Proc. Nati. Acad. Sci. USAVol. 91, pp. 11290-11297, November 1994

Review

The emergence of mass spectrometry in biochemical researchGary SiuzdakThe Scripps Research Insthtte, Departent of Chemistry, 10666 North Torrey Pines Road, La Jolla, CA 92037

ABSTRACT The initial steps towardroutinely applying mass spectrometry inthe biochemical laboratory have beenachieved. In the past, maw spectrometrywas confined to the realm of small, rela-tively stable molecules; large or thermallylabile molecules did not survive the desorp-dion and ionization processes intact. Elec-trospray ionization (ESI) and matrix-assisted laser desorption/ionization(MALDI) mass spe met allow for theanalysis of both small and large biomole-cules through "mild" desorption and ion-ization methods. The use of ESI andMALDI mass spectrometry extends beyondsimple characterization. Noncovalent in-teractions, protein and peptide sequencing,DNA sequencing, protein fdding, in vitrodrug analysis, and drug discovery areamong the areas to which ESI and MALDImass spectrometry have been applied. Thisreview summarizes recent developmentsand major contributions in mas spectrom-etry, focusing on the applications ofMALDI and ESI mass spectrometry.

Advances in chemical technology havebeen the engine powering the biotechnol-ogy industry. Analytical chemists haveadded fresh impetus to bioresearch withtwo new mass spectrometry ionizationtools: electrospray ionization (ESI) andmatrix-assisted laser desorption/ioniza-tion (MALDI). Commercial availability ofthese instruments has made routine theanalysis ofcompounds including proteins,peptides, carbohydrates, oligonucleo-tides, natural products, and drug metab-olites. ESI and MALDI mass spectrome-try offer picomole-to-femtomole sensitiv-ity, enabling the direct analysis ofbiological fluids with a minimum amountof sample preparation. These techniquescan be used to measure the mass of bio-molecules >200,000 Da, to provide struc-tural information, and to detect noncova-lent complexes with molecular weight ac-curacy on the order of ±0.01%. Theysignify another dimension in molecularcharacterization through a new level ofsensitivity, accuracy, and mass range.Mass spectrometry is based on produc-

ing, differentiating, and detecting ions inthe gas phase. The transfer of small mol-ecules into the gas phase has traditionallybeen accomplished by thermal vaporiza-tion; however, thermal vaporization forbiopolymers and other nonvolatile or

dry gasand/or

A. heat

and/ordry gas

vacuum pumps

FIG. 1. ESI source.

thermally unstable molecules has littleuse. In fact, the search for ionizationsources that would transfer large, ther-mally labile molecules into the gas phasewithout degradation has occupied massspectrometrists for many years. It haslargely been the development of ESI andMALDI that have made routine whatwas, until recently, impossible.The trick has been forming ions in the

gas phase; once formed, the ions can bedirected electrostatically into a mass an-alyzer that differentiates the ions accord-ing to their mass-to-charge ratio (m/z).Although the analyzers have not changedsignificantly in the past two decades, thechanges that have been made have beenlargely stimulated by the development ofthe ESI and MALDI ionization sources.Both ESI and MALDI-MS support

biochemical research, each havingunique capabilities, as well as some fun-damental similarities. Although there aremany exciting examples of how they arebeing used as bioanalytical tools, theirbasic utility in molecular characterizationshould not be underestimated. ESI andMALDI-MS offer a rapid, simple, andaccurate means of obtaining molecularweight information on a wide range ofcompounds. Their utility for mass mea-surement meets the needs ofchemists andbiologists alike, facilitating routine char-acterization in small molecule synthesis,protein synthesis, and compounds ob-tained directly from biological matrices.

ESIThe utility ofESI (1-9) lies in its ability toproduce singly or multiply charged gas-eous ions directly from an aqueous oraqueous/organic solvent system by cre-

ating a fine spray of highly charged drop-lets in the presence of a strong electricfield (Fig. 1) (8). The sample solution istypically sprayed from the tip of a metalsyringe maintained at -4000 V. Dry gas,heat, or both are applied to the highlycharged droplets, causing the solvent toevaporate. Evaporation causes the drop-let size to decrease, while surface chargedensity increases. Ions are transferred tothe gas phase as a result oftheir expulsionfrom the droplet and then directed into amass analyzer through a series of lenses.

