In dian Journal of Bi ochemistry & Bi ophys ics Vo L 39. August 2002. pp. 205-2 16

Minireview

Mass spectrometry and protein structure

Kapil Maith al' and K Muralidhar2* 'Dr. B R Ambedkar Center for Biomedi cal Research. 2Department of Zoology. Uni ve rsity of Delh i. Delhi 11 0007, India

Received 24 April 2002; accepled 6 lillie 2002

Historical Overview ha s led to the usage of cesium (Cs) instead of atom Today's mass spectrometer is based on the seminal gun . wo rk performed by Sir J. J. Thomson of the Another method that all owed analys is of large, Cavendi sh Laboratory (Uni versity of Cambridge), non-volatil e orga nic molecul es was plasma­ whi ch led to the di scovery of the electron in 1897 and desorpti on (POMS) developed by 5 6 also led to the first mass spectrometer whil e he was Torgerson et al. in 1974 . . Wi th the advent of th is measuring the effects of electric and mag neti c fie ld s tec hnique analysis of proteins also became poss ible 7 on ge nerated by residual gases in cathode ray and in 1982 first ma ss spectrum of in sulin wa s tubes. Thomson noti ced that the ions move th rough recorded. This was soon foll owed by FAB mass 8 9 pa raboli c trajectori es proporti onal to their "mass-to­ spectra of in sulin . . In spite of these ac hievements. charge" rati os. Thomson received the 1906 Nobel both the techniques had maj or limitations, li ke it wa s Pri ze in Phys ics "in recogniti on of the great merits of difficult to ex tend the detecti on mass range beyond 25 his theoreti ca l and experimental in vesti gati ons on the kOa and it was useful for a few ideall y behaved conduct of electric ity by gases". The time period proteins onl y. Further, sensiti vity of pi comole ran ge from the late 1930's can be consi dered the era, which was not as good as desired and analys is of mi xtures saw major breakthroughs in the field of mass was difficu lt due to suppression effec ts. spectrometry. But it was onl y around 1950 that mass This led to constant development and in mid j 980's spectromet ric (MS) analysis of peptides came into two new techniques were developed viz. electrospra y exi stence. The onl y shortcoming at that time wa s that ion ization (ESJ) and matri x assisted laser the onl y method ava il able was electron desorpti on/ioni za ti on (MALDI), whi ch had a impact and it requi red extensive derivatizati on of the sign ifica nt impact on the capabilities of mass peptide to make it vo lat il e. Inspite of this , many spectro metry. ESI was first conceived by Malcolm groups led by M. Shemya kin in USSR', K. Biemann Dole in the 1960's and by in corporatin g the in MIT, USA" and E. Lederer in France) were act ive ly tec hn ology that had become ava il abl e over the yea rs, in vo lved in research. John Fenn put ESI into use for biomolec ul e analysi s ]n J 970s, MS analys is of peptides progressed in the j 980'SI0. On the other hand MALOI uses a laser slowly, alth ough its potential was demonstrated by to desorb sampl e mol ecules from a solid or liqu id many groups for structure elucidation of modified matrix contain ing a hi ghl y UV -absorbing substance peptides. A rea l breakthrough came when Mi chael an d was developed by Franz Hillenk amp and Michael (Mickey) Barber discovered a new techn iqu e fo r Kara s". desorption and ionization of non-vo latil e organic Both ES I and MALOI fac ilit:lted adva ncements in compound s called (FAB( the app lications of mass spectrometry in the fi eld s of 1n FAB , a beam of 3- 10 keY argon atoms was Ll sed to biology and med icine. In the j 990's, coupli ng of mass effec t desorption and ioni za ti on . Later it was found spectrometry with liquid chromatography syste ms that prima ry ions work just as we ll as atoms and this add ed a new dime nsion to the research in and bi oc hemistry. The limitati ons of mass *A uth or for correspondence spectro metry have yet to be defin ed as larger and 206 INDIAN J. BlOCHEM., BlOPHYS ., VOL. 39, AUGUST 2002

