Mass spectrometry

introduction and principle of Mass spectrometry with its components.
ionization , accelerators deflection and detection, types of MS, different types of ion sources , types of mass analyzers , advantages and disadvantages of different types of ion source and mass analyzers, different types of detectors for the ions dectections

1. Mass Spectrometry Hari Sharan Makaju M.Sc. Clinical Biochemistry 1 st Year

2. Introduction Mass spectrometry Extremely valuable analytical technique in which the molecules in a test sample are converted to gaseous ions that are subsequently separated in a mass spectrometer according to their mass-to-charge (m/z) ratio and detected Mass spectrum The mass spectrum is a plot of the (relative) abundance of the ions at each m/z ratio.

3. Introduction Powerful qualitative & quantitative analytical technique Use to measure wide range of clinically relevant analytes Universal detector & detects everything . Coupled with either gas or liquid chromatography results in expanded analytical capabilities with widespread clinical applications

4. Types of Mass Spectrometry AMS (Accelerator Mass Spectrometry) Gas Chromatography-MS Liquid Chromatography-MS ICP-MS (Inductively Coupled Plasma-Mass spectrometry ) IRMS (Isotope Ratio Mass Spectrometry ) Ion Mobility Spectrometry-MS MALDI-TOF SELDI-TOF

5. Basic Principle In this technique, molecules are bombarded with a beam of energetic electrons. The molecules are ionized and broken up into many fragments, some of which are positive ions. Each kind of ion has a particular ratio of mass to charge , i.e. m/z ratio(value). For most ions, the charge is one and thus, m/z ratio is simply the molecular mass of the ion. The ions passes through magnetic and electric fields to reach detector where they are detected and signals are recorded to give mass spectra.

6. The ions are separated in the mass spectrometer according to their mass-to-charge ratio, and are detected in proportion to their abundance. A mass spectrum of the molecule is thus produced. Provides: Molecular Weight Fragmentation information Nature and S tructure of their precursor molecule.

7. Principle and Instrumentation

8. 1. Sample Inlet Sample stored in large reservoir from which molecules reaches ionization chamber at low pressure in steady stream by a pinhole called “Molecular leak” Solid samples with lower vapor pressure: Directly inserted into ionization chamber & volatilization is controlled by heating the probe Liquids : are handled by hypodermic needle injection through a silicon rubber dam Gaseous samples : are leaked into the ionization chamber directly by the help of mercury manometer

9. Ionization Loss of electrons from a molecule leads to radical cation The ion sources is the part of MS that ionizes the material under analysis (the analyte ) The ions are produced: By removing or adding the electron By removing or adding the proton (H+) By addition of entities s/a NH4+ or CH5+ 11

10. 2. Ion source Liquids and solids are first converted into gases from the gaseous sample, ions are produced in a Box like enclosure called Ion Source. Function Produces ion without mass discrimination of the sample . Accelerates ions into the mass analyzer . Molecular ions are formed when energy of the electron beam reaches to 8-15 eV. Fragmentation of the ion reaches only at higher bombardment energies at 70 eV.

11. 2. Ion source Electron ionization and chemical ionization are ionization techniques used when gas phase molecules can be introduced directly into the analyzer from a gas chromatography In HPLC-MS, following ionization sources are used Electrospray ionization Sonic spray ionization Atmospheric pressure chemical ionization Atmospheric pressure photoionization Other ionization technique Inductively Coupled Plasma Matrix Assisted Laser Desorption Ionization (MALDI) Atmospheric pressure-MALDI Fast Atom Bombardment 14

12. 2. Ion source Classification of ion sources Gas Phase Sources .(volatile sample) Electron Ionization ( EI). Chemical Ionization (CI). Field Ionizations (FI ). Desorption Sources (non volatile sample) Field Desorption ( FD) Electrospray Ionization ( ESI). Matrix assisted desorption Ionisation (MALDI). Plasma desorption (PD). Fast Atom Bombardment (FAB). Thermospray Ionization (T S). Secondary Ion Mass Spectrometry ( SIMS).

