mass spectrometry: an essential tool for genome and...

Indian Journal of BiotechnologyVol 2, January 2003, pp 48-64

Mass Spectrometry: An Essential Tool for Genome and Proteome Analysis

Pushpendra Kumar Gupta* and Sachin Rustgi

Molecular Biology Laboratory, Department of Genetics and Plant Breeding, Ch. Charan Singh University,Meerut 250 004, India

Mass spectrometry (MS), in its various forms, has become an essential tool for genome and proteome analysis. Itinvolves gaseous ionization of the analyte to be examined, followed by separation of ions according to mass-to-charge(mlz) ratio and determination of molecular masses of iOIlSfrom mass spectra obtained after mass spectrometry ofanalyte. Several methods for ionization, mainly including MALDI and ES, each coupled with a specific mass spectralanalysis system (e.g. TOF-MS and quadrupole MS) are available. MS/MS is devised particularly for thedetermination of amino acid sequences of small peptide. The advantage of MS over other techniques is its speed,since gel electrophoresis and labeling of the analyte, needed in other techniques used for genome/proteome analysis,can be dispensed with. Applications of mass spectrometry for genome analysis include DNA sequencing and SNPdetection, the latter involving PinPoint assay (minisequencing), PNA hybridization, invader cleavage, "MALDI on achip", etc. Similarly, its applications for proteome analysis include peptide sequencing, determination of molecularweights of proteins and protein identification by database search. Protein modifications and protein-proteininteractions can also be examined by coupling mass spectrometry with database search. In this manner, massspectrometry has become an essential tool for genome and proteome analysis.

Keywords: mass spectrometry, genome analysis, proteome analysis, MALDI-TOF MS

IntroductionMS involves separation of charged atoms or

molecules according to their mlz ratio and thereforehelps in the determination of relative molecularmasses of organic compounds and biomolecules withvery high precision and sensitivity. This has led to avery wide range of applications of MS ininvestigations involving study of biomolecules.Although mass spectrometry had its beginning in theearly years of the zo" century, it was only in 1980sand 1990s, that mass spectrometry was extensi velyused for research in various fields of biologicalsciences. Application of MS for the study ofbiomolecules actually took long time because itrequires charged gaseous molecules for analysis, andthe polymeric biomolecules, being large and polar,cannot be easily transferred into the gaseous phaseand ionized. However, the availability of ionizationtechniques like matrix-assisted laserdesorption/ionization (MALDI) and electro spray (ES)in 1980s and the major advances made in samplepreparation for MS led to powerful instrumentation(sample preparation methods/protocols are out ofscope of this article, while an extensive amount ofinformation is given on PennS tate College of

* Author for correspondence:Tel: 0121-2768195; Fax: 0121-2767018E-mail: [email protected]

Medicine's website). This made it possible to obtainpolymeric biomolecules in gaseous state and in anionized form, so that MS has been utilized extensivelyfor the study of biomolecules. Starting in early andrnid-1990s, software algorithms also becameavailable, which allowed study of correlations of thedata collected from MS with the data available inmassive databases/databanks. Thus, during the lastdecade of the zo" century (1990-2000), MS becamean important technique for genomics and proteomicsresearch, leading to the 2002 Nobel Prize in chemistryto J B Fenn and K Tanaka. Currently it is used mainlyfor characterizatiop, identification and quality controlof a variety of nucleic acid and protein molecules.which is so important for genomics and proteomicsresearch. A vast amount of literature is available onmass spectrometry; the purpose of the present reviewis not to summarize all the literature but to provideresearchers with an overview of recently used massspectrometric techniques including the principles ofionization methods used in MS, the major MSinstruments currently in use for the study ofbiomolecules, and the various applications of MS inbiological sciences

Ionization MethodsAs mentioned above, MS requires that the

molecules to be examined should be available incharged gaseous form. Therefore, methods were

GUPTA & RUSTGI: MASS SPECTROMETRY: A TOOL FOR GENOME AND PROTEOME ANALYSIS 49

developed to obtain biomolecules in the form ofgaseous ions to be used for MS. Following commonmethods are available for ionization of biomolecules.

Matrix Assisted Laser Desorption-Ionization(MALDI)

MALDI, developed in the year 1988 by Karas andHillenkamp, involved co-precipitation of large excessof a matrix material (a small organic molecule) withthe analyte molecule (the molecule to be analyzed).This is achieved by pipetting a submicrolitre volumeof the mixture of matrix and analyte onto a metalsubstrate, where it is allowed to dry. The dried solidhaving matrix and analyte is then irradiated bynanosecond laser pulse, usually supplied by a smallnitrogen laser with a wavelength of 337 nm, which isspecific for the absorbance of the selected matrixmaterial. The irradiation causes energy transfer anddesorption, producing gas phase matrix ions. The non-absorbing intact analyte molecules are also desorbedinto the gas phase and get ionized with the help ofmatrix ions (Fig. 1). The charged molecular ions ofthe analyte, generated during a gas-phase protontransfer reaction with the matrix molecules, aredetected and analyzed by MALDI- TOF MS (seelater). The matrix used with biomolecules is generallymade up of one of several available substances (Table1), which differ in the energy they impart to thebiomolecules during desorption and ionization andtherefore, also differ in the degree of fragmentation(unimolecular decay) that they cause. The DHBmatrix, which gives highest sensitivity in MALDI, ispreferred when stability of the ions for milliseconds isrequired as in trapping experiments. In time of flight(TOF) experiments, where stability for microsecondsis required, other matrix substances like CHCA canalso be used. Several methods are available forsample preparation also. For instance, matrix may belaid down in microcrystalline thin film on thesubstrate leading to better adherence and providinglarge crystalline surface from which the ions can bedesorbed. Sometimes admixture of analyte with thematrix can also be beneficial. MALDI is generallyused for the study of molecules with mass above 500Daltons. However, proteins undergo fragmentationduring MALDI, resulting in broad peaks and loss insensitivity. As a result, MALDI is mostly applied tothe analysis of oligonucleotides and peptides.Surface Enhanced Laser Desorption-Ionization(SELDI)

The patented SELDI based protein chip(Ciphergen® Biosystems, USA)/biochips (LumiCyte,

0°0°0°0° tP0°0°0°0°0°0°0°0°

"E 008~•• g8000CD ogoga. .°0°0 <\jE 0°0°0•• °0°0 0'" 000°0 ° (M+W)

°8°~0 0 0 ° 0 • matrix(protonation)o 000 --°0°00 ° ° 0 o analyte(M-H+)°0°00(deprotonation)

Fig. l-Schematic diagram showing mechanism involved inmatrix assisted laser desorption-ionization.