more complex molecules are being successfully production of ions fl~om collision with the analyte is characterized. Applications that were once stable. Even at a stable rate of ion production, inconceivable are In use today and include, ionization efficiency is low with EI techniques. In sequencing of peptides and proteins; studies of general, only one molecule in a thousand will undergo noncovalent complexes and immunological ionization. molecules; DNA sequencing; and the analysis of Ionization efficiency can be infl uenced by the intact viruses, to name a few. degree of interaction between the analyte and the electrons. The amount of interaction, and therefore the Introduction ionization efficiency, can be increased by increasing Mass spectrometry is one of the most important the anode current, increasing the width of the electron physical methods in analytical chemistry today beam, and increasing the sample pressure in the ion 2 bas ically because of its high sensitivit/ . The MS source. As always, there are practical limitations on performs primarily three functions; ionization of the extent to which one can increase each of these analyte molecules, resolve analyte molecules on the parameters. bas is of their mlz ratio and detection (Fig. 1). During the interaction of the energetic electron and the analyte molecule, a sufficient amount of energy is Ionization Methods transferred to the analyte to overcome its ionizati on The sarvple ions necessary for mass analysis are potential. The electron may transfer additional energy produced in the ion source. A good ionization method to the analyte, which may lead to the molecule should offer high ionization efficiency, should not be shedding two or even three electrons or to sensitive to the ion source conditions (e.g. , pressure or fragmentation of the initial molecula r ion. After temperature) or sample impurities, should produce ionization occurs, the ions are accelerated and then ions quickly, and should only produce one molecule focused through a potential field and sent on to the (structure) per ion (not isomeric mixtures). mass analyzer.

1. Electron Ionization (EI) A-B ---1~. [A-Bt + e' In EI the sample is vapourized usually by thermal [A-Bt ~ A + + B desorption. Once a sample is in the gas phase it passes into an electron impact ionization chamber. This small chamber contains a metal filament that is heated Electron impact is one of the most popular through a CUITent of 3-4 A to a temperature at which it ionization techniques for organic mass spectrometry. emits free electrons. The energy of the electrons is It is an easy technique that is very reproducible and controlled by the potential difference between the can be used for quantitati ve studies. Thi s method fil ament and the anode. tends to produce large amounts of fragments, whi ch The energy of the electrons is variable from 20-120 can either be a benefit or a di sadva ntage depending on e V (electron volts; 1 e V = 23 kcal), but reference the system. EI works best for molecul es under 400 Da spectra are typically obtained at 70 eV. At this energy because larger molecul es tend to be non- vo latil e and level, fragmentation patterns are reproducible and the decompose during vaporization.

Analyte Mass Filter/ Molecules/ Ionization Source Analyzer Detector Sample

+ + + + +, + 00 + o + + + + t + +'"t"++ + + + ++ ++ + ++ 08~ ~ ++-It- + + ---+ 00 0 + + + ++ ... 00 + ++-f+ + +++ o 0 o + ++ + + + + + + + ~~Il~ ______~I~--~ ______~

Fi g. I- A schemati c di agram, showing th e bas ic design of a mass spectro meter. MAITHAL & MURALlDHAR : MASS SPECTROMETRY AND PROTEIN STRUCTURE 207

2. Chemical Ionization (CI) 3. Fast Atom Bombardment (FAB) CI is known as a soft ionization technique because The particle desorption methods were developed in the analyte is ionized without the transfer of excessive the late 1970's and early 1980's, superceding the energy to the resultant ions. Since the sample ions earlier field desorption method. The first particle have little excess internal energy, fragmentation is desorption method was plasma desorption, whi ch much less frequent in CI than in EI. CI is therefore made use of the 252Cf fission process to initiate useful for molecular structure and molecular weight desorption. The technique was fairly successful, but it studies. In addition, CI spectra can often reflect fine was soon overshadowed by the development of 4 14 I6 differences in structural isomers. CI is typically used FAB . ,IS and LSIMS , for the same types of samples as EI and will often The techniques of FAB and LSIMS invol ve the produce more abundant molecular ions than E11 3. bombardment of a solid analyte + matrix mixture by a