Typically, ESI is interfaced with qua-drupole mass analyzers because quadru-poles tolerate high pressures (10-5 torr; 1torr = 133.3 Pa) and have good resolvingpower (=2000). An intrinsic property ofall mass analyzers (including quadrupolemass analyzers) is that they separate ionsaccording to their mass-to-charge ratio(m/z), not the mass. This is often a pointofconfusion, especially with the ESI tech-niques, which typically generate multiplycharged ions (Fig. 2). An advantage ofmultiple charging is that a mass analyzerwith a relatively small m/z range (quadru-pole instruments) can be used to observevery large molecules. The data generatedvia multiple charging can then be used todetermine molecular weight. Another ad-vantage of multiple charging is that moreaccurate molecular weight can be ob-tained from the distribution of multiplycharged peaks. The mass spectrum ofeggwhite lysozyme (in Fig. 2) illustrates theability of the ESI technique to generatemultiply charged ions. It is especially in-

Abbreviations: ESI, electrospray ionization;MALDI, matrix-assisted laser desorption/ionization; FTMS, Fourier-transform ion cy-clotron resonance mass spectrometry.

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10+1432

11+1302

12+1194

1250 1500

m/z

egg white tysozyme

9+

1592

8+

A..~~191750

HPLC Chromatogram C0.25. A B

S

2000

FIG. 2. ESI mass spectrum of egg whitelysozyme. The molecular weight can be cal-culated from the equation: Mr = (mass-to-charge ratio x total charge) - total mass ofcharging species or Mr = (m/z x z) - z (ifcharging species are protons). Sample calcu-lations for egg white lysozyme are as follows:m/z= 1302.5, z = 11 (+), Mr= (1302.5 x 11)- 11 = 14,317; m/z = 1432.4, z = 10 (+), Mr= (1432.4 x 10) - 10 = 14,317; m/z = 1591.7,z = 9 (+), Mr = (1591.7 x 9) -9 = 14,317.

teresting to note that an instrument with amass range of <m/z 2000 was used toobserve a protein ofmass 14,317 Da. Theequation and sample calculations shownwith Fig. 2 illustrate how molecularweight information can be calculated fromthe multiply charged peaks. If the chargestates are not known and adjacent peaksdiffer by a charge of one, the molecularweight can easily be calculated from them/z values (9). Fortunately, all of themanufactured ESI-MS instrumentationcome with programs that rapidly performthese calculations.HPLC Mam Spetner and Tandem

Mass Spectrometry. In the past, attemptswere made to couple HPLC analysis witha variety of mass spectrometry tech-niques. These attempts resulted in somesuccess; however, until the advent ofESI, HPLC-MS applications could nothave been considered routine. The abilityof ESI-MS to directly analyze com-pounds from aqueous or aqueous/organic solutions has established thetechnique as a convenient mass detectorfor HPLC. ESI also allows for MS anal-ysis at relatively high HPLC flow rates(1.0 ml/min) and high mass accuracy(±0.01%), adding a new dimension to thecapabilities of HPLC. In fact, usingESI-MS as a detector for HPLC was oneof its first obvious applications. Numer-ous reports (10-19) have been publishedon this application; special emphasis hasbeen placed on peptide and protein anal-ysis. HPLC combined with ESI-MS isexcellent for routine and reproduciblemolecular weight determinations on awide variety of compounds, whetherthey are positively (i.e., peptides andproteins) or negatively (i.e., oligonucle-otides) charged. One such example isshown in Fig. 3. Besides its use forHPLCanalysis, ESI-MS detection has also beensuccessfully applied to capillary zoneelectrophoresis (20).

HPLC-MS100

0~

a).e-Atto

0.

10.0 15.0time (minutes)

20.0

scan number

Molecular Weight Spectra

c

a1)

MdolurWeight

100 B to

Moea550Molecular Weight

W

100 C 543

05200 Molecuilar Weight 5700

FIG. 3. HPLC chromatogram, HPLC-MS, and MS data acquired from a single run of amixture of three oligonucleotides. The HPLC-MS data show the extracted ion currentcorresponding to a 17-mer, and two 18-mer oligonucleotides, respectively. The molecularweight spectra were reconstructed from multiply charged ions observed from each of the threepeaks shown (unpublished data, Brian Bothner, Kelly S. Chatman, Molly Sarkisian, and G.S.,The Scripps Research Institute).

In addition to characterizing com-pounds by molecular weight, it is alsoimportant to gain structural information.Most chemists are familiar with the frag-mentation observed with electron ioniza-tion mass spectrometry; however, be-cause ESI often produces only a minimalamount of fragmentation, it has beennecessary to induce fragmentation on theions formed by ESI. In general, the abil-

parent ion

daughter ion

ity to induce fragmentation and performsuccessive mass spectrometry experi-ments on these fiagment ions is known astandem mass spectrometry (abbreviatedMS"; where n refers to the number ofgenerations of fragment ions being ana-lyzed) and is illustrated in Fig. 4.

