AS) SETH tech 1 Levtatignes ard Ucaenaald Lecture 11 Mass Spectrometry: I Mass spectrometry (abbreviated as MS) has slowly emerged as a very powerful (wot in analyzing the organic molecules incliding biomolecules. A- mass. spectrometer separates the molecules based on their mass and charge. The underlying principle conceptually very simple: a moving charged particle can be deflected by applying electric and magnetic fields. The deflection caused by the electric and magnetic fields depends on the mass and the charge of the particle. Let us see what happens to a charged particte in an electric field (Figure 11.1) by stationary charged particles (A) and charged pasicles wih fon when the eharged particles are in vsifrm motion with sie ial velocity vectors perpendicular tthe electri eld vector.receleration of the partiéhe depends on the mass 10 jon HE shows that ths ier particle carrying ratio, 2 A lighter particle 18 accelerated more than a fe ‘ the same charge. Similarly. x particle with higher charge is ‘accelerated more as compared to the particle of same mass but having lesser charge in-a magnetic field, a moving charged particle experiences a force, F that is given by the Lorentz. force law: F=q@ xB) » is the velocity of the moving charged particle and B is the magnetic (4) where, field strength “The direction of the force can be determined using the right hand rule; If the fingers represent the magnetic field (B) and the thumb represents the. velocity (v), then the direction of the force is given’ by the direction of the palm (Figuré 11: force is always perpendicular to the Velocity, the deflected particle moves in a circular path (Figure 11.2B).NETEE ties Ficaty teat Tevtuigse Rearranging in terms ob rs oS q aaa (11.6) ae sind ae ay where, 0 is the angle between the velucity vector, » and the magnetic field vector, B. In amass spectrometer, v and B are generally orthogonal to each other; in that ease (11.8) Equation 14.8 shows that the deflection caused by a magnetic field ina moving - we charged particle is proportional to the mass to charge ratio. For the two ‘particles having same charge but different masses, the one with lesser momentum deflects ‘more (r «mv and smaller r means larger deflection). In mass spectroscopy, the charge is usually represented as z and we shall be sticking to the same convention. A ‘mass spectrum is a two dimensional plot betweeri ion abundance and 7 ratio (Figure 413) Relative abundanceAGHA tainoctinolagy HHicanodgnieat Lectin anal (ier nmoter (riguce (14). THE) Bie «y mass spectrometry is that i the Let us see the design of a typical mass spec requirement for an analyte molecule to be studied ust however + the molecules. The spectrometer +f. may not be charged has to be charged. A large number of molecule: first step in an MS experiment is therefore to ions ‘ed are then separated by one oF therefore has an ionization source. The ions generat more mass analyzers which are then detected by a detector: Detector Ionization source Mass analyzer(s) Figure 11.4 The components ofa mass spectrometer Ionization/ionization source The first. step. in an MS experiment is to obtain the ions in gas phase. The mass spectrometers, therefore have Ion mode Mass. spectrometric analyses are usually performed’ in. the positive ion mode ie. only cationic species are detected. It is, however, ‘an ionization chamber (also | POssible:ta:stuidy the molecules in negative ion Biel nen 5 thee anode: :svell, where anions are detected. Unless ; i it is usually assumed that in positive ion mode. molecule, M giving Mte > Mi" is roferred toSome ese Oo > Frngnon ime Moleete ga phase Eleciron generating Hite Figore 11.5 Design of am electron ionization source ‘The kinetic energy of the electrons is usually 70 eV in the electron ionization method. Typically 10-20 eV energy is transferred to the molecules. Around 10 eV energy is sufficient to cause ionization of most organic molecules; the radical cation is therefore left with an excess energy. Electron fonization, therefore often causes extensive fragmentation of the radical cation. Detection of these fragments can provide useful structural information about the molecute but can complicate the data for larger molecules. In some cases, molecular ion may not even be detected at all. The fragmentation is usually hemolytic, resulting in an even-electron cation and a neutral radical (Equation 11.10). Fragmentation into a neutral miolecule and a smaller radical 1.10) quay - Gaseous and highly Liquid andivan tie boetniques aon! Hie PIPL, Bites Chemical tonizenion (CD) .