> The CI ion source is essentially the same as the EI fast particle beam. The matrix is a small organic source. A similar metal filament is used to produce an species (glycerol or 3-nitrobenzyl alcohol, 3-NBA) electron beam, which then ionizes the reagent gas. which is used to keep a 'fresh' homogeneous surface Typically, methane, isobutane, or ammonia are used for bombardment, thus extending the spectral lifetime as reagent gases and are introduced at a relatively and enhancing sensitivity. In FAB, the particle beam hi gh pressure of 1 torr. Analyte molecules, however, is a neutral inert gas, typically Ar or Xe, at are introduced into the chamber at about 10'6 tOIT. bombardment energies of 4-10 KeV, whereas in Since the reagent gas molecules, greatly outnumber LSIMS, the particle beam is an ion, typically CS+, at the analyte molecules, the electron beam bombardment energies of 2-30 Ke V. preferentially ionizes the reagent gas. The particle beam is incident at the analyte surface, Once the ionization by the energetic electrons has where it transfers much of its energy to the occurred, the reagent gas ions undergo ion-molecule surroundings, setting up momentary collisions and reacti ons with the reagent gas neutrals. Some of the disruptions. Some species are ejected off the surface products of these ion-molecul e reactions can then go as positive and negative ions by this process, and on to react with the analyte molecules to form sample these 'sputtered' or secondary ions are then extrac ted ions. Typically, the ionization of methane through EI, from the source and analysed by the mass fo ll owed by the ion-molecule reaction to form the spectrometer. The polarity of the source extrac ti on molecular ion of the neutral sample molecule (M) is can be switched depending on what species are to be shown as: analysed. Both FAB and LSIMS are comparatively soft CH.j+ + e' ~ CH + + 2e' 4 ionization techniques, and are thus well suited to the CH/ + CH.j CHs+ + CH ~ 3 analysis of low volatile species. CHs+ + M ~ CH4 + [M+Ht

The degree of fragmentation in CI can be 4. Matrix Assisted Laser Desorption Ionization controlled by the choice of reactant gas. In most (MALDI) cases, a proton is donated from a reagent gas ion to MALDI mass spectrometry (Fig. 2), first the analyte, thereby forming a protonated molecule introduced in 1988 by Hillenkamp and Karas, has (molecular ion). The amount of excess energy become a widespread analytical tool for peptides, transferred to the molecular ion when it is formed is proteins and most other bi omol ec ul es determined by the relative proton affinities of the (oligonucleotides, carbohydrates, natural products and reagent gas ion and the analyte molecul e. The greater lipids) 17,18. It is similar to FAB, but uses pulsed laser the proton affi nity of the conjugate base (i.e. , the li ght and solid . matrix. The efficient and di rected greater the acidi ty of the reagent gas), the greater the energy transfer during a matrix-assisted laser-induced energy that is transferred to the protonated molecule desorption event provides hi gh ion yields of the intact and the greater the degree of fragmentation. analyte, and allows for the measurement of A complex mixture of neutra l and ionic species are compounds with high accuracy and sub-picomole formed during either chemical or electron impact sensitivity, ion iza tion. In general, only posi tive ions are sent on to MALDI provides for the nondestructi ve the ma ss analyzer, wh il e the neutral and negati ve vaporization and ionization of both large and small species are discarded. bi omolecules. In MALDI analysis, the analyte is first 208 INDIAN J. B10Cl-lEM ., BIOPHYS., VOL. 39, AUGUST 2002 co-crystalli zed with a large mol ar excess of a matrix 5. Electrospray and [on-spray Ionization (ESI) compound , usuall y a UV-absorbing weak organic Electrospray ioni zation l 9 (Fi g. 3) ge nerates ions acid, after which pulse UV laser radiation of this directly from solution (usually an aqueous or analyte-matrix mi xture results in the vapori zation of aqueous/organic solvent system) by creating a fin e the matrix which canies the analyte with it. The spray of highly charged droplets in the presence of a matri x therefore pl ays a key role by strongly strong electric fi eld (typically 3.5 kV). As th e dropl et absorbin g the la ser li ght energy and causing, decreases in size, the electric charge density on its indirectly, the analyte to vaporize. The matrix al so surface increases. The mutual repul sion between like se rves as a proton donor and receptor, acting to ioni ze charges on thi s surface becomes so great that it the anal yte in both positive and negati ve ioni zation exceeds the forces of surface tension, and ions begin modes, respectively. to lea ve the droplet through what is known as a MALDI has had its biggest impact on the fi eld of "Tay lor cone,, 20 (Fig. 4). The Ion s are then protein research. The ability to generate MALDI-MS electrostaticall y directed into the mass ana lyze r. da ta on whole proteins and proteolytic fra gments is Vaporization of these charged droplets results in the ex treme ly useful for protein identi fication and production of singly or multiply-c harged gaseous characte ri zation. ion s. The number of charges relained by an analyte