Fragmentation is usually achieved byinducing ion/molecule collisions. Thisprocess, known as collision-induced dis-

mass select parent ion(MS)

fragmentation of parent ion andanalysis for daughter Ions(MSZ)

fragmentation of daughter ion andana~sls for granddaughter ions

granddaughter ion

Fio. 4. Generation of firgment ions via collision-induced dissociation (CID) and the massanalysis (MS") of the progeny fiagment ions. The terms parent, daughter and granddaughter

ions (21) were used throughout this review to facilitate understanding, especially with the MS3discussion. However, precursor, product, and second-generation product ions are also com-monly used (21).

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collision gasinto Q2

electrospray 01 I03

ioa.I I =.I

Ms MMS +~rafrag

MMS2 +M frag

me M~+MM3 +MS Mtfrag

Q10 02 and 03 set to allow all ions to pass

Q1 Q2fragmentation

-* M+ -

(collision gas)

Q1 Q2fragmentation

-** M+f 00frag Ar

Q3daughterfragment -* %ions

Q3granddaughterfragment -* %ions

IDetector

I

mass spectrum

M+ M+

m/z

--~~~~~~~~~~~~~~~~~~~

m/z

FIG. 5. A triple-quadrupole ESI mass spectrometer with ion selection and fragmentationcapabilities. Each quadrupole has a separate function: The first quadrupole (Q1) is used to scanacross a preset m/z range or to select an ion of interest, the second quadrupole (Q2), also knownas the collision cell, transmits the ions while introducing a collision gas (argon) into the flightpath of the ion selected by Q1, and the third quadrupole (Q3) serves to analyze the fragmentions generated in the collision cell (Q2). For an MS experiment Q1 scans a over a selected m/zrange, and all the ions are observed. In the MS2 experiment the molecular ion M+ can beselected by Q1, which results in its fiagmentation at Q2. Analysis of the fragments occurs atQ3. An MS3 experiment can be performed if a daughter fragment ion is generated in ESI-i.e.,Mf . Q1 can be used to select the daughter ion, Q2 will generate granddaughter fragment ions,and Q3 will mass analyze for the granddaughter fragment ions.

x4 x2 x4 | x6 Ixl I

(M+H) +

C"Oo

._a

a)

1200

m/z

x3 y3 z3 x2 Y2 Z2 xI Y1 Z1

+TH-

+2H1 +2H1Ctria

N-terminal RI R2 R3I 1 I 11 11

NH2 -C- -N- -C--C--N--C--C--N--C-CO2HH H H H H

--j--jtm I +2 HI +211

al bI cl a2 b2 c2 a3 b3 c3

FIG. 6. An MS2 experiment (Upper) done on peptide (M+H)+ ions at m/z 1121 using100-300 fmol of material derived from 2 x 108 cells. The fragmentation processes (Lower) ofprotonated peptides occur through bond cleavages resulting in different classes of fragmentions. Fragment ions of type a", b5, and c. are generated if the charge is retained on the Nterminus of the peptide. The x,,, yn, and z,, ions are formed when the charge is retained on theC terminus. The figure is reproduced with permission from ref. 25 (copyright AmericanAssociation for the Advancement of Science, Washington, DC) and ref. 4 (copyright AnnualReviews, Inc., Palo Alto, CA).

sociation (CID) and/or collision-acti-vated dissociation (CAD), is accom-plished by selecting an ion of interestwith the mass analyzer and introducingthat ion into a collision cell. The selectedion will collide with a collision gas such asargon, and the collision may result infragmentation. The fragments can thenbe analyzed to obtain a daughter ionspectrum. The information obtained fromtandem mass analysis is primarily used toobtain structural information. ESI typi-cally uses a triple-quadrupole mass spec-trometer (22) with collision-induced dis-sociation to perform these analyses. Abrief description of MS2 and MS3 exper-iments with a triple-quadrupole mass an-alyzer is given in Fig; 5. ESI with asingle-quadrupole mass analyzer is alsocapable of generating and analyzing frag-ment ions; however, this is generallyapplicable to only very pure samples.

