Mass spectrometry.

Transcription

1 Mass spectrometry Mass spectrometry provides qualitative and quantitative information about the atomic and molecular composition of inorganic and organic materials. The mass spectrometer produces charged particles that consist of the parent ion and ionic fragments of the original molecule, and it sorts these ions according to their mass/charge ratio.

2 What does a mass spectrometer do? 1. It measures mass better than any other technique. 2. It can give information about chemical structures. What are mass measurements good for? To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers

3 What is a Mass Spectrometer? All Instruments Have: 1. Sample Inlet 2. Ion Source 3. Mass Analyzer 4. Detector 5. Data System

4 In mass spectrometry molecules in a test sample are converted to gaseous ions. Gaseous ions are then separated according to their mass to charge (m/z) ratio, and detected.

5 e - ionise accelerate 4000 V 0 V separate e - Magnetic and/or electric field vapourise e - v a c u u m light heavy e - A sample B Mass spectrometry C A B C 5

6 The mass spectrum is a plot of the (relative) abundance of the ions at each m/z ratio

7 In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. In mass spectrometry three essential functions are performed by three associated components: 1. A small sample is ionized, usually to cations by loss of an electron. Component: The Ion Source 2. The ions are sorted and separated according to their mass and charge. Component: The Mass Analyzer 3. The separated ions are then measured, and the results displayed on a chart. Component: The Detector

8 Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum. Atmospheric pressure is around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10-5 to 10-8 torr (less than a billionth of an atmosphere). All mass analysers operate under vacuum in order to minimise collisions between ions and air molecules. Without a high vacuum, the ions produced in the source will not reach the detector. At atmospheric pressure, the mean free path of a typical ion is around 52 nm; at 1 mtorr, it is 40 mm; and at 1 mtorr, it is 40 m.

9 Ionisation methods in mass spectrometry Ion sources have the dual function of producing ions without mass discrimination form the sample and accelerating them into the mass analyzer with a small spread of kinetic energies prior to acceleration. Ions may be produced from a neutral molecule by removing an electron to produce a positively charged cation, or by adding an electron to form an anion. Both positive and negative-ion mass spectrometry may be carried out.

10 Ionisation When a high energy electron collides with a molecule it often ionizes it by knocking away one of the molecular electrons (either bonding or non-bonding). This leaves behind a molecular ion (colored red in the following diagram). Residual energy from the collision may cause the molecular ion to fragment into neutral pieces (colored green) and smaller fragment ions (colored pink and orange). The molecular ion is a radical cation, but the fragment ions may either be radical cations (pink) or carbocations (orange), depending on the nature of the neutral fragment

11 Electron impact ionisation (EI) Electron impact ionisation (EI) is widely used for the analysis of metabolites, pollutants and pharmaceutical compounds. Neutral molecules are diffused in a gaseous-phase The chamber is maintained at a pressure of torr and a temperature of 200 ± 0.25 C.

12 Electron impact ionisation (EI) A stream of electrons from a heated metal filament is accelerated to 70 ev potential. Sample ionisation occurs when the electrons stream across a high vacuum chamber into which molecules of the substance to be analysed (analyte) are allowed to diffuse. Electrons emitted from a glowing filament are drawn off by a pair of positively charged slits through which the electrons pass into the body of the chamber. Electron beam anode Ion accelerating region

13 Electron impact ionisation (EI) Interaction with the analyte results in either loss of an electron from the substance (to produce a cation) or electron capture (to produce an anion). The positive ions formed in the ionization chamber are drawn out by a small electrostatic field between the repeller plate (charged positive) behind them and the first accelerating slit (charged negative) ahead of them. The analyte must be in the vapour state in the electron impact source, which limits the applicability to biological materials below ca. 400 Da.

14 Chemical bonds in organic molecules are formed by the pairing of electrons. Ionisation resulting in a cation requires loss of an electron from one of these bonds (effectively knocked out by the bombarding electrons), but it leaves a bond with a single unpaired electron. This is a radical as well as being a cation and hence the representation as M, the ( ) sign indicating the ionic state and the ( ) a radical. Such radical ions are termed molecular ions, parent ions or precursor ions and under the conditions of electron bombardment are relatively unstable. Their energy in excess of that required for ionisation has to be dissipated. This latter process results in the precursor ion disintegrating into a number of smaller fragment ions that may be relatively unstable and further fragmentation may occur. This gives rise to a series of daughter ions or product ions, which are recorded as the mass spectrum.

15 Almost all possible bond breakages can occur and any given fragment will arise both as an ion and a radical. For the production of a radical cation, as it is not known where either the positive charge or the unpaired electron actually reside in the molecule, it has been the practice to place the dot signs outside the abbreviated bracket sign,.