Inc., Fermont, CA) system, is a unique combinationof affinity chromatography and time of flight massspectrometry on a single platform-a combinationthat is designated SELDI-TOF MS (Surface EnhancedLaser Desorption/Ionization Time-of-Flight MassSpectrometry). This system enables protein capture,purification, ionization and analysis of complexbiological mixtures directly on protein chips arraysurface and detection of the purified proteins by LaserDesorption/Ionization Time-of-Flight MassSpectrometric analysis (Weinberger et al, 2000).

SELDI system can be divided into three parts (i)protein chip arrays, (ii) the protein chip reader (LDl-TOF MS) and (iii) specialized software. The array haseight 2-mm diameter spots, each with surfacesdesigned to select proteins from crude extractsaccording to protein properties. The reader is an LDl-TOF MS (Laser/Ionization Time-of-Flight MassSpectrometer), which utilizes pulsed nitrogen laserenergy, to ionize proteins from the array and measuresthe mass of each protein species based on its velocitythough reader's ion chamber i.e. TOF. Data is finallycollected in the computer with the help of specificsoftware. This system can detect and accuratelycalculate the mass of compound ranging from smallmolecules and peptides of less than 1 kDa to proteinsof 500 kDa or more (Weinberger et al, 2000). Using

Table i-Some common MALDI matrices having maximumabsorbance at 337 nm and the corresponding analytes that can bestudied

Matrix

a-Cyano-4-hydroxycinnamicacid (CHCA)

3,5-Dimethoxy-4-hydroxycinnarnic acid(sinapinic acid)

2,5-Dihydroxybenzoic acid(DHB)

Analytes

Peptides, proteins. lipids andoligonucleotides

Peptides, proteins andglycoproteins

Peptides, proteins. lipids andoligosaccharides

50 INDIAN J BIOTECHNOL, JANUARY 2003

SELOI-based protein chip technology, one canproduce differential maps of protein expression'phenotypic fingerprints' - directly from crudesamples such as serum, urine or tissue extracts. Itsgeneral applications include disease diagnosis,toxicological screening, target-ligand interaction,peptide mapping and immunoassay development.

Recently Nelson & coworkers (2000) havecombined surface plasmon resonance-biomolecularinteraction analysis (SPR-BIA) with MALDI- TOFanalysis (BIA-MS analysis). SPR-BIA is also a chipbased technique that uses an immobilized receptor tomonitor biomolecular interactions. Since the SPRdetection is nondestructive, proteins may be furthersubjected LO TOF analysis for identification. Thisapproach has detection limits at or below 20 fmol anda mass range up to 150 kOa (for a review see Hayduket at, 2002).

Electrospray (ES)The technique of electrospray mass spectrometry

(ESMS) was developed in late 1980s by J B Fenn,who was awarded the 2002 Nobel Prize in chemistry(Fenn et al, 1989; Yamashita & Fenn, 1984). In thistechnique, the sample is dissolved in a liquid withmobile phase (water: acetonitrile or water:methanol,1:1) and is pumped (at the rate lower than microlitreper-minute) through a hypodermic needle at highvoltage in order to either electrostatically disperse, orelectrospray small droplets measuring up to amicrometre in size. These droplets rapidly evaporateand impart charge onto the analyte molecules. Thisionization takes place in the atmosphere and thereforeis very gentle, causing no fragmentation of analyteions in the gas phase. It is, therefore, also described asan atmospheric pressure ionization (API) technique. A

stream of nitrogen gas (the nebulizing gas) that flowsthrough a tube co-axial to the main capillary helps inthe spraying process (Fig. 2).

ES produces many more ions than MALOI andthese ions are transferred into mass spectrometer withhigh efficiency for analysis. A wide range ofmolecules including proteins, oligonucleotides, sugarsand lipids can be analyzed by ESMS. Electrospray isperformed in either of the two different modes. First.is the nanoelectrospray, which is a miniaturizedversion and operates without pumps at a very lowflow rates (few microlitres per minute), facilitated byglass capillaries with an inner diameter of onemicrometre at the tip, and second, is the high-performance liquid chromatography (HPLC), whichmay also be combined with mass spectrometry (LC-MS), so that MS analysis of components takes placeon line, as they elute from chromatographic column(Vestal,1990).

Fast Atom Bombardment (FAR) or LiquidSecondary Ionization (LSI)

In this technique, the sample is first dissolved in aliquid matrix, which is a viscous, low-pressure liquid,like glycerol or 3-nitrobenzyl alcohol. The mixture isnot crystallized as done in MALOI, but instead a fewmicrolitres of this liquid are placed on a small metaltarget at the end of a probe, which is inserted into MS.The liquid surface is then bombarded with a beam ofhigh kinetic energy atoms (xenon or argon) or ions(caesium) rather than laser used in MALD!.Molecules are sputtered from the surface, enter thegas phase and ionize. This method of ionization hassome inborn advantages over other ionizationmethods viz. use of liquid matrix instead of solidsurface, which if used is permanently damaged by a

drying gas at counter samplingatmosphericpressure electrode cone/orifice

nebulisinggas ~ Ij}'~ capillary, droplet. skimmer~~~~~~~~~ 3-4 evaporating

analyte-""".""~;, ..•",....,.,."""•..,:.,...,,,..",.,.,tJ~--~1II:::~:::= 1041105 mbar

droplet ions evaporatingcontaining fromthe surface pressureions of the droplets = , mbar

Fig. 2--Schematic diagram of standard eiectrospray ionization source.


beam of high energy atoms, leading to short livedsamples and spectra; the mobility of liquid matricesused allows the surface to be continuously replenishedand provides long life to sample and spectra. This hasdistinct advantages for the study of many medium tolarge biological molecules (Caprioli, 1989; Barber etal, 1981).

Plasma Desorption Mass Spectroscopy (PDMS)PDMS utilizes 252Cf fission fragments to desorb

large molecules from target. The target containing theanalyte is made up of a thin aluminium foil, oftencovered with a layer of nitrocellulose. The desorption-ionization involves hydrophobic interactions, whichallow salts to be washed off and chemical reaction tobe carried out on the target. Alternatively, the samplecan be electrosprayed directly onto Ni or Al foil. Thistechnique is more efficient for the analysis of smallpeptides. PDMS has a reasonably good sensitivity forpep tides and small proteins, and typically about 10picomoles are needed for molecular massdetermination of such a protein. The main virtue ofthis technique is its simplicity, easy usability andeasily interpretable spectra, which can be easilyutilized by non-experts in mass spectrometry (forreview see Gordan, 2000).