Perhaps the most well-known applica-tion of ESI tandem mass analysis (with atriple-quadrupole mass analyzer) is thework done by Don Hunt and colleagues(23-26). In these studies ESI tandem massspectrometry was used in conjunction withHPLC separation techniques to identifymajor histocompatibility complex-boundpeptides (Fig. 6). The quantity of peptideused to obtain structural information wason the order of tens of femtomoles, ulti-mately resulting in the identification of apeptide with a high affinity for cytotoxic Tlymphocytes (killer T cells) (23).Many biological problems are now be-

ing addressed at the molecular level;therefore, the ability to characterize acompound or compounds from biologicalmedia has taken on additional impor-tance. Don Hunt and colleagues havedemonstrated the ability to characterize9-residue peptides at very low quantities,thus addressing the characterization ofmajor histocompatibility complex-boundpeptides. Three laboratories at TheScripps Research Institute have also uti-lized ESI capabilities to investigate sleep(27) and the compounds associated withthis event. Lerner and colleagues (27)have performed studies on cerebrospinalfluid, extensively using HPLC-MS, MS2,and the MS3 (Fig. 7) capabilities of ESItriple-quadrupole mass analysis (Fig. 6).In these studies two novel lipid moleculeshave been isolated.ESI-MS is generally thought of as a

tool for macromolecule analysis; how-ever, as Hunt and Lerner have demon-strated, it is also useful in the character-ization of small molecules.Noncovalent Interactions. Another ap-

plication that has generated a great dealofexcitement involves the ability ofESIto produce and mass-analyze biologicalnoncovalent complexes in the gasphase. Since the first two papers (28, 29)describing ESI as a tool for the obser-vation of noncovalent complexes, the

daughter fragment Ions

m / M+

nlz

fragment ions

M+

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o 75.

0c 500Cua

) 25

0

parent282ion

daMS3

granddaughterions

. 6781 1 121 1 149V V 1~I- I I... 163 17719150 100 150

mhz

MS2

ughterion247

200 250 300

FIG. 7. Electrospray MS3 data on the daughter ion (m/z 247) of a cerebrodiene (27). Theparent ion, m/z 282, is added to the spectrum for clarity. The mass data, in conjunction withNMR and independent synthesis, have aided in the characterization of this compound.

possibility of correlating condensed-phase intermolecular interactions withmass spectrometry has captured the at-tention of many researchers. For thefirst time, mass spectrometry can beused as a tool to observe complexes inthe gas phase taken from an aqueousenvironment, thereby providing insightsinto specific noncovalent associations insolution. The selectivity of mass spec-trometry may eventually help avoid im-purity problems associated with immu-noaffinity procedures and also facilitatedrug screening. Examples include theobservation of the heme globin complex(29), the ternary complex of dimerichuman immunodeficiency virus type 1protease and an inhibitor (30), oligonu-cleotide duplex (31, 32), calcium-mediated cell-surface carbohydrate as-sociation (33), catalytic antibody-hapten interactions (34) (Fig. 8), andleucine zipper peptides (35).Recent work demonstrates that the

gas-phase intermolecular interactions ofbiomolecular ions observed with ESI-MSmay be related to the interactions ofthesecompounds in solution. Smith and Light-Wahl (36) have raised some interestingquestions concerning noncovalent com-

plexes observed with ESI and the rele-vance of these observations with respectto the solution-phase interactions. Re-cent data have suggested that, at least forsome biopolymers, the gas-phase ionsmay indeed reflect their solution-phaseproperties (37-41), with answers to thesequestions being obtained, in part, fromexperiments on protein folding.

Protein Folding. Protein folding repre-sents another example of a noncovalentinteraction: an intramolecular interac-tion. Brian Chait, one ofthe innovators inapplying ESI mass spectrometry (37),quickly recognized ESI as a tool for ob-serving noncovalent intramolecular in-teractions. One of Chait's and Katta'sinitial experiments (37) qualified ESI asan effective new method for probing con-formational changes of proteins in solu-tion. The method is based on the massspectrometric measurement of hydro-gen-deuterium exchange that occurs indifferent protein conformers over time;as a protein's tertiary structure changes,so does the number of its exchangeableprotons. Specifically, the method wasused to probe bovine ubiquitin confor-mational changes induced by the additionof methanol to aqueous acidic solutions

of the protein. The information obtainedfrom hydrogen-deuterium exchangemeasurements has also been used as acomplement to nuclear magnetic reso-nance (NMR) spectroscopy experiments(41). A combination ofESI-MS andNMRspectroscopy was used in experimentswith hen lysozyme to distinguish be-tween alternative mechanisms of hydro-gen exchange, providing insight into thenature and populations of transient fold-ing intermediates (Fig. 9). Further study(40) of other gaseous protein ions sug-gests their compactness in vacuo corre-sponds directly to known conformerstructures in solution. These studies sug-gest the nature of proteins in the gasphase is, to some extent, a reflection ofthe solution-phase structure.The ability to observe a basic protein

characteristic (native structure versusdenatured) has also been addressed byusing ESI-MS. ESI-MS has been identi-fied as a simple and effective device forinvestigating the denaturation ofproteins(37, 39) where denaturation has beenobserved as a function of temperature,solution pH, and solvent.The utility of ESI-MS in molecular

characterization, as a detector for HPLCand capillary zone electrophoresis, in thestudy of noncovalent complexes, and inobtaining structural information, all illus-

