CD4 both show a high degree of oligosaccharideheterogeneity. The complex pattern of the non-covalent com-plex of gp 120 and CD4 poses difficulty in spectral deconvolu-tion during ESI-MS analysis. Recently MALDI-MS wasutilized in the characterization of a non-covalent complex ofrecombinant gp120 with the extracellular part of its primarycellular receptor, CD4 (Borchers and Tomer, 1999). MALDI-MS experiments clearly showed a 1:1 stoichiometry betweengp120 and CD4, with a combined mol. wt of ~145 kDa. Thisapproach yielded higher resolution and accuracy in comparisonwith sugar gradient sedimentation experiments. However, todetermine the stoichiometry of a macromolecular non-covalentassembly by MALDI-MS, several key issues need to beconsidered. In MALDI-MS of intact non-covalent complexesthe nature of the matrix, the laser power and the laserpenetration depth in the target sample are among the mostcritical parameters. In the presence of organic solvents andacidic MALDI matrices denaturation of protein complexes islikely to occur (Glocker et al., 1996; Little et al., 1997a;Jespersen et al., 1998). In addition, repeated laser pulses on agiven spot may dissociate the non-covalent complex. Fig. 12. ESI-MS spectra of (a) transthyretin indicating the presence of
The analysis of specific interactions of nucleic acid monomers (T) and tetramers (4T); (b) signals arising from non-covalentcomplexes of transthyretin containing one (4TR) and two (4TRR) moleculessequences with repressors and other regulatory proteins hasof retinol and retinol-binding protein (R). ESI charge states corresponding tocommonly been performed using electrophoretic gel mobilityfree R were also detected. (Kindly provided by Prof. C.V. Robinson and Dr
shift assay. However, this technique requires radiolabeled DNA A.A. Rostom, Oxford Center for Molecular Sciences, Oxford, UK.)and does not provide accurate (i.e. within 0.01% of thetheoretical value obtained from the known sequence) mol. wtdeterminations. In recent years MS techniques have provided
carrier of retinol (vitamin A). Transthyretin (~55 kDa) is aa complementary approach to the existing methods forserum protein which plays a role in transport of thyroiddeciphering the mol. wt and stoichiometry of DNA–proteinhormones. Non-covalent complexes of transthyretin and Rcomplexes (Veenstra, 1999). For example, an innovative(binding affinity ~1.1�10–7–1.5�10–7 M) have been identifiedmethod involving a high throughput determination of DNA-in both human and chicken. Generally, a few microliters ofbinding proteins using an immobilized DNA probe and sub-the solution (~1–5 µM) of interest sufficed for obtainingsequent read-out by MALDI-MS was recently reported (Nord-satisfactory ESI-MS results. Signals corresponding to thehoff et al., 1999). Specific sequences of DNA strands weretransthyretin tetramer (4T) and non-covalent complexes withimmobilized (i.e. using biotin) on Dynabeads, incubated withone (4TR) and two (4TRR) molecules of retinol-bindingprotein mixtures, washed and subjected to MS analysis. Severalprotein were reflective of previously known solution chemistryknown DNA-binding proteins, such as cAMP receptor protein,observations.rat retinoid X receptor and poly(ADP-ribose) polymerase, were
Although the majority of published MS work has been onidentified.previously characterized non-covalent complexes, it is clearThe specific binding of aminoglycoside antibiotics to rRNAthat MS offers enormous possibilities (in conjunction withsubdomains was examined by Griffey et al. (1999) using aNMR and X-ray crystallography data) for the investigation ofhigh resolution mass spectrometer equipped with an ESIless well defined macromolecular complexes. Nonetheless, itinterface. Aminoglycoside antibiotics (i.e. apramycin, ribosta-is imperative to perform proper control experiments to rulemycin, tobramycin, bekanamycin, paromomycin and lividomy-out the possibility of false positive observations during thecin) are a class of compounds that inhibit protein synthesisMS analysis of non-covalent complexes. Variations in solutionand RNA splicing both in vivo and in vitro. Elegant MSpH, instrument parameters (ionization interface temperatureexperiments demonstrated specific binding of several anti-and voltages) and chemical modification of the analyte underbiotics to rRNA subdomains, provided estimated bindinginvestigation are some of the measures that can be takenaffinities and determined their respective binding sites.to distinguish between specific interactions and non-specificRobinson and co-workers (Last and Robinson, 1999; Rostomaggregations (Loo, 1997).and Robinson, 1999) presented a collection of elegant work