Thermospray and Particle Hearn (PH)Thermospray ionization involves coupling of

HPLC to mass spectrometry at conventional flow rate(0.5 to 1.5 ml/min). The eJutriate from HPLC columnis released and partially vaporized under reducedpressure by heating a stainless steel tube of 0.10 to0.15 mm inner diameter. The resulting small dropletsvaporize further due to hot gas in this low-pressureregion of the ion source. When an auxiliary filamentor low-current discharge device is used, completeevaporation of the solvent from the liquid dropletsproduces gas phase ions from ionic compounds in thesample solution (Blakeley & Vestal, 1983).

The particle beam (PB) interface, derived from theMAGIC interface (Monodisperse Aerosol GenerationInterface for Chromatography), has elements incommon with thermospray, but gives spectra withmore fragment ions. It involves an initial stepinvolving formation of an aerosol, followed bydispersion caused by a gas stream (usually helium),and desolvation. This leads to formation of a beam ofcharged particles, which makes the ion source. In theoriginal interface, a momentum separator thenseparates the lighter dispersion gas and the vaporized

solvent from the sample particles having highermomentum, thus facilitating entry of ions into themass spectrometer. PB is less sensitive thanthermospray or electrospray and is not best suited forthe analysis of substances like ionic components, highmolecular mass samples, and thermally labilecompounds.

Electron Ionization (EI) and Chemical Ionization(CI)

In this technique, a beam of energetic electrons isgenerated inside a chamber by heating a metalfilament to a sufficiently high temperature (approx.2000 K). The electrons are attracted toward a plate(away from the filament), called the trap, bymaintaining the trap at a relatively positive potential.A relatively volatile analyte, in gas phase, is directedinto this high-energy beam traveling from filament tothe trap, and an interaction with electron beamgenerates charged molecular ions of the analyte.Depending on the compound and the ionizationenergy, the molecular ions may then fragment. Thespectra, usually containing many fragment-ion peaks,are useful for structural characterization andidentification. Small impurities in the sample are easyto detect (McCloskey, 1990).

Chemical ionization (CI) is very similar to EI but inthis case a reactive ionized reagent species is usedinstead of a beam of electrons and the interactioncauses ionization of the analyte. Many such reagentsare gases (e.g. isobutene, methane and ammonia, etc.),which are used to enhance the abundance ofmolecular ions. For both these ionization methods,range of molecular weight of the analyte is 50 to 800Da. In rare cases, it is possible to analyze samples ofhigher molecular weight also (Harrison, 1992).

Electron-capture IonizationElectron-capture, sometimes called negative Ion

chemical ionization (NICI), is used for moleculescontaining halogens, N02, CN, etc. It usually requiresthat the analyte be derivatized to contain highlyelectron-capturing moieties (e.g., fluorine atoms ornitrobenzyl groups). Such moieties are generallyinserted into the target analyte after isolation andbefore mass spectrometric analysis. The sensitivity ofNICI analysis is generally two to three orders ofmagnitude greater than that of positive ion chemicalionization (PCI) or EI analysis. Little fragmentationoccurs during NICI, and this mode of ionization isgenerally employed for quantitative analysis of trace


amounts of compounds of known structure, where aheavy isotope-labeled compound is used as an internalstandard.

Mass Spectrometers and Mass Spectral AnalysisEssentially, a mass spectrometer measures mlz

ratios of analytes such as nucleic acids,oligonucleotides, proteins, peptides or peptidefragments. It has three components, ionization source,the mlz analyzer and the detector (Fig. 3). Followingthree principles are available for separation ofmolecules that differ in mass: (i) separation of ions onthe basis of time of flight (TOF MS); (ii) separation ofions by quadrupole electric field generated by metalrods (quadrupole MS) and (iii) separation of ions byselective ejection of ions from a three-dimensionaltrapping field (ion trap or Fourier transform ioncyclotron MS). Any of the ionization methodsdescribed above, including MALDI, ESI or FAB canbe coupled to any of the above three methods of ionseparation. However, since MALDI produces shortbursts of ions in vacuum, it is coupled with TOF MS,while electrospray, producing continuous beam ofions in atmosphere is coupled with quadrupole andion trapping MS.

In 1900, J J Thomson introduced the first massspectrometer, which employed fixed magnetic andelectric fields (described as electric sector andmagnetic sector) to separate ions of different massand energy. He recognized that charged particles thatdiffer in momentum behave differently in anelectromagnetic field, and used this property forseparation of ions with different masses. Using thisinstrument, he was able to prove that the noble gas Ne(Neon) was composed of two different isotopes ofmass 20 Da and 22 Da (Thomson, 1913). These socalled 'sector instruments' are not very sensitive incomparison with recent mass spectrometers and arenow more or less of historical importance. Someadvantages, although not unique, are their relativelyhigh mass range, sensitivity and resolving power, and

their compatibility with wide range of ionizationtechniques. The disadvantages are their size and costcompared to most other mass spectrometersdeveloped later.

MALDI-TOF MSMALDI-TOF mass spectrometers utilize MALDl

for ionization and the time of flight (TOF) of the ionsto reach the detector as a parameter to estimate mlzratio. Since the velocity of ions will depend on itsmass and energy, the time taken by an ion to travel aspecified distance will depend on mlz ratio and isdescribed as TOF. Several types of MALDl-TOF MSare now available (Gordan, 2000).

(a) Linear-TOF MS. In linear spectrometers usingMALDI and TOF, samples are deposited on a metalsubstrate, capable of holding several hundred analytespots. Irradiation of these spots by a laser pulsegenerates a short burst of ions, which are acceleratedto a fixed amount of kinetic energy in order to traveldown a flight tube (Fig. 4). The small ions have ahigher velocity than the larger ones, thus producing aTOF spectrum.