DctcclOr

Detector

Fi g. 2- A schematic diagram of MALDI showing the formation of ions by lase r deso rption and pass ing of th e ions through mass ana lyzer 10 the detector.

Ion formation Ion transmi ss ion Ion de tcc ti oll

Skl mmers lligh Energy Dynode Dete ctor

+v

Lenses/

Atmospheric Pressure Vaccum

f-i g. 3--A schelllatic diagram o f ESI sho wing the formation of chargcJ ion,. \\ hic h arc pushed th ro ugh a mass analyze r to th e detector. MAITHAL & MURALlDHAR : MASS SPECTROMETRY AND PROTEIN STRUCTURE 209 can depend on such factors as the composition and pH 6. Atmospheric Pressure Chemical Ionization of the electrosprayed solvent as well as the chemical (APCI) nature of the sample. For small molecules « 2000 Similar to electrospray ionization, liquid effluent is Daltons) ESI typically generates singly or doubly introduced directly into the Atmospheric Pressure charged ions, while for large molecules (> 2000 Chemical Ionization (APCI) source, however the Daltons) the ESI process typically gives rise to a similarity with electrospray stops there. The APCl series of multiply-charged species. Because mass source contains a heated vaporizer which facilitates 2 1 spectrometers measure the mass-to-charge (m/z) ratio, rapid desolvation/vaporization of the droplets • the resultant ESI mass spectrum contains multiple Vaporized sample molecules are carried . through an peaks corresponding to the different charged states ion-molecule reaction region at atmospheric pressure. (Fig. 5). The ionization occurs through a corona discharge, ESI allows for very sensitive analysis of small, creating reagent ions from the solvent vapour (Fig. 6). large and labile molecules such as peptides, proteins, Chemical ionization of sample molecules is very organometallics, oligosaccharides, and polymers. efficient at atmospheric pressure due to the hi gh Another advantage of ESI-MS is that ions are formed collision frequency. Proton transfer (protonation MH+ directly from solution (usually an aqueous or reactions) occurs in the positive mode, and either aqueous/organic solvent system), a feature that has electron transfer or proton transfer (proton loss, [M­ establi shed the technique as a convenient mass Hn in the negative mode. The moderating influence detector for high performance liquid chromatography of the solvent clusters on the reagent ions, and of the (HPLC). high gas pressure, reduces fragmentation during

A \£JJ

Hi ghl y charged droplet Tay lor' Cone Formati on of small er droplets

Fig. 4--Electrospray ioni zation and the formation of Taylor cone.

100 A: 15126 4E6 3.5E6 80 3E6 2.SE6 B:15867 2E6 . 60 I .SE6 IE6 500000 40 0 - J 15000 15500 16000 20

I 0 Illl , , , , , , , , J, J, , , , 1000 1500 2000 m/z

Fig. 5-Chargc state di stribution of hemoglobin in a typi cal electrospray mass spectrum. [Inset shows th e deconvoluted masses of alpha and beta chains of hemoglobin.] 210 INDIAN J. BIOCHEM ., BIOPHYS., VOL. 39, AUGUST 2002

Reagent gas

Corona Discharge Detector

Sample

Mass Analyzer

Fig. 6--Atmospheri c pressure chemical ioni zation (A PC!) mass spectrometry showing the ionization of the analyte sampl e through a cororu discharge.