¢ with the primary “The ions are produced through the collision of the sample molecule: ions produced by # gas (called a reagent 884 gas is ionized through electron ionization he radical cations generated fragmentations and reactions. ‘The mo: transfer from a gas cation (GH') to the molecule. Methane, are the most eominon reagent BSCS. how chemical ionization occurs: A Electron ionization: CH, + e7 > CH4* + 2e7 CH;t > CH3 + H" cH;* > CH3* + He ‘The radical cations and the.carbocations, can react with the reagent gas to give various ) in the ionization chamber. ‘The reagent will undergo st common reaction generating, ions is a proton isobutane, and ammonia Let us take miethané as an’ example to understand Fragmentation: protonated species: Reactions: CH; + CH, > CHE + CH3 CH3 + CH, > C,Hi + H2 “The analyte molecules acquire the protons from any one of these cations: /MHt + CHy,HHL Hiqtevtanen Fast liom Bombardment (1B) Fast atom bombardment is it soft ionization technique Le, it causes little fragmentation ‘of the molecular ions generated. In fast atom bombardment ionization methods. the sample is dissolved in a non-volatile liquid and the ions are extracted by bombarding the sample with a beam of high energy atoms (~5 keV), usually argon (sometimes xenon). The commonly used liquid matrices include glycerol, thioglycerol, and m- nitrobenzyl alcohol. Fast moving Argon atoms are uenerated as shown in the Figure 11.6. The Argon radical cations (Ar), generated through electron ionization, are ‘accelerated and focused as a sharp beam. ‘The high energy Ar” ions are allowed to collide with the Ar atoms resulting in the neutralization of some of the Ar’ ions in the beam. The residual Ar™ in the beam are’extracted out by applying an electric field, thereby resulting in a beam of fast moving atoms. The atoms collide with the sample dissolved in the liquid matrix extracting the ions into the gas phase. Figure 11.6 Diagram showing the Jointi ae ial Ns tonnes cant tastes APHEL sted n Arie on jy ube FAB eases little oF no ionization bat desorbs the ions already extstns info the gas phase, FAB causes desorption of the ions, preston she. surface matrix: the compounds having higher surface activity are therefore detected bente” FAB is particularly good tor polar molecules with large molecular weights and molecules up to 10,000 Da can be detected. It is, therefore possible to detect biomolecules like peptides, oligonucleotides, and oligosaccharides using FAB ionization. Liquid secondary ion mass spectrometry (LSIMS) LSIMS is similar to FAB. with only difference that an ion is used for bombarding the ‘samples. Usually argon, xenon, or caesium ions are used for the LSIMS. Laser desorption Laser desorption or laser ablation is the ionization method wherein’ an intense laser beam is focused on a solid sample resulting in ablation of mass from the surface. The laser pulse causes both desorption and ionization of the molecules. The ions generated are short-lived and therefore detected simultaneously. Laser desorption, however, ‘causes fragmentation for large molecules (Molecular mass >500 Da) therefore restricting its use to small molecules. bead a (amino acids, peptides, Trot of much use for revolutionized theNEIL iota sy Wield Tet et stcibersaies Pisieinds pepindes oligonucleotides Proteins, pep 2,5-Dihydroxybenzoic acid (DHB) oligosaccharides FE-Dimethoxy-a-hydroxyeinhamic acid | Proteins, peptides (Sinapinic acid) J Hiydroxypicolinic acid (HPA) Oligonucleotides *Trihydroxyacetophenone (THAP) Oligonucleotides, ol igen | “The sample for MALDI is usually prepared in one of the following ways: i. mixing the analyte solution with the matrix: solution — deposition of the ‘mixture on a metallic plate + complete drying of the sample ii. Deposition of the matrix on the metallic plate —> drying of matros —~ addition of analyte solution —+ drying of analyte solution Desorption and ionization is achi red by applying the laser pulse on the dried sample. “Although the exact mechanism behind MALDI is not completely understood, it is believed that the absorption of light by the matrix molecules causes s iblimation of matrix crystals carrying along with them the ‘analyte molecules into the gas phase Cigue 4.7). = z Page 76 of 99se rogt oli’ ad (Teri AEat tWtestnohray _Hshsael em jety oF non-volatile iy generate tre was phase 10" MALDL ean very efticien j nucleic acids. and shohydrate as proteins. carbol bile molecules seh ge as 300 kia and therm and ionize the morectes a s only one charge ¢ molecule (MH"). The ften resulting in synthetic polymers, MAL DI can d i 1 positive MALDI usually resuits in the molecular spectes havin ion mode,'a quasimolecutar ion is formed by protonation of th ts of alkali metal ions, 0! samples or the matrices can have trace amount (Figure 11-8). Mu ly charged the quasimolecular species, MNa’ or sometimes MK’ species are also observed sometimes. 