(b) DE MALDl-TOF MS. Linear MALDI-MSdescribed above lacks resolution and sensitivity toanalyze complex mixtures like those of Sanger'ssequencing reactions. This is because the initialvelocities and therefore also the range of flight tiIllesof ions of the same mass extracted in conventionalMALDI-TOF MS involving continuous ion extractionmay differ due to a variety of reasons thus reducingthe resolution of the technique. Improvements havebeen made by varying the time between formationand extraction of ions from the source. The ion sourcemay be field free during ionization and only after aspecified time delay, a high amplitude pulse ofirradiation extracts ions. This results in the arri val ofions of the same mass simultaneously at the detectorand helps in correlating the velocity differences inions with their masses thus improving the resolutionof a time of flight (TOF) mass analyzer. The

components of MS

II I I

ionisation source e.g. analyser mass-to-charge, detector e.g. photomultiplier.electrospray (ESI), matrix rnIz e.g. quadrupole, microchannel plate,assisted laser desorption time-of-flight, magnet, electron multiplier(MALDI) FT-ICR

Fig. 3--Simplified schematic diagram of a mass spectrometer (MS).


technique is described as delayed extraction MALDI(DE MALDI) and has been used for DNAsequencing.

(c) Reflectron TOF (RETOF)-MS. In thisinstrument, a single stage or a dual stage reflectron isused at the end of the flight tube, which leads toimprovement in mass resolution. The reflectron,located at the end of the flight tube, is used tocompensate for the difference in flight times of ionswith same m/z, but different kinetic energies (Fig. 5).

Quadrupole Mass Filter InstrumentsA quadrupole is a mass filter consisting of four

rods, to which an oscillating electric field is appliedand which lets only a certain mass pass through, theother masses being on unstable trajectories, do notreach the detector. By scanning the amplitude of theelectric field and recording the ions at the detector,one obtains a mass spectrum. Quadrupole MS is

capable of resolving mass accuracy of 0.1 to I Da andthus excels in quantitative measurements. Mostexperiments are performed on triple quadrupoleinstruments, consisting of three sections: two massseparating quadrupole sections separated by a centralquadrupole (or a higher multipole) section, whichcontains the ions during fragmentation.

Quadrupole MS can sometimes be combined withTOF, so that the dissociated ions enter the TOFanalyzer for getting the mass spectrum. For instancein a quadrupole TOF instrument, the first quadrupole(qO) focuses the ions, second quadrupole (q I)separates the ions and the third quadrupole (q2)dissociates the ions to enter the TOF analyzer, wherethe ions are pulse accelerated into the reflector andthen to the detector (Fig. 6). Quadrupole TOFanalyzer may sometimes be used with MALDI ionsource as done in MALDI-quadrupole TOF (MALDl-q TOF) (Fischer, 1959).

accelerationregion

laser

G1W;LB __ ----{0~-----Ier___-------~mfield freedrift region

for continuous ion extraction: G1> G2'

r--l pulse voltage--l bias voltage

Idela*uls~time duration

for delayed ion extraction: G1 VOltage: G2 voltage = bias voltage

detector

Fig. 4--Schematic representation of basic components of a linear TOF mass spectrometer.

•

decelerating reflectingvoltage voltage

\ \0 \ \\. o \• \ \

0\ \

laser

1~1~:o~ ~o~ ~ __~o ~~~~ri i·· · ·

same mlz Ions• slow ionso fast ions

Reflecting TOF mass spectrometry

•odetector

o

Fig. 5--Schematic representation of basic components of a reflecting TOF mass spectrometer.

S4 INDIAN J BIOTECHNOL, JANUARY 2003

focusing ring -ionsource 1--+--IE+.iii

modulator focusing gridI accelerator columnI, -:'iiiii~~ii.l Ir,.----::,..:...,/."II' -.--~~-=-=q2 .)

(ion (ionseparated) dissociated)

4 anodedetector

curtainplate

orifice plate I field freedrift region

eflector

Fig. 6--Schematic diagram of quadrupole TOF instrument, representing different sections of quadrupole (modified from Mann et 01.2001).

Trapping InstrumentsIn these instruments, ions in a continuous beam are

trapped in three-dimensional electric fields. Thetrapped ions are ejected one after another from thetrap by application of additional electric field and areanalyzed to produce a mass spectrum. Ion trapinstruments are compact, versatile and highlyautomated, but in practice the accuracy and resolutionachieved by these instruments do not reach the levelof quadrupole instruments.

Another version of the trapping principle isemployed in Fourier transform ion cyclotron MS(FTMS), which involves trapping of ions formed byEl, CI, MALDI, and ESI and uses Fourier transformdetection, permitting simultaneous detection of awide-range of masses. The method has severaladvantages over other methods, such as highestresolving power, a high upper mass limit of analytethat can be examined, high sensitivity, nondestructivedetection, and high accuracy for mass measurement.

Tandem Mass SpectrometersTandem mass spectrometry (MS/MS) is utilized,

where a mixture of peptides is used for getting aminoacid sequences of peptides one at a time. The first partof tandem spectrometer selects one peptide on thebasis of mass, which is then dissociated or fragmentedby collision with an inert gas like argon or nitrogen.In the second part of tandem mass spectrometer, theresulting fragments are separated and tandem massspectra or MS/MS spectra are obtained (Fig. 7). Thefragments differ in size depending upon the position

of amide bond fragmentation and can be C-terminalor N-terminal, the former being predominant. Mostpeptides thus examined are tryptic peptides (obtaineddue to digestion with trypsin enzyme) having arginineor lysine residues at their C-terminus (Busch et al,1988).

Tandem mass spectra are usually interpreted withcomputer assistance or are matched against databases,while interpreting the data of C-terminal or N-terminal fragments. One will notice that C-terminalfragments start with masses 147 (1ys) or 17S (arg) andthe next fragment peak differs by mass of a singlesecond amino acid, which can be identified by massdifference. Consecutive amino acids will be identifiedusing this principle thus giving the amino acidsequence of a small peptide. Similar will be the casewith the N-terminal ion series.

Other Mass SpectrometersIn addition to four basic types of mass

spectrometers described above, following are otherinstruments, which are relatively less important.