16 Fragmentation pathways in n-butane and the EI spectrum.

17 The highest-mass ion in a spectrum is normally considered to be the molecular ion, and lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound. Ex. CO 2 Since a molecule of carbon dioxide is composed of only three atoms, its mass spectrum is very simple. The molecular ion is also the base peak, and the only fragment ions are CO (m/z=28) and O (m/z=16).

18 Ex. propane The molecular ion of propane also has m/z=44, but it is not the most abundant ion in the spectrum. Cleavage of a carbon-carbon bond gives methyl and ethyl fragments, one of which is a carbocation and the other a radical. Both distributions are observed, but the larger ethyl cation (m/z=29) is the most abundant, possibly because its size affords greater charge dispersal.

19 Ex. cyclopropane A similar bond cleavage in cyclopropane does not give two fragments, so the molecular ion is stronger than in propane, and is in fact responsible for the the base peak. Loss of a hydrogen atom, either before or after ring opening, produces the stable allyl cation (m/z=41). The third strongest ion in the spectrum has m/z=39 (C 3 H 3 ). Its structure is uncertain, but two possibilities are shown in the diagram. The small m/z=39 ion in propane and the absence of a m/z=29 ion in cyclopropane are particularly significant in distinguishing these hydrocarbons.

20 Electrospray ionisation (ESI) It involves the production of ions by spraying a solution of the analyte into an electrical field. It is a soft ionisation technique and enables the analysis of large intact biomolecules, such as proteins and DNA. The electrospray (ES) creates very small droplets of solvent-containing analyte. The essential principle in ES is that a spray of charged liquid droplets is produced by atomisation or nebulisation. ESI is the result of the strong electric field (around 4 kev at the end of the capillary and 1 kev at the counter electrode) acting on the surface of the sample solution.

21 Sample dissolved in polar, volatile buffer (no salts) is pumped through a stainless steel capillary ( mm) at a rate of ml/min. Strong voltage (3-4 kv) applied at tip along with flow of nebulizing gas causes the sample to nebulize or aerosolize Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges) Pressure = 1 atm Inner tube diam. = 100 um Sample Inlet Nozzle (Lower Voltage) Partial vacuum N 2 MH Sample in solution N 2 gas MH 2 MH 3 High voltage applied to metal sheath (~4 kv) Charged droplets

22 Electrospray (Detail) As the solvent evaporates in the high-vacuum region, the droplet size decreases and eventually charged analyte (free of solvent) remains.

23 Electrospray Ionization Can be modified to nanospray system with flow < 1 ml/min Very sensitive technique, requires less than a picomole of material Strongly affected by salts & detergents Positive ion mode measures (M H) (add formic acid to solvent) Negative ion mode measures (M - H) - (add ammonia to solvent)

24 Positive or Negative Ion Mode? If the sample has functional groups that readily accept H (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-proteins If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-dna

25 Masses of large intact proteins, DNA and organic polymers can also be accurately measured in electrospray MS although the m/z limit of measurement is normally 2000 or 3000Da. The distribution of basic residues in most proteins is such that the multiple peaks (one for each M nh) n ion, are centered on m/z about 1000.

26 Multiple Charging Consider a peptide with MW of With ESI-MS, charges by H addition M nh MnH n Resultant ions formed are :- When z = 1 m/z = (100001)/1 = When z = 2 m/z = (100002)/2 = 5002 When z = 3 m/z = (100003)/3 = When z = 4 m/z = (100004)/4 = 2501 When z = 5 m/z = (100005)/5 = 2001

27

28 Multiple Charging Advantage in that allows measurement of high mass ions with instruments of limited m/z range. Particularly true for ESI-MS Advantage for analysis of high mass samples that take multiple charges brings sample m/z down into measurable range of MS Computer Algorithms deconvolute m/z to original mass. Figure from The Expanding Role of MS in Biotechnology G. Siuzdak

29 Calculation of Molecular Mass of a lysozyme using following mass spectrometry data: m/z = (MW nh)/n where, m/z = the mass-to-charge ratio MW = the molecular mass ; n = the integer number of charges on the ions H = the mass of a proton = Da. Usually the number of charges for each m/z signal is not known. However, two adjacent m/z signals in the series of multiply charged ions differ by one charge. For example, if the ions appearing at m/z in the above figure have n charges, then the ions at m/z will have n 1 charge.