7 290.4680 ana Relative itesiy (4) ‘Sigel teal techniques such as shall be discussing theAPTEE tictectie Ninatysical Veen and baie Electrospray ionization (SD) vn elambers ine electrospray fonization, the analyte sotution enters ME jonization eh se eapitry The pea OM" Es ried between the capillary field, the maintained at atmospheric pressure, through @ are ~1-20 l/min. A potential difference of ~3-6 kV is app! and the counter-electrode that is ~0.3 ~ 2 em away: Under this electric sample droplets appearing at the capillary end accumulate large amount of charge. If the potential of the capillary is above a threshold voltage, the drop will be dispersed rary through which into a very fine spray. A coaxial sheath is present around the ca dry nitrogen is supplied for better nebulization and restricting the dispersion of the spray in space. Evaporation ofthe solvent causes He droplets to diminish in size and their charge density to increase: (Figure 11.9). The high charge density on these droplets can further result io the production of smaller droplets. The droplets keep fosing the solvent ultimately resulting, in the desorption of molecular ions fr urface, a surface active molecule will be om the surface. As the ions are generated from the su detected better. For large molecules such as proteins, the molecules do not desorb from the droplets but become ionized through complete ‘evaporation of the solvent. Meal plate =100V eel. species. Biological consecutive peaks the mass of the Page 78 of 99Lecture 12 Mass Spectrometry Hl ‘¢ aecelerated towards the mass analyzers. the gas phase ion: fs are utilized in the mass spectrometers. All_of these Following ioni A great variety of mass analyze! analyzers separate the molecules using static or dynamic electric fields and magnetic fields, alone or in combination. After mass analysis, the ions are detected and the mass spectra are generated. Imagine what would happen if the ions collide with the air molecules in the MS tube. This can lead to the loss of charge to the moleculés in the air, deviation in the ion trajectory which might lead to the collision with the MS tube, reactions with the air molecules, ete. A mass spectrometer, therefore operates under very high vacuum to ensure that the ions reach the detector without colliding with the air molecules. The mean free path of a particle is the average distance a particle travels before colliding with other particles and is inversely proportional to the pressure of the. gas and the size of the colliding molecules. The mean free path, according to the kinetic théory of gases is given by: kr. Vinay 21) where, 2 is the mean free path, kis the Boltzmann constant (1.38 x 10 JK", Tis the temperature, dis the sum of the radii of the ion and the colliding molecule, and p is the pressure. . few assumpti is Peay nitrogen (~78%) and oxygen (21%. The Van der Walls radii for nitrogen and oxygen are 155 pm and 152 pm, respectively. As the Van (12.3) Page 79 of 99Feet ical cehigucs nd Nisierret: Nine 24) 3.26 x 10m © 32.6 Fem). Wis In mass spectrometer. the ions have to travel lare distances (usually 1 the mean tree path n for incre: theretore absolutely essential to apply large vacu by several orders of magnitude. Let us now have look at some of the important mass analyzers: ‘Magnetic sector Figure 12.1 shows a diagram of the magnetic sector analyzer mass spectrometer. ‘The ions (say, cations) generated in the ionization chamber are accelerated under a strong electric field. The accelerated ions are allowed to pass through a narrow slit resulting in a sharply focused ion beam. The ions in the beam can be deflected by applying 4 magnetic field perpendicular to the velocity of the ions. Figure 12.1), which (Equation 11.8), c within a small = sequentially allow all t magnetic field, A: will move straightSELLE Roantowdiy here seu! Teste es ee wit lowest mamentinn ane! highest charge will appear Fret white Arighes somemtum and lowest charge il apne lst Time of igh (TOP) Jn atime of fight mass analyzer, the time taken by the fons to reach the detector is measured. The ions are generated in bundles eg. by MALDI. The ions are then ‘ accelerated towards the flight tube. The flight tube does not have any electric field and the ions drift in the flight tube according to their velocities (Figure 12.2). 