ESI-MSA B

26,769single-chain catalyticantibody/hapten complex

single-chaincatalvtin antihodv

21

26,000

6, I0IUVUY!6,420

26,500 27,000 27,500Molecular weight

FIG. 8. Noncovalent single-chain catalytic antibody-hapten complex as observed withpneumatically assisted electrospray (ion spray) mass analysis. Reproduced with permissionfrom ref. 34 (copyright American Chemical Society, Washington, DC).

molecular weight

FIG. 9. Time evolution of the ESI-MSspectra of lysozyme monitoring hydrogen-deuterium exchange (A) and from pulse-labeling studies ofprotein refolding (B). ForA,deuterated lysozyme was dissolved in 2H20and equilibrated. After specified lengths oftime, hydrogen exchange was quenched by therapid cooling. For B, the samples were pre-pared such that buffer exchange was made intoH20 (as in A), and exchange was performedbefore the samples were mass analyzed. Re-produced with permission from ref. 41 (copy-right American Association for the Advance-ment of Science, Washington, DC).

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metal surface20,OOOV

ultraviolet laser pulses

4Hf+ mass analyzer

protein ion desorbedfrom matrix

FIG. 10. MALDI source.

trate its capacity toward probing and solv-ing biological problems. ESI does havelimitations in that it is not very tolerant ofthe presence of salts (>1.0 mM) or of theanalysis ofmulticomponent samples. For-tunately, in several aspects of mass anal-ysis where ESI is not very useful,MALDI-MS has proven very effective.The combination of these two techniquesprovides great versatility in sensitivity,mass range, and applicability.

MALDI-MSMALDI-MS has also emerged as an ef-fective bioanalytical tool (4, 5, 42-48).The MALDI ionization technique (Fig.10) typically uses a pulsed UV laser beamto desorb and ionize cocrystallized sam-ple/matrix from a metal surface. Thematrix (e.g., 2,5-dihydroxybenzoic acid)serves to minimize sample damage fromthe laser beam by absorbing the incidentlaser energy, resulting in the sample and

100phosphorylated

C : N: Y: :LGpS: R:R:L

0-700 1200 1700 2200

1000 nonphosphorylated

.2; N N Y LGSA R R L

.CD ...

700

matrix molecules being ejected into thegas phase. The sample ion may be pre-formed in the condensed phase or resultfrom molecules that have reacted withthe matrix or other molecules to formeither singly or multiply charged ions (thesingly charged species are typically thedominant ions in the MALDI mass spec-trum). Once ions are formed in the gasphase, they can be electrostatically di-rected to a mass analyzer.MALDI is typically used in conjunction

with time-of-flight mass analyzers. Time-of-flight analysis is well-suited to thepulsed nature of laser desorption inMALDI (49). In addition, because time-of-flight analysis has virtually no uppermass range, it is compatible with MALDI,which can produce very high m/z ions.However, MALDI-MS is not limited tolarge biopolymers. It allows for the anal-ysis of both small and large molecules onthe femtomole level and has proven usefulfor both qualitative and, recently, quanti-tative analysis (50-54). In addition, theutility of MALDI-MS for heterogeneous

100

1200 1700 2200m/z

FiG. 11. Protein ladder sequencing of a 16-residue synthetic peptide that is phosphorylated(Upper) and not phosphorylated. A partialamino acid sequence is noted above the massspectra. Reproduced with permission from ref.58 (copyright American Association for the Ad-vancement of Science, Washington, DC).