30 Thus, = (MW nh )/n and = [MW (n1)h ] /(n1) After rearrangement of the equation to exclude the MW term: n(1431.6) - nh = (n1) (n1)h and so: n(1431.6) = n(1301.4) H therefore: n( ) = H and so: n = ( H ) / ( ) Charges on the ions at m/z = /130.2 = 10. Once the number of charge is calculated, molecular mass can easily be calculated as explained: m/z = (MW nh )/n = MW (10 x 1.008)/ x 10 = MW (10 x 1.008) MW = 14, = MW = 14,305.9 Da

31 MASS ANALYSERS Once ions are created and leave the ion source, they pass into a mass analyser, the function of which is to separate the ions and to measure their mass to charge ratio. In the majority of instruments, a particular type of ionisation is coupled to a particular mass analyser that operates by a particular principle. That is, ESI and its derivatives with quadrupole (or its variant ion-trap) and MALDI is coupled to TOF detection.

32 Magnetic sector analyser The ions are accelerated by an electric field. The electric sector acts as a kinetic energy filter and allows only ions of a particular kinetic energy to pass, irrespective of the m/z. The ions emerge from the electrostatic analyser (ESA) with the whole range of masses but the same velocity. A given ion with the appropriate velocity then enters the magnetic sector analyser. It will travel in a curved trajectory in the magnetic field with a radius depending on the m/z and the velocity of the ion.

33 Magnetic sector analyser Easiest Conceptually to understand Separate electromagnetically Electromagnetic Prism Usually combined with ESA (energy focusing device) - enables high mass resolution (Double Focusing Instrument) makes high accuracy mass measurements possible Large (Heavy!!), Expensive to operate Comparatively slow scan rates High Skill level required to operate and maintain Self-service use by users not possible

34 Quadrupole mass spectrometry Four parallel cylindrical rods: A direct current (DC) voltage and a superimposed radio frequency (RF) voltage are applied to each rod, creating a continuously varying electric field along the length of the analyser. Once in this field, ions are accelerated down the analyser towards the detector. The varying electric field is precisely controlled so that during each stage of a scan, ions of one particular massto-charge ratio pass down the length of the analyser. Ions with any other mass-tocharge value impact on the quadrupole rods and are not detected. By changing the electric field (scanning), the ions of different m/z successively arrive at the detector.

35 Quadrupoles have variable ion transmission modes m2 m4 m1 m3 m4 m3 m2 m1 mass scanning mode m2 m1 m4 m3 m2 m2 m2 m2 single mass transmission mode

36 Mass Analyzers - the quadrupole vs. magnetic sector Quadrupole: Changes DC and RF voltages to isolate a given m/z ion. PRO: cheap, fast, easy Magnetic Sector: Changes B and V to focus a given m/z into detector. PRO: turn in geometry means less dark noise, higher precision,

37 Ion trap mass spectrometry Ion trap mass spectrometers use ESI to produce ions, all of which are transferred into and subsequently measured almost simultaneously (within milliseconds) in a device called an ion trap. The ion trap contains three hyperbolic electrodes which form a cavity in a cylindrical device in which the ions are trapped (stored) and subsequently analysed.

38 Ion trap The ring electrode has an alternating potential of constant radio frequency but variable amplitude. This results in a three-dimensional electrical field within the cavity. The ions are trapped in stable oscillating trajectories that depend on the potentials and the m/z of the ions. To detect these ions, the potentials are varied, resulting in the ion trajectories becoming unstable and the ions are ejected in the axial direction out of the trap in order of increasing m/z into the detector. A very low pressure of helium is maintained in the trap, which cools the ions into the centre of the trap by low-speed collisions that normally do not result in fragmentation. These collisions merely slow the ions down so that during scanning, the ions leave quickly in a compact packet, producing narrower peaks with better resolution.

39 ESI ion trap mass spectrometers have found wide application for analysis of peptides and small biomolecules such as in protein identification by tandem MS; liquid chromatography/mass spectrometry (LC/MS); combinatorial libraries and rapid analysis in drug discovery and drug development. Structural analysis, MS n in an ion trap. The MS n procedure in an ion trap involves ejecting all ions that are stored in the trap, except those corresponding to the selected m/z value. To perform tandem MS (MS 2 ) a collision gas is introduced (a low pressure of helium) and collision-induced dissociation (CID) occurs. The fragment ions are then ejected in turn and the fragment spectrum determined. The process can be repeated successively where all the fragment ions stored in the trap except those fragment ions corresponding to another selected m/z value are ejected. This fragment ion can then be further fragmented.

40 In this example, of a steroid-related compound, the structure can be analysed when the (MH) ions at are selected to be retained in the ion trap. These ions are subjected to collision-induced dissociation (CID) resulting in loss of the aliphatic sulphonate from the quaternary ammonium group and partial loss of some hydroxyl groups in the tandem MS (MS 2 ) experiment. The major fragment ions (561.2 and 579.6) are further selected for CID (MS 3 ) resulting in subsequent losses of more hydroxyl groups from specific parts of the steroid ring.