420K Drift region ‘igure 12.2 Separation of theions ina POF:tube. Ife ic it a s ‘particle wih charge, q (ex) and mass, is accelerated towards the flight tube by «lectrostatic potential, Y, the kinétic energy (KE) of the particle can be given by: KE= qv a Amv? = zmv? = zeVSPILL ttsctn Equation 12.4 shows thot te time ken fy the price ws reel the de Hire tlh the detector is irc The F ratio oF the particle cam therefore be calculate! fc the time of flight of the particle: Rearranging equation 12.8 (129) A serious problem with the linear TOF analyzers is their poor resolution. “This happens due to difference in the flight times among the ions having same ratio. The factors responsible for the poor resolution include length of the ionization laser pulse, space distribution of the ions formed, and spread in the initial kinetic energies of the ions. In MALDI-TOF, for example, these factors severely affect the resolution. Two techniques have considerably improved the resolution in MALDI-TOF: i, Delayed extraction: In continuotis extraction, the ions generated are continuously extracted towards the TOF tube. An ion with higher initial kinetic energy reaches the detector earlier than the ion with smaller initial kinetic “energy. Delayed io proves this situation substantially. tion, the ions are allowed to move in the field free region ra ion Following i according to their kinetic energies during a short delay. An extraction pulse is then applied; the pulse gives more energy to the ions that are nearer to the jontegenectes compared to those that have moved away. The ions with small initial kinetic energies, therefore gain more energy and catch up the ions Page 82 of 99APIEAL Monstering Hisacals ical Totus al informatics is Extraction lonization source Flight tube! . +IBkV +1820KV +18Kv wy Site +18kV ~ 20k +18KV TLL Patse amplitude Time delay Figure 123 A dingrammatic presentation of layed extraction TOFna Hisinkormnaties Retlectrons: A. simple rette urids. The Nis Composed Uf a series uf equally spaced rellectron is placed atthe tube end! opposite 10 the ion source. A Potential is applied to the retlectron so as (0 reflect the incoming ions. A reflectron therefore acts as am, ion mirror. The ions with higher kinetic: energy Will travel longer distance before reflecting back than those have smaller Kinetic energy. The ions with higher kinetic energy are therefore made to travel longer distances thereby correcting for the spread in the peaks (Figure 12.4). sien oo TH ee = Pee UL UUULLLE ges sep ed pupae of cE ¥| i madi pote analyzers ; 1 to cach other as. four rodls arranged parallel ur rods arranged paral i c : posed by ! described as is made up oF ie of a quadrupole was Pro} e routs WAS tion have also proved A quadrupole mays analyze shown in Figure 12.5. The principl ; c t Steinwegen in 1953 wherein hnyperbolic cross-section OF ross sec rods with circular 6r necessary. In practice, however, : bolic cross-section in modern effective and have replaced the rods with hyper quadrupole detectors. @) vir te poentia ac potentat de persist sh penta 3c potentiat Sete potential Figure 125 A qeadrupole mass analyzer (A) andthe potentat on the rods 28 a function of time (B)- ‘Consider the rods in x-z plane to be ata positive potential, U and the rods in y-z plane to be al a negative potential -U, An ac. potential is subsequently applied to the rods such that the ae. potential on the rods in x-z plane is 180° out of phase than that applied on the rods in the y-z plane. ‘The potential on the rods in x-z and y-z plane can therefore be repre tea : The values of U gas phase ions To understand in the x-z plane positive dc. pthe cation remains largely ivatt \ causes the ion lo get Focused towards the centre, I a vatic Light. it will readit respond to the changes in the potential and can accelerite towards the rods during ERATIVE pote tential and collide with them. Collision of the cation with the quadrupol rods during negativ Ng Negative poteneial depends on the magnitude of the potential on the rod frequency of the ac. potential, mass of the cation, charge on the cation, and the Position of the cation in the quadrupole. Let us now ttirn our attention towards the rods in the y-z plane. These rods have a negative average potential; the heavier cations will therefore get accelerated towards the rods and collide with them. Lighter cations will respond to the a.c. potential and get focused towards the centre. We car igher say that the x-2 rods filter out the lower masses while y-z rods filter out the. hi .d to allow the passage of a very masses. The four rods together can therefore be u: small range of masses. A quadrupole therefore acts as a.mass filter Jon trap As the name suggests, ion trap mass analyzers trap the ions inside them. An ion trap can either be a 2D or a 3D ion trap. A 3D ion trap is basically a quadrupole in 3 dimensions. It has a circular electrode (also called a ring electrode) with two ellipsoid electrodes as its caps (Figure 12.6). Ring electrode Figure 126A ramumatic representation of aa Joint initiative of ITs and 1Sc~ Funded by MHRD ‘ Page 86 of 99NeTEL Hien inlike linear quadrupole 8 surfaces slectrodes. have hyperbolic: sur All the