on protein folding and multi-protein complexes using ESI-Chip-based technologies and microfabricated devicesMS. Figure 12 depicts an example of nanoflow (2–10 nl/min)
ESI-MS spectra for human transthyretin with retinol-binding Chip and other miniaturized technologies are rapidly beingadapted to analyze changes in gene expression (Epstein andprotein (R) obtained from chicken plasma. Retinol-binding
protein (~21 kDa) is a plasma protein which is a specific Butow, 2000). DNA microarray technology (e.g. the Affymetrix
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GeneChip®) has shown great promise in toxicological studies, DNA polymerase (ThermoSequenase) and all four dideoxynu-gene mapping, gene polymorphism and transciptional analysis cleotide triphosphates. The resulting extension products were(DeRisi et al., 1997; Kurian et al., 1999; Voehringer et al., desalted using a POROS reversed phase column and subjected2000). DNA microarrays consisting of thousands of individual to MALDI-MS. The base at the polymorphic site was easilygene sequences can be printed in a high density format on a detected by the mass added onto the primer oligonucleotide.glass microscope slide or deposited on a miniature matrix by Similar approaches could be employed using ‘MALDI on aa photolithographic process (Knapp et al., 2000). These DNA chip’ technology in which only nanoliter amounts of samplemicroarrays can then be utilized to obtain a global view of are deposited on the target with piezoelectrical pipettes (Littlechanges in expression of genes during drug development and et al., 1997b; Griffin and Smith, 2000).provide a vivid ‘snapshot’ of how cells respond to a drug Although DNA immobilization through biotin–streptavidinin vivo (Afshari et al., 1999). (i.e. on magnetic or controlled pore glass beads) or via covalent
Many diseases stem from gene mutations, which may take linkage (i.e. on a silicon surface) is compatible with highplace via various routes such as replacement, deletion, insertion throughput MALDI-MS (Ekstrom et al., 2000) analysis, severalor duplication of a single or multiple base units that are the ESI-based microfabricated devices have also been presented.building blocks of the gene. In the area of gene expression, Lazar et al. (1999) designed a microchip nano-ESI device withwhere many human genes have already been characterized, a fluid delivery of 20–30 nl/min and sub-attomole sensitivitythe sensitivity of detection of mRNA at the level of one in for detection of several peptide and protein mixtures. Zhang,B.thousands is of interest. Driven by chemistry, the process of et al. (1999) reported the coupling of an electrophoreticdrug research and discovery is increasingly led in the post- device for on-chip CE (constructed using photolithographic/genomic era by advances in biotechnology and bioinformatics wet chemical etching techniques) separation followed by ESI-(Drews, 2000; Sander, 2000). The trend towards sensitive and MS analysis. A sample volume of 15 µl could be electrosprayedhigh speed microfabricated devices has been reinforced by a for up to 20 min at a flow rate of ~200 nl/min. Similarly,recent initiative to construct a mammalian gene collection Chan et al. (1999) used polydimethylsiloxane to design soft(Strausberg et al., 1999). In this regard, MS is becoming a polymer chips that were successfully employed in the identi-viable platform for rapid genomic DNA characterization (Laken fication of rat serum albumin separated by 2D gel electrophor-et al., 1998; Ross et al., 1998; Griffin et al., 1999; Tang et al., esis. A lower limit of detection of 100 fmol/µl was consistently1999; Cantor, 2000). achieved.