Gas Chromatography-Mass Spectrometry (GC-MS)It permits separation of a complex mixture into its

components, before ionization and mass analysis.This is particularly useful when analyzing relativelylow levels of target compounds derived from complexbiological mixtures. The target analyte must berelatively volatile or must be susceptible toconversion into a volatile derivative to permit GCseparation. In general, the derivatized analyte should


Argon.-------------..------------~~----, ~----~------------------

F1/m1 (TK)

F2Im2 (STK)

F3/m3 (NSTK)

F4/m4 (HNSTK)

00000 0---'o 000

electrosprayionization

detectormassspectrometer 1(MS-1)

P2Im

oo 0

o

collisioncell

detectormassspectrometer 2(MS-2)

P2IHNSTK

Fig. 7-Schematic representation of tandem mass spectrometry for determination of a peptide sequence (PI to P3, different peptides: FIto FS peptide fragments obtained from peptide P2 and ml-m-l corresponding masses determined by MS2: H, N. S, T and K = differentamino acids).

have a MW of less than 1000 Da to be used for GC-MS. In special cases, derivatized analytes with MWup to 1000-2000 Da can also be analyzed. EI and CIin positive and negative modes are employed forionization.

Liquid Chromatography and Tandem MassSpectrometry (LC-MS)

Liquid chromatography (LC) coupled with tandemmass spectrometry, called LC-MS/MS or popularlyknown as LC-MS, is a powerful technique foranalysis of proteins and peptides. In a typical LC-MS/MS experiment, the analyte is eluted by reversed-phase high performance liquid chromatography(HPLC) as applied elsewhere in protein chemistry; theonly difference is that the dimension and flow ratesare much smaller, that are achieved by nano-LCcolumns of internal diameter of 50-100 urn. Thesensitivity of this technique depends upon columndiameter, because the lower the flow-rate for thechromatographic separation, the higher will be thesensiti vity. The detection limit of few femtomoles (1-10 fmol) in this technique makes it compatible fordetection of proteins/peptides at low copy number percell (Vestal, 1990; Caprioli, 1994).

Complicated mixtures containing hundreds ofproteins can be analyzed directly by LC-MS evenwhen concentrations of different proteins differ. LC-MS/MS can be used alone or in combination with l-Dor 2-D electrophoresis, immunoprecipitation, orprotein purification techniques. In this manner, in a

single LC-MS/MS run, a large number of peptides canbe sequenced even if they have the same molecularweight but differ in hydrophobicity.

Isotope Ratio Mass Spectrometry (IRMS)Isotope ratio MS, which can be combined with GC-

MS is capable of very precise determination of13CI12C ratios. It is exploited principally inexamining trace enrichment of 13C in smallmolecular-weight analytes (e.g. protein-derived aminoacids) after biosynthetic incorporation of a !3C-labeled precursor. Applications of IRMS include thestudy of substrate disposition in humans after infusionof 13C-labeled precursors. Other elemental ratios (e.g.2HI1H, and 1801160) can also be measured althoughit is mainly used for detection of 13CIl2C and15N/14N ratios that are useful in proteomics research.

Applications of Mass Spectrometry forGenomelProteome Analysis

Mass spectrometry in the form of MALD[-TOF hasbeen extensively used for the analysis of nucleicacids, proteins and peptides, although initially thetechnique was more often used for analysis ofpeptides (Hillenkamp et al, 1991; Fitzgerald & Smith.1995; Nordhoff et al, 1996; Yates III, 1998; Li et 0/.2000). For biotechnology applications, we need tostudy both nucleic acids and proteins and thereforeuse of MS for the study of both nucleic acids andproteins has been briefly discussed.


Genome Analysis using MSSeveral methods that require neither the gel

electrophoresis nor labeling, utilize MALDI-TOF MSfor genome analysis. For instance, a study of DNApolymorphism using MS, depends largely on thedetermination of relative masses of individual DNAfragments with sufficient accuracy, so that even asingle base replacement (transition or transversion)can be identified due to unique mass of each base.However, the methods have been applied only torelatively short oligonucleotides, because theresolution and sensitivity of MALDI-MS generallyfalls off dramatically with increase in size ofoligonucleotides. Analysis of nucleic acid byMALDI-TOF MS has several advantages includingthe following: (i) The ionization, separation by size,and detection takes milliseconds to complete, so thatthe technology is fast, in contrast, electrophoreticseparation and detection takes hours, sometime days;(ii) The results are more accurate than theelectrophoresis-based and hybridization-basedmethods, which are influenced by secondarystructures formed by nucleic acids; and (iii) Completeautomation involving both sample preparation andacquisition/processing of data is possible (Leushner,2001).

(a) DNA sequencing by DE-MALDI-TOF MS. Aswe know, 'Sanger's dideoxy termination' method ofDNA sequencing depends on resolution to allowsequential distribution of the termination fragmentsdiffering by single nucleotides. These terminationproducts of Sanger's termination reactions can beexamined by delayed extraction MALDI-TOF MS. Inthis approach, mass spectra of each of the fourspecific dideoxy termination reactions generated fromSanger's chemistry are overlaid and each sizedproduct correlated to one of the four base terminationreactions, since mass of each base is known. A majoradvantage of this approach is the fast acquisition ofdata by MALDI-TOF MS, which needs only minutesper sample. An additional advantage is that there is noneed for either the gel electrophoresis or for anyradioactive or fluorescence labeling. Short sequencesof oligonucleotides were distinguished from eachother by this technique (Smith, 1993; Fitzgerald et al,1993; Williams, 1994). But the actual analysis ofreaction products of Sanger's sequencing reactionsproved difficult firstly, due to availability of onlysmall amount of each fragment in the mixture andsecondly, due to interference by buffer salts and othercomponents of the reaction. Delayed extraction (DE-

MALDI) allowed further improvement in thesensitivity and resolution of the technology ofMALDI-MS (Colby et al, 1994; Vestal et al, 1995). Itinvolved improvement of the resolution of time offlight (TOF) mass analyzer by correcting for thevelocity distribution of ions (by altering the timebetween formation and extraction of ions) formed inthe ion source. DE-MALDI is coupled with high yieldcycle sequencing protocol to make it suitable foranalysis of sequencing mixtures of oligonucleotides.40-50 bases long (Roskey et al, 1996) (Fig. 8). Sincefragments larger than 100 bases cannot be utilized forsequencing (Nordhoff et al, 1992; Fitzgerald et al,1993; Mouradian et al, 1996; Koster et al, 1996;Monforte & Becker, 1997; Taranenko et al, 1997;1998; Fu et al, 1998; Kirpekar et ai, 1998), thetechnique can not be employed for genomesequencing, but can certainly be used forminisequencing required for single nucleotide poly-morphism (SNP) genotyping (Ross et ai, 1998;Timothy & Smith, 2000).