samples makes it very attractive for bio-logical samples (43).MALDI is unique in its capacity to

analyze complex mixtures (55), produc-ing spectra uncomplicated by multiplecharging or significant fragmentation.For example, MALDI provides a routinemeans of analyzing glycoproteins (45, 56)and tryptic digests (57), which typicallyhave a high degree ofheterogeneity. ESI-MS, on the other hand, is useful foranalyzing mixtures after they have beenseparated by HPLC; yet its ability todirectly analyze very heterogeneoussamples is limited.A dramatic demonstration ofthe ability

ofMALDI to analyze heterogeneous sam-ples is shown with "protein ladder se-quencing" (58). Protein ladder sequencinginvolves the simultaneous analysis of amixture of peptides/proteins that haveundergone a stepwise Edman degrada-tion. Ladder-generating chemistry is ini-tially required, whereby a family of se-quence-defining peptide fragments aregenerated by using wet chemistry tech-niques. Each of these fragments differsfrom the nextby one amino acid. Once themixture of peptides is obtained, analysisby MALDI-MS can be done, thus gener-ating a sequence ladder (Fig. 11). Eachamino acid in this sequence is identifiedfrom the mass difference between succes-sive peaks. The protein ladder sequencingmethod has also been shown to be usefulin locating posttranslation modifica-tions-e.g., phosphoserine residues in aphosphopeptide (see Fig. 11 Upper).DNA Sequencing. The analysis of oli-

gonucleotides by MALDI has enjoyed areasonable degree of success (59-63),especially with the development of new

Prmer

6000 8000

C

m/z10,000 12,000

FIG. 12. Negative-ion MALDI mass spectra of synthetic oligonucleotides corresponding tomock A, C, G, and T sequencing reactions. The order ofthe peaks corresponds to the sequence.Reproduced with permission from ref. 64 (copyright Wiley, Sussex, U.K.).

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FiG. 13. Uptake of tetraphenylphospho-nium (TPP) by carcinoma cells as a function ofexposure time, as observed for unlabeled TPPwithMALDI-MS (e) and for tritiated TPP withscintillation counting (0) (52). Note the signif-icantly smaller error bars (representing 95%confidence limits) observed for MALDI-MS.In these experiments methyltriphenylphos-phonium was used as an internal standard.

matrices. Several groups (59, 64) havefurther demonstrated that MALDI mayalso be useful in DNA sequencing. Oneapproach is similar to protein ladder se-quencing and uses the time-dependenceof exonucleases (59) to generate a se-quence ladder. MALDI-MS has alsoshown some utility in the analysis ofDNA fragments generated in conven-tional dideoxynucleotide chain-termina-tion sequencing reactions (see Fig. 12)(64). These experiments have evaluatedMALDI-MS for oligonucleotide analysisin terms of resolution, sensitivity, multi-ple charging, and adduct formation, sug-gesting that MALDI-MS may have po-tential as a DNA sequence analysis tool.

Combinatorial Libraries. In addition tothe use ofMALDI as a sequencing tool, itscapability toward the analysis of combi-natorial libraries has also been explored(65). Specifically, Thomas Keough andcolleagues (65) have used MALDI-MS inan interesting set of experiments for therapid sequence determination of biologi-cally active peptides. These peptides havebeen isolated from support-bound combi-natorial peptide libraries. Sequence deter-

100

o,-ea)

Caa:

time-of-flight mass analyzerresolution =

quadrupole mass analyzerresolution =

/Ix 2500 i

Fourier-transform mass analyzerresolution250,000 ,

2535

mination is accomplished by encodingeach resin bead with the informationneeded to establish the sequence of theproducts also on the beads. MALDI-MScould then be used to read the sequences.In an example ofthe use ofthis technique,an anti-gpl20 monoclonal antibody wasscreened against a hexapeptide library,and six of the eight peptides isolated wereshown to possess the exact recognitionsequence for the antibody.The applicability of MALDI-MS to

proteins, peptides, complex samples, andcombinatorial libraries is evidence thatthis technique has achieved the prmaryfunction of an analytical device, to ....derive a measurable signal which is char-acteristic of the identity and/or amountof the species" (Fred W. McLafferty).The hundreds of papers that have beenpublished on MALDI typify its utility forcharacterization.

Quantitative Aspects ofMALDI-MS. Inaddition to producing a "characteristicsignal," quantitative information is oftennecessitated from analytical equipment.Recent papers have demonstrated theability of MALDI to yield quantitativedata. The use of MALDI-MS as a tech-nique for molecular weight determina-tions on biopolymers has been demon-strated repeatedly, yet until recently onlya limited amount of work has been pre-sented on its applicability to quantitationand the characterization of low-molecu-lar-weight compounds. Recent researchon the quantitative aspects ofMALDI hasincluded both small and large molecules.These initial studies have been done withcarbohydrates (50, 66, 67), peptides (51,68-70), proteins (53, 68), small molecules(51), and drugs (52, 66). In general theseresults suggest that the use of an internalstandard, similar to the molecule of inter-est, will enable quantitative information tobe obtained. Fig. 13 illustrates the appli-cability ofMALDI-MS to quantifying celldrug uptake using an internal standard ascompared with scintillation counting.MALDI-MS has an inherent problem

in obtaining quantitative information sim-

molecular formulaC1011.H145.N34.044

monoisotopic massl 2538.0153 Da

average mass2539.4832 De

2538 2540 2542 2545m/z

FIG. 14. The mass spectra of a singly protonated peptide calculated at a resolution of 250,2500, and 250,000. The respective resolving powers correspond to the resolution typicallyobtained for time-of-flight, quadrupole, and Fourier-transform mass analyzers. Notice that theindividual isotope peaks can be distinguished at the higher resolution.