In one strategy Tang et al. (1999) demonstrated the immobil- Affinity isolation is undoubtedly the most specific of separa-ization of reduced thiol DNA strands on silicon chips using tion techniques and, when coupled with MS, offers anN-succinimidyl(4-iodoacetyl)aminobenzoate chemistry. Each extremely powerful method for the selective isolation andsilicon chip contained 36 wells with a surface area of 6.25 concentration of a desired ligand. Recently, ProteinChip™mm2/well (~1 µl/well). DNA fragments containing the poly- technology based on surface-enhanced laser desorption/ioniza-morphic sites (i.e. codons 163 and 33 in human platelet alloan- tion (SELDI)-MS was introduced by Hutchens and co-workerstigens 2 and 1, respectively) were amplified by PCR and (Kuwata et al., 1998; Paweletz et al., 2000). In SELDI-MSdetected by this method. Parallel primer annealing, extension
affinity chips are tailor designed to capture a specific smalland termination were performed on a 1 µl sample scale andmolecule or biopolymer. An antibody, a receptor or a DNAdirectly subjected to MALDI-MS.fragment with defined arrays of binding surfaces is selectedOne of the emerging disciplines of human genetics is theto modify a chip surface. Subsequently, a crude protein sampledetection of DNA polymorphisms to identify the componentsor a combinatorial ligand library is applied to the chip surface,involved in complex genetic diseases. To put this in perspective,washed and subjected to SELDI-MS analysis. The resultingconsider that the nucleotide sequence difference betweenmass spectrum typically corresponds to all the analytes capturedhumans and chimpanzees is estimated to be ~1.5% (Liyanageby the affinity chip with minimal sample clean up. SELDI-and Xanthopoulos, 2000). Clearly, this seemingly small vari-MS was recently applied to profiling the site of in vitroation has led to profound differences between the two species.phosphorylation of caspase-9 (Cardone et al., 1998). FigureThe so-called single nucleotide polymorphism (SNP) refers to13 depicts an application of the ProteinChip™ in conjunctiona position at which two alternative bases occur at a frequencywith SELDI-MS that was successfully utilized in ‘fishing out’of ~1% in humans. This corresponds to approximately 1 ina high affinity ligand from a proprietary combinatorial ligandevery 1000 nt. The significant implications of SNP includelibrary. An immobilized ‘receptor’ ProteinChip™ array (Figurethe presence of a particular SNP allele that could be the13, top) and a control ProteinChip™ array (Figure 13, middle)underlying reason for a genetic disorder along with variationswere incubated with a combinatorial library of 100 compoundsin protein coding sequences. Furthermore, SNPs could beof mol. wt � 600 Da. Only one of the ligands was retaineduseful as genetic markers for mapping purposes and localizationspecifically on the receptor and not the control chip. The bottomof important functional genes. A combination of MALDI-MSpanel (Figure 13) depicts the differential ligand binding plot.and microfabricated devices for DNA minisequencing and
Other novel approaches, such as matrix-free MALDI usingcharacterization could obviate the need for labeling studies.a porous silicon target (Wei et al., 1999) and chip-based surfaceSeveral laboratories have recently reported the use of MALDI-plasmon resonance biomolecular interaction mass spectrometryMS for detection of SNPs for direct genetic analysis (Haff(Nelson and Krone, 1999; Williams,C. and Addona, 2000),and Smirnov, 1997; Griffin et al., 1999). For example, Haff andhave been reported. Furthermore, microfluidic chip technologySmirnov (1997) discussed an assay for single base variations atfor direct analysis of proteins using ESI-MS (Licklider et al.,specific locations within the DNA sequence using MALDI-2000; Pinto et al., 2000; Wen et al., 2000) has shown potentialMS. In this approach a primer oligonucleotide was annealedin characterization of minute (i.e. femotmole quantities)to a target DNA upstream of the polymorphic location and
was extended by a single base in the presence of a thermostable amounts of samples in an automated fashion.
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tion using a combination of MALDI, ESI and whole cell stableisotopic labeling (Oda et al., 1999). Cells were separatelycultivated in media enriched with 14N and 15N, combined andsubjected to SDS–PAGE. Gel spots of interest were thenexcised, digested by a protease and analyzed by MS. The ratioof 14N- to 15N-labeled peptides reflects their abundance. Proteinexpression and in vivo phosphorylation in the wild-type versuscells mutant for PAK-related yeast Ste20 protein kinase wereinvestigated utilizing this approach. This technology is some-what analogous to that described by Gygi et al. (1999b) andyields a quantitative visualization of cellular protein phenotypeexpression and modification (i.e. phosphorylation).
Cortez et al. (1999) conducted CID experiments usingcapillary LC-MS to locate the phosphorylation sites (Ser1423and Ser1524) of the breast cancer gene 1 tumor suppressorprotein. Likewise, automated sequence database searchingalgorithms were used on the resulting CID marker productions to map the in vivo phosphorylation sites of endothelialnitric oxide synthase by Figeys et al. (1999). An immobilizedmetal affinity chromatography method was used to obtainfractions of tryptic digests, which were subsequently subjectedto CE-MS and CID. A broader experimental set-up capable
Fig. 13. An immobilized ‘receptor’ ProteinChip™ array (top) and a control of identification of 22 different types of post-translationalProteinChip™ array (middle) were incubated with a combinatorial library ofmodification was reported by Wilkins et al. (1999). MALDI-100 compounds of mol. wt �600 Da. Only one of the ligands was retained
specifically by the receptor and not by the control. (Bottom) The differential MS analysis of proteins from a 2D gel of E.coli and sheepligand binding plot. (Courtesy of Drs E.A.Dalmasso and M.Sha, Ciphergen wool were studied using 5153 entries of post-translationalBiosystems, Palo Alto, CA.) modifications recorded in the SWISS-PROT data bank. This
approach also shows promise in high throughput mapping ofmodified regulatory proteins.