(b) SNP detection using MS. A variety of methodsutilizing MS have been developed for analysis ofpoint mutations that are due to SNP (Gupta et al,2001).

(i) PinPoint assay for SNP detection using MALDI-TOF MS of peR products--One of the methods thatwas described as PinPoint assay (Taranenko et al,1996; Tsuneyoshi et al, 1997; Paracchini, 2002) isbased on using a primer directly upstream of a knownpoint mutation or SNP, so that in PCR, the primer isextended by a single ddNTP. Such primers maysometime anneal several bases downstream of SNPand extend more than one bases before incorporatinga ddNTP, which terminates the reaction. The PCRextended primers are' solid-phase purified anddetected by mlz value specific for the nucleotideadded in the extension reaction. The technique isdescribed as minisequencing, since it involvessequencing of a segment few bases long. SNP inseveral genes like p53 gene, apolipoprotein E geneand RET proto-oncogene have been identified usingthis approach. Multiplex SNP analysis has also beenpossible using MALDI-TOF MS, as shown for PCRamplification from BRCAI exon 13 locus (Haff &Smirnov, 1997) having five SNPs. This is facilitatedby using mass-tagged minisequencing primers (extranon- complementary dT nucleotide at the 5' end) foreach SNP position, so that all the five primers couldbe extended in a single reaction and PCR productscould be simultaneously analyzed by MALDI-TOF


Primer GATGCAGAATTC

(T-2H)2

26

AGCGCTATGTACGACGT

4000 6000 8000 10000 12 00 14000mlz

Fig. 8--Diagram showing negative ion mass spectra of four dideoxy reactions generated by primer extension on a 50-base template.Numbers indicate peaks that correspond to expected termination products numbered according to the total DNA fragment lengthincluding primers; sequence deduced is given at the top of the figure (modified from Roskey et at, 1996).

MS. Mass tagged ddNTPs have also been used toincrease the mass difference between peR products,so that this mass difference can be resolved byMALDI-TOF MS (Fei et at, 1998). In another study,multiplex genotyping of 12 different SNPs (selectedfrom human genomic SNPs) was possible usingminisequencing (Ross et at, 1998). The products canbe analyzed by Voyager DE-MALDI-TOF platform.Mass spectrometry has been used both for thediscovery of SNPs and for genotyping SNPs at theknown position (Nordhoff et al, 1992; Fitzgerald etat, 1993; Mouradian et al, 1996; Koster et al, 1996;Monforte & Becker, 1997; Taranenko et al, 1997;1998; Fu et al, 1998; Kirpekar et at, 1998). Forinstance, peR products from p53 tumour suppressorgene have been sequenced and analyzed by MALDI-TOF MS (Fu et al, 1998).

(ii) SNP detection using PNA probes-Peptidenucleic acids (PNAs) are being used for a variety ofpurposes, including their use as hybridization probes.They are actually preferred over DNAIRNA probes,due to (i) their ability to hybridize under low ionic

strength, (ii) increased hybridization specificity forcomplementary DNA, and (iii) the increased thermalstability they provide to the hybrid duplexes formed.PNAs can also be more easily analyzed by MALDI-TOF MS, since peptide backbone does not fragmentduring MALDI. For SNP analysis, two allele specificPNA probes, one for each SNP allele are annealed tobiotinylated single-stranded peR products that areimmobilized on streptavidin coated magnetic beads.After annealing, the beads are stringently washed sothat only perfectly matched probes remain hybridized.The entire bead solution is then spotted on MALO Iprobe tip and acidic matrix solution is added to thebeads, which dissociates the PNA probe fromimmobilized DNA. The mixture is then irradiated sothat PNA is desorbed and ionized and detected by itsmass; each unequally mass-labeled PNA probedetected corresponds to a specific allele (Fig. 9). ThePNA probe may be mass labeled either by (i)incorporating a variable number of 8-amino-3,6-dioxaoctanoic acid residues on the N-terminal end ofthe PNA probe, as done for the detection of SNPs in


1'2 Where M2 > M'

PCR (("

am~ PNA ~ rt ~MALDI matnx

.~ probes ~.~ ~Sh ~ ~(JD-(if)i-crD - ~,;~~~~"st~vidin-coated magnetic bead \Ali.ll.lMWllfIIW~~'fAAJ "~IJJ)I't'V\'ollJfIo.w~~",,""""

(b)(a)

WTl2620

WT43097

WT3WT2 29902874

VAR43370

Fig. 9--{a) Schematic diagram of steps involved in the mass-spectrometric analysis of peptide nucleic-acid hybridization probes. (b)Representation of composite mass spectra representing four hypothetical wild types (WT - 1-4) and a variant (VAR) allele for a uniquesingle-nucleotide polymorphism. In this case sample was heterozygous for WT 4 allele and homozygous for other three WT alleles(modified from Griffin et al, 1997).

human tyrosinase gene (Griffin et al, 1997) or (ii) byadding extra non-complementary dT to the 3' end ofPNA oligomer, as done for detection of SNPs athuman leucocyte antigen DQ a locus (Jiang-Bucom etal, 1997).

(iii) SNP detection using invadercleavage--Invader cleavage is a technique that allowsSNP detection without PCR and has been discussedelsewhere (Taranenko et al, 1996; Gupta et al, 2001).

(iv) SNP detection involving minisequencing using'MALDlon a chip' technology-In this technology,nanolitre amount of sample from mini sequencingSNP-analysis reactions are piezoelectrically pipettedonto a silicon chip. This silicon chip is directlyinserted into mass spectrometer (MS) and eachseparate sample spot is automatically analysed usingMALDI-TOF MS approach. High quality massspectrum from 100 separate spots (each containingonly 8 femtomoles of an oligonucleotide) could beobtained successfully using this approach, within sixminutes (Little et al, 1997).

MALDI-MS is used generally to achieve highthroughput and automation in typing SNPs from alarge number of genotypes in a fraction of time that isneeded in other approaches. Krebs et al (2001)reported its utility in typing of short tandem repeats(STRs or SSRs) also. The approach of typing SSRsusing MALDI-MS has many advantages over theelectrophoretic discrimination of alleles, whichdepends largely upon relative mobility of fragmentsduring electrophoresis; in this case, allele sizing isrelative and generally prone to error because ofartefacts. These assays are also time consuming, havepoor resolution and are non amenable to automation

(Braun et al, 1997; Ross & Belgrader, 1997; Ross etat, 1998).