io ni za ti on and results in primarily molecular ions. APCI is widely used in the pharmaceutical industry to analyze relatively nonpolar, semi volatile samples of less than 1200 Daltons and it is an especially good ion izati on source for liquid chromatography. Mass Analyzers Immediately following ionization, gas phase ions enter a reg ion of the mass spectrometer known as the mass analyzer. The mass analyzer is used to separate . ""'1 ions within a selected range of mass-to-charge (mlz) raLios. The analyzer is an important part of the in strument because of the role it plays in the Fi g. 7- Longitudinal view of a quadrupole mass anal yzer.. rTh e in strument's accuracy and mass ran ge. Ions are "A" rods are connected and are at the same DC and superimposed Lypically separated by magnetic fields, electric fields, RF voltages. The same is true of the " B" rods; however, they ha ve an opposite DC vo ltage with respect to the "A" rods, and th e RF or by measuring the time it takes to travel a fixed fi eld is phase shifted by 180".] distance. 1. Quadrupole Analyzer distribution below mlz 3000. Finally. the relatively In a quadrupole analyzer; a voltage made up of a low cost of quadrupole mass spectrometers makes DC compon ent , U and a RF component, Vcos(wt) is them attracti ve as electrospra y anal yzers. app li ed between adjacent rods of the quadrupole as sembly, whereas, opposite rods are connected 2. Double-focusing Magnetic Sector Mass Analyzer electrica lly (Fig. 7). With a correct choice of voltages, In magnetic sector analyzers, the Ion s are only ions of a given mlz value can traverse the accelerated (using an electric field) and are passed 22 analyzer to the detector . into a magnetic field. A charged particle tra ve lling aL Quadrupole mass analyzers have been used in high speed through a magnetic field will ex perience a conjunction with electron ionization sources since the force, and travel in a circular motion wi th a radius 1950s and are the most common mass spectrometers depending upon the mlz and speed of the ion. A in ex istence today. Quadrupoles have three primary magnetic analyzer separates ions accord in g to their advantages. First, they are tolerant of relatively poor radii of curvature, and therefore onl y ion s of a given 5 vacuums (-5 x 10- torr), which make them well mlz will be able to reach a point detector at any given suited to electrospray ionization since the ions are magnetic field. produced under atmospheric press ure conditions. Secondl y, quadrupoles are now capable of routinely In order to improve resoluti on , single-sector anal yz ing up LO a rnlz of 3000, whic h is usefu l magnetic in struments have been replaced with beca use electrospray ionization of proteins and other double-sector in strumen ts by combin in g the mag netic biomolecu les commonly produces a charge mass analyzer with an electrostatic analyze r. The MAITHAL & MURALIDHAR: MASS SPECTROMETRY AND PROTEIN STRUCTURE 21 1 electric sector acts as a kinetic energy filter allowing (a) Ion Source only ions of a particular kinetic energy to pass . I . 23 through its field, thus en hancmg mass reso utIOn (Fig. 8).

3. Time-of-flight (TOF) Analyzer A time-of-flight (TOF) analyzer is one of the simplest mass analyzing devices and is commonly used with MALDI. Time-of-flight analysis is based on accelerating a set of ions to a detector with the same amount of energy. Because the ions have the same energy, yet a different mass, the ions reach the detector at different times. The smaller ions reach the Ion Source detector first because of their greater velocity and the (b) larger ions take longer, thus the analyzer is called time-of-flight and the mass is determined at the ions' time of arrival.

The arrival time of an ion at the detector is dependent upon the mass, charge, and kinetic energy of the ion.