three” -electrodes have hy a shied Je with four parallel rods) wherein electrostalic forces act in Wwo ax! (quadrupole with four parallel ro% ays: rc at the stable trajectories o} forces act in all the three axes in an ion tap This implies that the stable traj higher sensitivity and are useful the ions cause them to be trapped. fon traps provide ions of desired mass can selectively be allowed to in tandem mass spectrometry; escape the trap by varying the ac potential; the escaped ions can then be analyzed by another mass analyzer attached in tandem. Orbitrap Orbitrap is an electrostatic ion trap. It has a barrel containing a spindle-shaped electrode at the centre. The spindle electrode is held at a ‘constant negative voltage (- 3200 V) for positive ion mode MS. ons enter the orbitrap tangentially and get trapped by revolving around the spindle shaped electrode (Pigiire’ 12:7). The ions can be ejected out by applying the radiofrequencies of suitable frequency to the central electrode. Figure 127 A schematic diagram of Mass spectrometry coupled with chromatography _ Mass spectrometers have been successfully ‘coupled with “the “liquid and gas chromatographic methods, Chromatographic-methiods ‘separate the compounds based on the differences in their physico-chemical popes. A ‘pomplex mixture of compounds can be resol ved info pure components u Using one or niore chromatographic methods. Though excellent in separating the molecules, chromatographic methods do not allow identification of the unknown compounds in the mixture. The separated Joint initiative of ITs and I1Se ~ Funded by MHRD © Page 87 af 99components can be eotlected 4 sing NV s pa couple the iP nllected and identified using MS. 1 is also possible & he th i“ i also possible to coupte the mass spectrometers with the chromate: raphic methods. Gas chromatograph ca jas chromatography (GC) and liquid chrom: y tography (LC) coupled with mass spectrometers have emerged ay very powerful” analyficdt” tools. GC allows easy interfacing with’ the mass spectrometers; a gas chromatographic column can directly be coupled to the ionization source of the MS. Interfacing the LC with MS, however, is not as straightforward. We shall not be discussing the different types of interfaces of LC and MS. As it allows studying the large, polar, non-volatile, and thermolabile compounds. LC-MS is more widely used compared with GC-MS. Electrospray ionization is one of the softest ionization methods and probably the most widely used ionization method for LC-MS. LC-MS and GC-MS therefore allow determining the molecular masses of the eluants in real-time. The ions.separated by MS can be fragmented in a collision cell; identification of these fragments by another mass analyzer allows identification ‘of the ‘compounds (tandem mass spectrometry). We shall see in the next lecture how fragmented ions help in the identification of the compounds. “Tandem mass spectrometry _ Taedese mance, spectrmeniy eles Known as MS/MS) involves more than one mass analyzer. As we have just seen, incorporating more than ‘one mass analyzer greatly enhances the capabilities of a mass spectrometer: Furthermore, it improves the sensitivity, mass resolution, and the mass accuracy of the spectrometer. The most ‘common MS/MS experiment involves Selecting, ‘an ion using first mass analyzer, which is then fragmented os ae a ee are principle possible to do powerful tandem mass. biomolecules.SPH Biagelmoloe Detectors ch are: resenily exist, some of whICR A variety of ion detectors pt j. cormoniyueedin ‘An electron multiplier is perhaps the most Con monly ust It consists of a series of electrodes (dynodes). When lectrons from the dynode (the first sts the ion signal into electrons) Electron multiplier detector in mass spectrometers: an ion strikes the first dynode, it causes release of el dynode, therefore is a conversion dynode that conve , that strike the second dynode releasing more electrons and so on. This cascading effect causes a large amplification in the electrical current (Figure 12.8A). Another design of the electron multiplier uses a continuous dynode (12.88). (A) =, Amplified Clectronic signal Figure 12.8 A diagrammatic represeotation af current amplification in an electron moltiplier Faraday cup: Faraday cup consists of a metallié cup that is connected to the earth through a resistor. An incoming ion strikes the cup and gets neutralized. This results in an electric current through ‘the resistor that is proportional to the ion abundance.PS Weal i The nominal and average wolecular ma ethane are 6 Da and 16.0428 Da, respeeti S spec ectively. A mass spectrometer. however, detects the exe se8 OF the ions Isotopic Hess 1 Wie abundances: Isotopic abutances. are rellected in the high resolution