Proteome Analysis using MSIn the post-genomic era, proteome analysis will be

crucial for understanding different biologicalprocesses. To achieve this objective, massspectrometry has been and will be extensively utilizedfor (i) peptide sequencing, (ii) identification ofproteins, (ii) study of protein expression in differenttissues and under different conditions, (iii)identification of post-translational modifications(phosphorylation, glycosylation, etc.) of proteins inresponse to different stimuli and for (iv)characterization of protein interactions that includeprotein-ligand, protein-protein and protein-DNAinteractions. Recent improvements in MS havesignificantly improved its applications in the study ofprotein structure and function (Loo et al, 1995; Yates,1998). Much of the work with proteins falls into twocategories; (i) analysis of intact proteins and (ii)analysis of peptides derived from chemically orenzymatically cleaved proteins.

To-date, majority of systems used for proteinidentification from complex mixtures generallyinvolves proteolytic digestion followed by some formof chromatographic separation of tryptic peptidescombined with MS analysis. This approach of proteinidentification is referred to as the 'bottom-upapproach' (Chalmers & Gaskell, 2000; Pandey &Mann, 2000; Mann et al, 2001). In contrast, 'top-down approach' initially described by Mortz et al(1996) and recently by Stephenson et al (2002)involves starting with intact proteins and obtaining


sequence information directly from a massspectrometric experiment without resorting first toproteolytic digestion (Kelleher et at, 1999;Stephenson et at, 1999; Cargile et at, 2001; Reids etat, 2001; Wells et at, 2001; Forbes et at, 2001).

(a) Peptide sequencing. The sequence of a peptidecan be determined by interpreting mass spectrometrydata resulting from either of the following threetechniques: (i) tandem mass spectrometry of peptidefragments ionized using electro spray from a mixture(Li & Assmann, 2000); (ii) TOF-MS of a mixture offragments resulting from chemical degradation of apeptide from N-terminus or C-terminus, a techniquedescribed as ladder sequencing (Chait et at, 1993;Patterson et al, 1995), or (iii) RETOF-MS of the post-source decay (PSD) fragmentation of metastable ionsof a peptide produced with MALDI (Spengler et at,1991). The basic principle involved in the approach,where MS/MS is used for peptide sequencing hasbeen described earlier (Fig. 7).

(b) Determination of molecular weight of intactproteins. Mass spectra of intact protein fragmentsallow detection of precise molecular weights of majorand minor proteins, although larger proteins, beingheterogeneous, make determination of their precisemolecular weights difficult. However, high-resolutionFourier transform ion cyclotron resonance massspectrometry (FTMS) has been used for accuratedetermination of mass of small intact proteins (Hornet at, 2000 a b c; Kelleher et al, 1998; Kelleher et al,1999; Stephenson et al, 1996). But the molecularweight alone may not be enough for proteinidentification, and peptide sequencing by tandemmass spectrometry, as described above, may beneeded.

(c) Protein identification using MS followed bydatabase search. Mass spectrometry has beencombined with database search to create a valuableand automatic protein identification tool. Mainly threetypes of databases (genomic DNA, cDNA/EST andnonredundant protein database) are searched by massspectrometric data. In this application, intact proteinsare degraded into pools of peptides whose masses aredetermined by mass spectrometry and then searchedagainst genomic DNA, cDNAIEST database entries.Nonredundant protein database contains the knownset of full-length protein sequences, devoid ofduplicates. These databases can also be searched bothby mass fingerprint and tandem mass spectrometricdata. Recently, Weiller et al (2001) developed aspecialized database for the analysis of MALDI-TOF

MS data derived from tryptic peptides ofSinorhizobium meliloti proteins. This databasecontains amino acid sequence data of proteinspredicted from the complete chromosome. It allowsaccessing and comparing mass spectrometric datawith already available protein profiles in the database.Matches can quickly be annotated via links toannotated databases such as Swiss-Prot, etc. ESTdatabases, such as dbEST at the National Centre forBiotechnology Information (NCBI), and EMEST atEuropean Bioinformatics Institute (EB!), containmillions of short single-pass sequences from randomsequencing of cDNA libraries. These can be searchedusually by translating into the six reading frames(tBLASTn). However, EST databases are highly errorprone and each covers only a part of the gene, but it isexperienced that, all proteins encountered in aproteomics project can be correlated to theirrespective ESTs based on minimal mass spectrometricinformation (Mann et al, 2001). Finally, genomedatabases can also be searched with massspectrometric data. Surprisingly it has been found thatpeptides can match a raw genomic sequence, withoutany information about the reading frame, codingregion and without translating it into amino acidsequence. The advantage of searching databases ofcompletely sequenced genomes are that often themass spectrometric data can help to define thestructure of the gene, with the help of start codon,stop codon and intron-exon junctions.

Protein modifications do not present an obstacle toidentification because, for a typical 50 kD proteingenerally 50 peptides are obtained after trypticdigestion and only a few are modified. Only a smallnumber of peptides are required for unique matchingto a database entry, especially in case of data fromtandem mass spectrometry; therefore very extensivemodification only marginally increases the difficultyof protein identification. There are three mainprogrammes used for database mining for proteinidentification using MS data (Table 2).

(i) Searching with peptide mass fingerprints-Inthis method, first a mixture of proteins is subjected to2-D gel electrophoresis. A specific protein is thensubjected to in-gel digestion with a sequence-specificprotease such as trypsin and a mass spectrumdescribed as 'mass fingerprint' is obtained byMALDI-TOF. The mass data is compared to thetheoretically expected tryptic peptide masses for eachentry in the database. The proteins in the databasesmay differ according to the number of peptide


Programme Web site

Table 2-Three main programs for protein identification using database mining

Mascot http://www.matrixscience.com

ProFound http://prowl.rockefelleLedu

Protein Prospector (MS-Fit) http://prospector.ucsfedu

Based on

It incorporates a probability based implementation of theMOWSE* algorithm.

It calculates the probability that a protein in a database is theprotein analyzed here and it uses Z-score as an indicator of thequality of the search result.

It uses MOWSE score to evaluate a hit in protein identification.