2 Since kinetic energy (KE) = 112 mv ; Electrostatic analyzer or velocity v = (2KE/m)/2;

ions will travel a given distance, d, within a time, t, where t is dependent upon their rnIz24. Fig.8-I1lustration of the improved resolution obtained wi th a two-sector double focussing instrument (b) over a sin gle focussing instrument (a). 4. Fourier Transform-Ion Cyclotron Resonance Fourier-transform ion cyclotron resonance mass 26 and detected as the radio frequency field is scanned . spectrometry (FrMS) offers two distinct advantages, Further, it is also possible to isolate one ion species by hi gh resolution and the ability to tandem mass ejecting all others from the trap. The isolated ions can spectrometry experiments. First introduced in 1974 by subsequently be fragmented by collisional activation Comjsarow and Marshall, FrMS is based on the and the fragments detected to generate a principle of a charged particle orbiting in the presence fragmentation spectrum. The primary advantage of 25 of a magnetic field . While the ions are orbiting, a quadrupole ion traps is that multiple collision-induced radio frequency (RF) signal is used to excite them and dissociation experiments can be performed without as a result of this radio frequency excitation, the ions having mUltiple analyzers. Other important produce a detectable image current on the cell in advantages include its compact size and the ability to which they are trapped. The time-dependent image trap and accumulate ions to increase the signal-to­ current can then be Fourier-transformed to obtain the 27 noise ratio of a measurement . component frequencies of the different ions, which correspond to their rnIz. Tandem MS Nowadays use of multiple mass spectrometers 5. lOll-trap (MSn) is being used in tandem. This provid es In an ion trap the ions are trapped in a radio structural data via MS analysis of parent ion frequency quadrupole field . The ions are then ejected fragmentation productsD~ . A Wt· d e variety. 0 f 212 INDIAN J. BIOCHEM., BIOPHYS., VOL. 39 , AUGUST 2002 instrumental setup for tandem MS are available. One MS of the most widely used is triple-quadrupole ESI MS Q2 and Q3 set to allow all ions to pass --- Spectra (Fig. 9), which works as shown below:

M" ~ Daughter --. Q3 set to allow --. Spectra fragment all ions to pass eID ions MS (select ions of interest) Ions

M' Q, Daughter Q3 Grand- collision-induced dissociation (Cm) • - fragment - daughter - . Spectra elD ions elD fragment (initiate fragmentation) MSfMS (analyze ions 1 for fragments) Fi g. 9--I1lustration showing the ionization processes ill a si ngle. Daughter Ions double and triple quadrupole mass spectrometer. cm electrons. These secondary electrons are then attracted MSfMSflvlS (analyze for fragments to the next dynode where more secondary electrons of fragments) are generated, ultimately resulting in a cascade of 1 electrons. Granddaughter Ion~ 3. Photomultiplier Conversion Dynode or Scintillation Counting or Daly Detector The photomultiplier conversion dynode detector is Ion Detectors similar to an electron multiplier where the ions Once the ion passes through the mass analyzer it is initially strike a dynode, resulting in the emi ss ion of next detected by the ion detector, the final element of electrons. However, with the photomultiplier the mass spectrometer. The detector allows a mass conversion dynode detector, electrons strike a spectrometer to generate a signal current from phosphorus screen. The phosphorus screen, much like incident ions by generating secondary electrons, the screen on a television set, releases photons once whi ch are further amplified. an e lectron strikes. These photons are then detected by a photomultiplier, which operates with a cascading action much like an electron mUltiplier. T he primary 1. Faraday Cup advantage of the conversion dynode setup is that the A Faraday cup operates on the basic principle that photomultiplier tube is sealed in a vacuum (photons a change in charge on a metal plate results in a flow pass through sealed glass), unexposed to the internal of electrons and therefore creates a current. One ion environment of the mass spectrometer. Thus the striki ng the dynode (a secondary emitting material) possibility of contamination is removed. induces several secondary electrons to be ejected and temporarily displaced. This temporary emission of electrons induces a current in the cup and provides for Applications a small amplification of signal when an ion strikes the The applications of MS are so many that it is cup. beyond the scope of this review to even li st them all. But the impact of modern mass spectrometry on chemistry and biochemistry will be discussed below. 2. Electron Multiplier Some of the applications of modern MS include An electron multiplier is one of the most common protein structure, drug metabolism (pharma­ means of detecting ions, achieving high sensitivity by cokinetics), perfumery (flavor and smell chemi stry), extending the principle used with a Faraday cup. An petroleum and petrochemicals, organic foss il s, electron multiplier is made up of a series of dynodes inherited metabolic diseases, atmospheric chemistry, maintained at ever increasing potentials. Ions strike the analysis of respiratory gases, viral identificati on, the dynode surface, resulting in the emi ss ion of forensics, and many other specialized subjects. MAITHAL & MURALIDHAR : MASS SPECTROMETRY AND PROTEIN STRUCTURE 213