41 MALDI, TOF mass spectrometry, MALDI-TOF Matrix-assisted laser desorption ionisation (MALDI) produces gas phase protonated ions by excitation of the sample molecules from the energy of a laser transferred via a UV light-absorbing matrix. The matrix is a conjugated organic compound (normally a weak organic acid such as a derivative of cinnamic acid and dihydroxybenzoic acid) that is intimately mixed with the sample. The sample is mixed with an excess of the matrix and dried on to the target plate, where they co-crystallise on drying. Pulses of laser light of a few nanoseconds duration cause rapid excitation and vaporisation of the crystalline matrix and the subsequent ejection of matrix and analyte ions into the gas phase. This generates a plume of matrix and analyte ions that are analysed in a TOF mass analyser.

42 MALDI ionisation mechanism and MALDI TOF sample plate. (a) The sample is mixed, in solution, with a matrix the organic acid in excess of the analyte (in a ratio between 1000 : 1 to : 1) and transferred to the MALDI plate. An ultraviolet laser is directed to the sample (with a beam diameter of a few micrometres) for desorption. The laser radiation of a few nanoseconds duration is absorbed by the matrix molecules, causing rapid heating of the region around the area of laser impact and electronic excitation of the matrix. The immediate region of the sample explodes into the high vacuum of the mass spectrometer, creating gas phase protonated molecules of both the acid and the analyte. The laser flash ionises matrix molecules: neutrals (M) and matrix ions (MH), (MH) and sample neutral fragments (X).

43 MALDI Ionization Matrix Laser Analyte Absorption of UV radiation by chromophoric matrix and ionization of matrix Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/ popping )

44 MALDI: Matrix Assisted Laser Desorption Ionization Sample plate hn Laser MH 1. Sample is mixed with matrix (X) and dried on plate. 2. Laser flash ionizes matrix molecules. 3. Sample molecules (M) are ionized by proton transfer: XH M MH X. /- 20 kv Grid (0 V)

45 MALDI Unlike ESI, MALDI generates spectra that have just a singly charged ion Positive mode generates ions of M H Negative mode generates ions of M - H Generally more robust that ESI (tolerates salts and nonvolatile components) Easier to use and maintain, capable of higher throughput Requires 10 ml of 1 pmol/ml sample

46 Principal for MALDI-TOF MASS peptide mixture embedded in light absorbing chemicals (matrix) pulsed UV or IR laser (3-4 ns) detector vacuum V acc strong electric field cloud of protonated peptide molecules Time Of Flight tube

47 Principal for MALDI-TOF MASS Linear Time Of Flight tube ion source detector time of flight Reflector Time Of Flight tube ion source detector reflector time of flight

48 Principle of time-of-flight (TOF) The ions enter the flight tube, where the lighter ions travel faster than the heavier ions to the detector. If the ions are accelerated with the same potential at a fixed point and a fixed initial time, the ions will separate according to their mass to charge ratios. This time of flight can be converted to mass.

49 A reflector (or reflectron) is a type of ion mirror that provides higher resolution in MALDI TOF. The reflector increases the overall path length for an ion and it corrects for minor variation in the energy spread of ions of the same mass. Both effects improve resolution. The device has a gradient electric field and the depth to which ions will penetrate this field, before reversal of direction of travel, depends upon their energy.

50 Q-TOF Mass Analyzer NANOSPRAY TIP MCP DETECTOR PUSHER ION SOURCE SKIMMER HEXAPOLE QUADRUPOLE HEXAPOLE COLLISION CELL HEXAPOLE TOF REFLECTRON

51 Mass Spec Equation (TOF) m z = 2Vt 2 L 2 m = mass of ion z = charge of ion V = voltage L = drift tube length t = time of travel

52 The time-of-flight of the ion is converted to mass using the following principles: An accelerating potential (V) will give an ion of charge z an energy of zv. This can be equated to the kinetic energy of motion and the mass (m) and the velocity (v) of the ion zv = 1/2mv 2 Since velocity is length (L) divided by time (t) then m/z = [2Vt 2 ]/L 2 V and L cannot be measured with sufficient accuracy but the equation can be rewritten m/z = B(t-A) 2 where A and B are calibration constants that can be determined by calibrating to a known m/z

53 Resolution & Resolving Power Width of peak indicates the resolution of the MS instrument The better the resolution or resolving power, the better the instrument and the better the mass accuracy Resolving power is defined as: DM M M is the mass number of the observed mass (DM) is the difference between two masses that can be separated

54 Resolution in MS

55 Resolution in MS QTOF

56 Atmospheric Pressure Ionization (API) Ion Source.