mass Spectra of the compounds and allow easy iclentification of small organic compounds Let us take an example of methane. The different possible isotopoloaues of methane are listed in table 13.2 along with their natural abundances. Table 132 Fotopoogoes of methane Isotopologue Relative abundances “CH, = 0.989 x 0.99985 * 0.99985 * 0.99985 = 0.99985 = 0.9884 = 98.84 % 5D = 0.989 « 0.99985 x 0.99985 * 0.99985 = 0.00015 = 1,483 x 10= 1.483 x 107% = 0.989 x 0.99985 * 0.99985 < 0.00015 0.00015 | =2.247x 1022.27 x 10°% = 0.989 0.99985 * 0.00015 x 0.00015 = 0.00015 = 3,337 x 10? = 3.337 x 10°°% = 0.989 x 0.00015 x 0.00015 = 0.00015 x 0.00015 “| = 5.007 x 107°= 5.007 x10"*% = D.0110 x 0.99985 * 0.99985 x 0.99985 = 0.99985¢ Hive tor 1 cclunigues int 1 NPTEL ioteete ees jsotopologues of methane. Other HO} are the two predonrinant eae methane mass spectrum will ect AN isotopologues are too small in quantities 10 del therefore look like as shown in Figure 13.1 "CH, L~ Relative abundance 0 24 6 8 10 12 14 16 18 20 22 24 26 2% 30 m/e ‘Figure 1.1 Electron ionization mass spectrum of methane showing the two predominant ieotopologues Fragmentation: We have already studied that electron ionization imparts large amount of energy to the cations that it generates. The radical cations thus generated undergo extensive fragmentation giving smaller cations. The fragments generated from molecular ions can provide important structural information about the molecules. Let us take ethanol as an example to see how this works:y availible which can provide the puss Provide the possible molecular formulae of th fed wit he teRen the compound when 3 8 Relative abundance a 8 0 4 & 12 16 20 24 28 32 36 40 44 48 52 56 60 mlz FFiguee 12 Electron fonization mass spectrum of ethanol Analysis of biomolecules Since the advent of MALDi and ESI ionization method, mass spectrometry has become a routine method for analyzing biomolecules. MS has been successfully — “utilized for obtaining-a-large-amount of information.about biomolecules. including... - information that is difficult to obtain using other tools. Let us go through the various applications of MS in biomolecular analysis: _ } “Molecular weight determination: Determination of molecular weight of a biomolecule : is the most straightforward application of M a oO Structure verification and, 0 synthesized molecules, such i oligonucleotides are often 4 liquid chromatography . O spectrometry. Suppose ‘ synthesized peptide, DAKLI MALDI mass spectrum as (6 peptide is 1250.63 Da. The isont Higinkortics Ficcuutasicat fectasame Neri ie 163 100 25 go 3 me 3 60 2 fee oS 3 x 0 4000 "1650 1100. 1150 12001250 1300 1350” 14007 1450" 1500 mlz Fitere 133.4 typed MALDL mast specrem of the spiked pelle, DAKLAYEWOP (iheoreticay ‘acute mas: 125263 Day Mentfication of chemical modifications: Biomolecules, especially proteins, can undergo a variety of chemical modifications such as Phosphorylation, acetylation, ‘methylation, fatty acylation, glycosylation, ete. These modifications are involved in ity, signal’ transduction, gene cxpression, etc. It is therefore important to idemtify these species for understanding their function: Owing to its sensitivity and Tesolution, mass spectrometry has emerged & the method of choice for identification of small molecule snodifications in biomolecules. se{iaolves ten paincieieaiy Protein sequencing: i Nesity Rela | L Ah \ =@| =e R i : Page 96 of 99 Joint initiative of ITs and tl Se ~ Funded by MHRDsand Buseetnae= rvtonsy Hiouneas sical Technine sri Pronein idemification: Wentification of # prover classically requires complete or partial protein sequence, © partial sequence can allow protein identification by ences of the proteins available in protein sequence re be identified by doing sequencing using MS. determine the sequence of a protein for its comparing it with the seq therefor databases. A protein However, it may not be required to identification. A typical scheme for protein identification is shown in Figure 13.5. rosin sasple lieiibebastisbiie] tes cree vans [ Psat of ate | Sregh of inne protein | Tea 1. match is food i the databases proenceg typsin | ane eee ate as discussed io Figire 13.4 | Relative intensity, -ces oF the fragments in the pregin Seujnen Ces databases, Protein idaiification using MS is central to the prowomic studies. A typ pical prowomie analysis is brieny summarized in Figure 13.6 2D gel electrophoresis Isolation of protein —_. to resolve the protein components in the sample Given biological sample —. Protein identification ___ Isolation of the spots _ as discussed in Figure 13.5, from the 2D-gel 4 " Figute 13.6 Outline ofa proteomics experiment |. ESI, however, has proyed to be sufficiently i ecrplccen tis howe, important to note