Table I-Landmarks in the evolution of mass spectrometry

Year Achievement Scientist 1897 Discovery of electron Sir J. J. Thomson -1918 Introduction of high re solution of mass spectrometers Francis W. Aston & Arthur J. Dempster -1925 Advancements in vacuum technology and electronics reduce the size of Arthur Neir mass spectrometers

1946 Concept of Time-of-Flight introduced Willi am E. Stephens -1956 Concept of quadrupole analyzers introduced Wolfgang Paul 1960s Concept of electrospray ionization mass spectrometers introduced Malcom Dole " 1974 Introduction of plasma desorption mass spectrometers (PD-MS) D. F. Torgerson

1976 Secondary ion mass spectrometer (SIMS) introduced Benninghoven

1981 FAB-MS introduced Michael Barber 1988 ESI-MS introduced John Fenn 1988 MALDI introduced &

process is involved; in exchange reactions it can show 1. Organic Chemistry that particular atoms of, for example, hydrogen Mass spectrometry has a critical role in orgamc undergo exchange between the reacting species. chemistry. Its utility in chemical analysis is Labelling is also widely used in mass-spectrometric tremendous. It can be used in determining the research to give information about the fragmentation structure of compl icated molecules using the high reactions occurring in the mass .spectrometer. sensitivity of modern mass spectrometers and Further, with the advent of combinatorial fr agmentatl.o n ana I·YSIS 30-33 . It IS . .Important to note t hat chemistry, mass spectrometry is playing an predicting definitively the fragmentation patterns for increas ingly important role in the mol ec ul ar orga nic molec ul es is still very difficult, but many characterization and actl vlty of combinatorial semi-empirica l rules of fragmentation are known, and libraries. Toward this end, electrospray ionization and MALDI-MS have been useful for the qualitative, and it is usually possible to pick out peaks in the spectrum more recently, the quantitative screening of that are characteristic of particular chemical groups34. combinatorial libraries. Moreover, the developmen t of The techniq ue is valuable in that it is generally not these two techniques has significantly extended MS necessary to know any details of the composition of application toward a wide variety of challenging the unkn own compound in order to deduce a complete problems in drug discovery and toward the or partial stru cture. identification of effective ligand-receptor binding, new catalysts, and enzyme inhibitors. In addition, Continuous sampling of the materials contained in because mass spectrometry does not in volve a reaction v ssel, fo llowed by analysis with a mass chromophores or radiolabelling, it provides a viable spectrometer, has been used to identify and measure alternative to existing analytical techniques which the quantit y of intermediate species formed during a typically require extensive sample preparation and reaction as a function of time. This kind of analysis is optimiza ti on time, the disposal of biohazardous waste, important , both in suggesting the mechani sm by or req uir. e a slg. m·f ·Icant amount 0 f samp Ie ·353 . 6 . which th e overall reaction takes place and in enabling the detail ed kinetics of reacti ons to be resolved. 2. Biochemistry Isotopi c labell ing is also widely used in such With introduction of electrospray ionization (ESI) studi es. It can ind icate whi ch particular atoms are and matri x-assisted laser desorption/ioni zation invo lved in the reaction ; in rearra ngeme nt reactions it (MALDI) mass spectrometry to the repertoire of ca n show whether an int ramolecular or intermolecul ar classical bi oc hemical research methods, the demand 214 INDIAN J. BIOCHEM., BIOPHYS., VOL. 39, AUGUST 2002