Post-translational modificationsConclusionsPost-translational modification of proteins plays a pivotal role
in functional activity and signal transduction in all living In the past decade dramatic progress in the field of MS hasorganisms. The mass changes due to post-translational modi- resulted in a large increase in the number of commerciallyfication (i.e. acetylation, farnesylation, glycosylation, phospho- available MS instruments. Based on the large number ofrylation, methylation and sulfation) can be easily detected by published manuscripts, it is clear that MS is becoming anESI (Verma et al., 1997; Abraham et al., 2000) or MALDI important bioanalytical tool in many biotechnology and bio-(Vinh et al., 1997; Kirpekar et al., 2000; Zhou et al., 2000). chemistry laboratories. MALDI-MS and ESI-MS allow theIdentification of the protein fragment of increased mass sub- characterization of a large number of small and large moleculessequent to enzymatic digestion allows determination of the site with high sensitivity, speed, accuracy and efficiency. MS-basedof modification. For example, in the case of phosphorylation techniques are becoming a permanent component of studiesradiolabeling with 32P is not required and low levels of involving functional genomics, proteomics, early drug discov-phosphopeptides (i.e. 200–300 fmol/µl) can be readily identi- ery, clinical diagnostics and combinatorial chemistry. In addi-fied with high efficiency and speed. Additional information tion to large pharmaceutical corporations, a number of smallcan be obtained by performing tandem mass spectrometry or start-up companies have begun to embark on the ambitiouscollision-induced dissociation (CID) experiments (Hop and path of establishing large scale MS-based proteomics andBakhtiar, 1997; Bakhtiar and Hop, 1999). Ease of application genomics facilities (Service, 2000; Stipp, 2000).with most types of mass spectrometers, along with its experi- The literature on the above subject matter is growingmental simplicity, account for the wide popularity of CID for exponentially. Consequently, specialized journals are nowstructural analysis. The interpretation of CID spectra of peptides devoted to the area of MS, including Journal of the Americanof unknown sequence is facilitated by the use of computer- Society for Mass Spectrometry, Rapid Communications in Massaided database searching/matching algorithms. In a typical Spectrometry, Mass Spectrometry Reviews, European MassCID experiment a beam of ions with a specific m/z (denoted Spectrometry, Journal of Mass Spectrometry and Internationalthe precursor or parent ion) is selected and collided with a Journal of Mass Spectrometry. Thus, some related topics [e.g.neutral and non-reactive gas phase target such as argon. These complex sequencing and characterization of polysaccharidescollisions result in subsequent fragmentation and product ions (Venkataraman et al., 1999) and lipopolysaccharides (Ernstthat are a direct consequence of dissociation of the precursor et al., 1999), integration of MS into drug development andion. Generally, the resulting fragmentation pattern is unique quantitative analysis (Lee and Kerns, 1999; Watt et al., 2000)]for all ions having a particular structure. CID experiments are have not been included in this review but citations are providedparticularly useful in peptide and DNA sequencing (Little for interested readers throughout the manuscript.et al., 1996; Roskey et al., 1996; Zhu et al., 1997; Kelleheret al., 1999). Acknowledgements
Recently, Chait and co-workers reported quantitative meas- R.B. is grateful to Prof. C.V.Robinson and Dr A.A.Rostom (Oxford University,Oxford, UK) for providing Figure 12, Drs E.A.Dalmasso and M. Shaurements of protein expression and site-specific phosphoryla-
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(Ciphergen Biosystems, Palo Alto, CA) for kindly providing Figure 13 and Chan,J.H., Timperman,A.T., Qin,D. and Aebersold,R. (1999) MicrofabricatedDr J.Ashby (Astra-Zeneca) for encouragement and support of this initiative. polymer devices for automated sample delivery of peptides for analysis byWe also thank the Finnigan Corporation (San Jose, CA) for providing Figure electrospray ionization mass spectrometry. Anal. Chem., 71, 4437–4444.4. The constructive comments of the reviewers and the Editor-in-Chief were Chapman,J.R. (ed.) (1996) Protein and Peptide Analysis by Mass Spectrometry.extremely helpful in improving the quality of this contribution. Humana Press, Totowa, NJ.
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Received on January 5, 2000; accepted on May 9, 2000