*The MOlecular Weight SEarch algorithm (MOWSE) capitalizes on information obtained through mass spectrometric (MS) technique.using both the molecular weights of intact proteins and those resulting from digestion of the same proteins with specific protease.Calculations are based on information contained in the nonredundant (nr) protein databases. The score value indicates how often themolecular weight of a fragment occurs in proteins within a certain range of molecular weight.

matches. More sophisticated scoring algorithms takethe mass accuracy and the proportion of proteincovered into account and attempts to calculate a levelof confidence for the match. When high massaccuracy is needed, as a rule at least 5 peptide massesneed to be matched to the protein and 15% of theprotein sequence needs to be covered for anunambiguous identification. After a match has beenfound, a second-pass search is performed to correlateremaining peptides with the database sequence of thematch, taking into account possible modifications.

(ii) Searching with tandem mass spectrometricdata--Database can also be examined for similaritieswith tandem mass spectrometric data obtained onpeptides from the protein of interest. Because tandemmass spectra contain structural information related tothe sequence of peptide, rather than only its mass,these searches are more reliable and specific. Severalapproaches exist; one of the most importantapproaches is the peptide sequence tag method. Itmakes use of the fact that nearly every tandem massspectrum contains at least a short run of fragment ionsthat unambiguously specify a short amino acidsequence. Such a peptide sequence tag will then beused to retrieve the corresponding sequence from thedatabase. The procedure can be automated and ishighly specific, especially when performed usinginstrument, such as quadrupole TOF (Little el al,1997).

(iii) Searching for protein modifications-Proteinmodifications do not interfere in protein identificationby database searching. For instance, a phosphopeptidecan be correlated to a peptide sequence in thedatabase with an additional mass increment due to thephospho group (80 Da). The algorithm used forpeptide sequence tag allows detection of mass

difference on either side of tag sequence. Forinstance, the sequence between the tag and C-terminus of the peptide could agree with the databaseentry, but the mass for the sequence from tag to theN-terminus could be larger by 80 Da. This wouldsuggest that there is modification due to a singlephospho group yielding a mass difference of 80 Dabetween the N-terminus of peptide and the start of thesequence tag.

(d) Analysis of post-translational modifications.Mass spectrometry is also used for the study of post-translational modifications in proteins. For instance,protein phosphorylation, which is so common with inthe cell, can be detected by mass spectrometry.However, it requires more time and additionalpreparative steps. When tryptic peptides are usuallyanalyzed in mass spectrometer, the phosphorylatedpeptides may need to be separated fromnonphosphorylated peptides, the presence of whichmay obscure the signal from the phosphorylatedpeptides. Since, detection of the exact phosphorylatedresidue may still be difficult, it might be worthwhileto identify the relevant peptides that arephosphorylated by treatment with phosphatase andobserve the mass difference of the peptides before andafter treatment.

Other modifications most commonly found inproteins during routine proteome analysis includeacetylation, glycosylation and the N- or C- terminalprocessing by peptidases. Acetylated peptides arequite useful because they help in determining theamino terminus of mature proteins that are formedafter post-translational processing. Generally, allmodifications that lead to a mass change can beanalyzed by MS (for review see Krishna & Wold,1993).


(e) Quantitative mass spectrometry. While thetranscriptome experiments (cDNA or oligonucleotidearray on chips) provide a comprehensive picture ofthe expression of individual genes in a given cell typeor cell state, there are additional levels of controlsduring maturation of gene products. This is whysometimes there is a loose correlation between mRNAand protein expression levels. This bottleneck hasbeen removed due to recent advances in MStechniques and instrumentation (Shevchenko et al,1996; Oda et al, 1999). Now 'differential displayproteomics' that makes use of MS is used to resolvemany problems in proteomics, viz. knowledge ofquantitative changes of protein expression in a cell,tissue, or body fluid, etc (Mann, 1999). Previouslythese changes used to be examined through strainingintensities of proteins on gels, a labour-intensivemethod that is prone to error. Recently, stable isotopemethods have been introduced and used successfullyto access quantitative changes in protein expression(Han et al, 2001). The principle involved in thismethod involves incorporation of a stable isotopederivative in one of the two states to be compared.Stable isotope incorporation shifts the mass of thepeptides by a predictable amount.

Mass spectrometry is not only used in genome andproteome analysis but also used routinely to trace theflow of organic compounds at the molecular level. Itis also used for detecting metabolic pathways and toidentify specific populations of microorganisms withthe help of lipid biomarkers (specific compounds),characteristic of those populations. MS can also usedfor a study of diversity among microorganisms. Forthese purposes GC-MS is generally used (Zhang et al,2002).

Genomics/Proteomics Research in India UsingMass Spectrometry

There are several centres in India, where massspectrometry is being used for the study ofmacromolecules. Keeping in view the importance ofthis tool for research, Indian Society for MassSpectrometry (ISMAS) was established in 1978, withits headquarters at Bhabha Atomic Research Centre(BARC), Mumbai. This society, which has as many as600 members, conducts a workshop on massspectrometry every year. The society has alsocompiled a directory on mass spectrometry, whichprovides useful information about the types of massspectrometers available in various laboratories inIndia and their various applications along with thenames of scientists engaged in research.

In India, facilities for MALDI- TOF MS have beencreated at several centres including Centre forCellular and Molecular Biology (CCMB), Hyderabad;Indian Institute of Science (IISc), Bangalore; BhabbaAtomic Research Centre (BARC), Mumbai; RajeevGandhi Centre for Biotechnology (RGCB),Thiruvananthapuram, etc. At CCMB, efforts are beingmade to characterize (i) differentially expressedproteins in cancerous cells, and (ii) antibacterialpeptides from the skin of Rana tigerina. Similarly,IISc is working on maltose binding protein and theproteomics of renal disorders (Ganesh et al, 1999;Kumar et al, 2002).

ConclusionMass spectrometry, and in particular MALDI-TOF

MS, has become an essential tool for genomics andproteomics research. It provides for accuracy and highthroughput, often avoiding the time-consuming gelelectrophoresis. PCR amplification, which is sopervasive in molecular biology, now can also bedispensed with in some applications of massspectrometry. The third generation molecular markersystem i.e. SNP, which is becoming the molecularmarker of choice in genomics research, and theprecise identification of proteins and protein-proteininteractions in proteomics research will make MSindispensable for future research involving genomeand proteome analysis. In India also, facilities formass spectrometry that are being developed and usedin some laboratories, will be increasingly used infuture for genomics and proteomics research.

AcknowledgementThis review was written during senior author's

tenure as CSIR Emeritus Scientist. Financialassistance provided by CSIR and the facilitiesprovided by Head, Department of AgriculturalBotany, CCS University are duly acknowledged.

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