ply as a function of signal intensity; thenonuniform nature of the matrix/analytesolid mixture and variations in detectorresponse often result in a nonreproduc-ible signal response. When MALDI-MSis used for qualitative work, this is notimportant, as the laser fluence is simplyadjusted for each sample to provide anacceptable signal. ForMALDI to be usedfor quantitative analysis, it is essential tocompensate for this variation. The incor-poration of an internal standard allowsfor controlled shot-to-shot and sample-to-sample variability. A prerequisite for asuitable internal standard is that it shouldmimic the behavior of the analyte. Inmost studies referenced, this has beenaccomplished by the addition of struc-tural analogues or stable-isotope-labeledanalogues as internal standards.Mark Duncan and colleagues (51) have

pointed out several reasons whyMALDI-MS is an attractive method forquantitative analysis in the low-molecu-lar-weight range. These reasons includerapid analysis time, high sensitivity, spec-ificity, low cost, and versatility. Theseadvantages also are true for biopolymers,as demonstrated by Randy Nelson andcolleagues (53), who have also illustratedthe ability ofMALDI to be used as a toolfor quantitative analysis of proteins.These examples further demonstrate

the capabilities of MALDI-MS as a toolfor characterization and quantitation.However, because MALDI is usuallycombined with time-of-flight mass anal-ysis, it typically suffers from low resolu-tion, which, in turn, adds difficulty tointerpreting the mass spectra accurately.Increasing accuracy has been a primarymotivating factor for MALDI-MS in thedevelopment of other higher-resolutionanalyzers, including the time-of-flight re-flectron and Fourier-transform ion cyclo-tron resonance mass analyzers. For in-stance, the time-of-flight reflection ana-lyzer can provide good resolution and hasrecently been demonstrated as a tool fortandem mass analysis (71, 72). The com-bination of time-of-flight and reflectronmass analyzers has significantly in-creased the resolving power of these in-struments for m/z c 6000. However,Fourier-transform ion cyclotron reso-nance mass spectrometry (FTMS), com-bined with both ESI and MALDI, arenow offering the greatest potential inhigh-resolution mass analysis.

High-Resolution ESI and MALDI-MS.An important characteristic of any massspectrometer is its resolving power. Res-olution is the ability of a mass spectrom-eter to distinguish between ions of differ-ent mass-to-charge ratios. Greater re-solving power corresponds directly to theincreased ability to differentiate ions. Forinstance, a mass spectrometer with aresolution of500 can distinguish betweenions of m/z = 500 and 501, whereas a

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100-

theoreticaldistribution

01

Z

29998 29025 29052

Molecular weight

FIG. 15. Single-scan ESI spectrum of carbonic anhydrase with a FTMS analyzer. Repro-duced with permission from ref. 75 (copyright Elsevier Science, Inc., New York).

mass spectrometer with a resolution of2500 can distinguish between ions ofm/z= 2500 and 2501. The most commondefinition of resolution is given by theequation: Resolution = M/AM.

This equation can be described in anumber of different ways (73). Perhapsthe most common is whereMis the massof a peak of interest and AM can bedefined as the width ofthe peak at 50%6 ofthe height or "full-width at half maxi-mum" (FNWHM).High resolution is important for

biopolymer analysis because it allowsfor high accuracy. Fig. 14 demonstrateshow resolution affects the peak shapein the mass spectrometer for a com-pound having the molecular formulaClolHI45N34044.ESI and MALDI-MS commonly use

quadrupole and time-of-flight mass ana-

lyzers, respectively. The limited resolu-tion offered by time-of-flight mass ana-lyzers, combined with adduct formationobserved with MALDI-MS, results inaccuracy on the order of 0.1% to a high of0.01%, whereas ESI typically has an ac-curacy on the order of 0.01%. Both ESIand MALDI are now being coupled tohigher-resolution mass analyzers, suchas the ultra-high resolution (> 105)FTMS. The result of increasing the re-

solving power of ESI and MALDI massspectrometers is an increase in accuracyfor biopolymer analysis.FTMS, first introduced in 1974 by