for this instrumentation has literally exploded. Now, complex. These initial cleavage sites are then the commercial availability of MS instruments which identified using accurate mass measurements offer picomole to attomole sensitivity and enable the combined with the protein's known structure and the analysis of biological fluids with a minimum amount known specificity of the enzyme. Computer-based of sample preparation has made analysis of a large sequence searching programs allow for the variety of compounds, including proteins, peptides, identification of each proteolytic fragment, which 111 . , 4546 carbohydrates, oligonucleotides, natural products, turn can be used to map t h e protem s structure . . 37 42 drugs and drug metabolites possible - . Another application that has generated a great deal The ability to analyze complex mixtures has made of excitement is to study biological non-covalent electrospray and MALDI very useful for the complexes. Ganem and Henion showed that examjnation of proteolytic digests, an application electrospray ionization can be used to study such otherwise known as peptide mass mapping. This helps interactions in gas phase that can be con-elated to 43 47 in identification of protein primary structure . Same condensed phase interactions . This application has approach is also used for the identification of found its way in studying hemoglobin complex, DNA unknown proteins with the patterns predicted for all duplex, cell-surface carbohydrate assocIatIon, proteins in the database. This forms the basis of the catalytic antibody-inhibitor interactions, and the . f hI' 48-50 much-talked about, "Proteomics" approach44. The ana IYSls 0 woe viruses . overall approach used in proteomics is shown in Further, in addition to being useful for large Fig. 10. molecules, ESI is an important tool for qualitative and Protein mass mapping has also been used for quantitative analysis of small biomolecules. ESI­ studying higher order protein structure by combining based methods have recently been developed to limited proteolytic digestion, mass analysis, and quantitatively examine small molecules (steroids) at 18 computer-facilitated data analysis. In the analysis of the attomole level (100 x 10- moles)5 1. protein structure, enzymes are used to initially cleave Another important application achieved by modern surface accessible regions of the protein or protein mass spectrometry is the routine acqui sition of

Excision of spots 2D PAGE

Di gestion on membrane t -' ! In-gel tryptic digestion ., ., ~ .r:~::"~ Amino acid composition; I,· I"""," / / )0 N-tcrmin:d sequcnce (Edman dcgradation) ~ ,--iL"'_'" _----, t ------l)OO~ (~e no lll jc Data base Scarch

MALDI-TOF1'1"1 ESll\.'1S fMS t Peptide Fingerprint TARGET Sequencing

Mass Spectrometry

Fig. IO-A schematic di agram showin g the complete protcomics approach. MAITHAL & MURALlDHAR : MASS SPECTROMETRY AND PROTEIN STRUCTURE 215

complete sequence information from biopolymers. relatively straightforward. Using mass spectrometry, More specifically, electrospray ionization tandem it is also possible to identify viral protein post­ mass spectrometry has been routinely used to generate translational modifications and has even been recentl y fragment ions from a selected precursor ion by used to study viral protein dynarrucs. Mass initiating ion/molecule which can then be mass spectrometry has also been applied on a global scale analyzed and used to obtain sequence information. via the mass measurement of entire intact viruses (7 Perhaps the most well-known applications of . million Oaltons)57. electrospray ionization tandem mass spectrometry are It is important to note that the most exciting thing the work performed by Hunt and colleagues to in modern MS is that the developmental stage of mass identify major histocompatibility complex (MHC) spectrometry has not stopped; innovations such as 52 bound peptides . Electrospray ionization tandem nanoelectrospray, curved reflectrons and electrospray " mass spectrometry has also been successfully applied with orthogonal spraying continue to expand its to the sequencing of small 0ligonucleotides53 . capability. Extending beyond simple molecular MALDI-MS is playing an equally exciting role in weight characterization, these "mjld" ionization biopolymer sequencing, used in conjunction with methods can be applied to many new applications, enzymatic or chemical digestion to generate including protein-protein interactions, dynamic viral sequence-specific ladders for proteins and analysis, high sensitivity protein sequencing, routine oli gonucleotides. This process, also known as "ladder DNA sequencing, protein folding, high throughput sequencing", was first pioneered by Chait et al. for 54 analysis in combinatorial chemjstry, and drug proteins and further developed for 55 discovery. 0li gonucleotides . 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Biophys Res Commlill 18 , 469-473 Mass spectrometry is also offering a new 4 Barber M, Bordoli R S, Sedgwick R D & Tyler A N (198 1) .J Chelll Soc Chem Coml11un 19 81 , 325-327 perspective on the solution and gas-phase properties 5 Torgerso n D F, Skowronski R P & MacFarlane R D (1974) of viruses. Viruses are particles designed to transport Biochem Biophys Res COmlll l/1I 60, 6 16-621 genes between hosts and the cell s of a host. Most 6 MacFarlane R D & Torgerso n D F (1976) Science 191. 920- 925 viruses are composed of two pal1s: genetic 7 Hakansson P, Kamensky I, Sundqvist B, Fohlman J, Peterso n information and packaging materia l. 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