Comisarow and Marshall (74), is basedon the principle of a charged particleorbiting in the presence of a magneticfield. While the ions are orbiting, a radiofrequency signal is used to excite theions. As a result of radio frequency ex-

citation, the ions produce a detectableimage current. The time-dependent im-age current can then be Fourier-trans-formed to obtain the component frequen-cies of the different ions. The componentfrequencies of the ions correlate to theircorresponding m/z. FTMS offers twodistinct advantages, high resolution andthe ability to perform multiple collisionevents (MS", where typically n < 4) togain structural information.Coupled with FTMS, ESI (75) (Fig. 15)

and MALDI (76, 77) promise to be im-portant research tools for biotechnology.Although both techniques are still in theirinfancy, their application provides valu-able molecular weight information on awide variety of samples. ESI producesmany multiply charged species of thesame mass, and isotopic peak resolutionprovides unequivocal charge-state as-signment from the spacing of the iso-topes. This capability is of special valuewhen the spectrum also has many ions,such as from fragmentation. Mass mea-suring errors not only are lower (<0.1 Daor < ±0.001% error) than when the iso-topic peaks are unresolved but also areindependent of variations in 13C/12C nat-ural isotopic abundances (the ability todistinguish the individual isotopes of aprotein is shown in Fig. 15). Further-more, larger errors are avoided that oc-cur when the measured peak envelopeincludes impurity or adduct ions. Thisattribute also benefits tandem mass spec-trometry of peptides, in that the disso-ciation of these ions yields fragmentmasses consistent (to <0.1 Da) with theiramino acid sequences (78). This technol-ogy has also been proven useful for therapid sequencing of oligonucleotides aslarge as 25 bases (79).

CONCLUDING REMARKS ANDFUTURE PROSPECTS

The development of new ionizationsources has been the platform for routine

Table 1. Comparison of ESI-MS and MALDI-MSMass limit, Da

(practical) Advantages Disadvantages Suitable compoundsESI-MS -200,000 HPLC/MS capable Multiple charging can be Peptides

(70,000) Multiple charging confusing with mixtures ProteinsResolution -2000 Typically need c mM salt CarbohydratesCapable of observing noncovalent concentrations for good Nucleotidescomplexes directly from water signal Oligonucleotides

Femtomole-to-picomole sensitivity Not tolerant of mixtures PhosphoproteinsGood accuracy -±0.01% (if sample too heterogeneous Small chargeable molecules

often little or no signal)MALDI-MS >300,000 Tolerant of mM concentrations Typically low resolution (s500) Peptides

(150,000) of salts Accuracy ±0.1%-0.01% ProteinsHighest mass capability Not amenable to LC/MS GlycoproteinsTolerant of mixtures CarbohydratesFemtomole sensitivity NucleotidesBeing developed as a tool for Oligonucleotides

sequence analysis PhosphoproteinsSmall chargeable moleculesHeterogeneous samples

LC, liquid chromatography.

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biomass analysis, resulting in the applica-tion of ESI and MALDI-MS toward awide range of biochemical problems. Thecomplementary nature of ESI andMALDI-MS (Table 1) has made it increas-ingly important that researchers have ac-cess to both types of instrumentation.Independently, ESI and MALDI-MS canhelp answer many questions; yet togetherthey represent a formidable research toolwith new levels of sensitivity, accuracy,and mass range. Fortunately, because thisinstrumentation is becoming more com-mercially available, chemists and biolo-gists may soon see these mass spectrom-eters as companions to their NMR spec-trometers and electron microscopes.

Significant design changes in the ionsource have redefined the applicability ofmass spectrometry for biotechnology re-search. Perhaps it is time to consider adevice that hasn't changed significantlyover the past 30 years: the ion detector.If larger or more complex systems are tobe observed, changes in the ion detectorwill likely be the next step in the devel-opment of mass spectrometry.

I thank the many talented scientists for theirinvaluable contributions to this research ef-fort-particularly John B. Fenn, Franz Hillen-kamp, Michael Karas, and Brian T. Chait. Ialso thank Richard A. Lerner and StephenB. H. Kent for valuable discussions beforeand during the course of this review. I alsoappreciate the help of Mark W. Duncan,Michael C. Fitzgerald, Susan D. Richardson,Jennifer Boydston, Thomas Knapp, andChristopher Claiborne in their careful reviewof this manuscript. I gratefully acknowledgefunding from The Lucille P. Markey Charita-ble Trust and National Institutes of HealthGrant 1 S10 RR07273-01.1. Fenn, J. B., Mann, M., Meng, C. K., Wong,

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