13. 2. Ion Source a. Electron Ionization ( EI ) Gas phase molecules are bombarded by electrons emitted from a heated filament & attracted to a collector electrode Occurs in vacuum to prevent filament oxidation A potential difference of 70eV is enough to bring ionization Positive ions are repelled or drawn out of ionization chamber by an electric field The cations are then electrostatically focused and introduced into the mass analyzer

14. a. Electron Ionization (EI) 17

15. 2. Ion Source a . Electron Ionization ( EI ) Advantages Gives molecular mass and also the fragmentation pattern of the sample. Extensive fragmentation and consequent large number of peaks gives structural information. Gives reproducible mass spectra. Can be used as GC/MS interface . Disadvantages Sample must be thermally stable and volatile. A small amount of sample is ionized (1 in 1000 molecules ).

16. 2. Ion Source b . Chemical Ionization Soft ionization technique In chemical ionization the ionization of the analyte is achieved by interaction of it’s molecules with ions of a reagent gas in the chamber or source. Chemical ionization is carried out in an instrument similar to electron impact ion source with some modifications such as:- Addition of a vacuum pump. Narrowing of exit slit to mass analyzer to maintain reagent gas pressure of about 1 torr in the ionization chamber. Providing a gas inlet.

17. 2. Ion Source b . Chemical Ionization

18. 2. Ion Source b . Chemical Ionization Typical reagent gases are methane, ammonia, isobutane & water An electron beam produces reactive sps . Such as CH5+ for methane Collision between the relative reagent gas and the analyte cause proton and energy transfer Because the protonated molecule is not highly excited in this process, relatively little fragmentation occurs More controlled than EI reduced fragmentation greater sensititivty typically ~5 eV energy transfer

19. 2. Ion Source b. Chemical Ionization Advantage For analyte molecular mass determination and for its quantification Negative ion electron capture CI has become popular for quantification of drugs such as benzodiazepines Used in Quadrupole Ion Trap in lower gas pressure Used for samples which undergo rapid fragmentation in EI Limitations Provides less information about structure Not suitable for thermally unstable and non-volatile samples. Relative less sensitive then EI ionization. Samples must be diluted with large excess of reagent gas to prevent primary interaction between the electrons and sample molecules

20. 2. Ion Source c . Electrospray Ionization (ESI ) A technique in which a sample is ionized at atmospheric Pressure before introducing into mass analyzer A sample is passed through a narrow metal or fused silica capillary to which 3 – 5 kV charge is applied The partial charge separation in between liquid and capillary results instability causing expulsion of a charged droplets series from a Taylor cone A coaxial nebulizing gas helps direct the charged droplets towards a counter electrode Labile compounds are analyzed using “cold electrospray” Unique feature: production of multiple charge ions from peptides/proteins

21. 2. Ion Source c. Electrospray Ionization

22. 2. Ion Source c. Electrospray Ionization (ESI ) In this method quassimolecular ions are produced by addition of a proton (hydrogen ion) to give (M+H)+ or other cations such as sodium ion ( M+Na )+ or removal of hydrogen ion (M-H ). Multiply charged ions are often observed Advantages Used for analysis of high molecular weight biomolecules such as polypeptides, proteins, oligonucleotides and synthetic polymers . Can be used along with LC and capillary electrophoresis . softest ionization technique. Disadvantage Multiply charged ions are confusing and needs careful interpretation. Sensitive to contaminants such as alkali metals or basic compounds. Not suitable for low polarity compounds .

23. 2. Ion Source Sonic spray ionization Coaxial nitrogen gas travelling at the speed of sound can be used to create the spray and cause ionization As the sonic velocity gas flows over the surface of mobile phase exiting the capillary, 2 effects: Droplet fission occurs as a result of shear stress created by sonic gas flow Ionization efficiency optimized by minimum droplet size at sonic velocity

24. 2. Ion Source d. Atmospheric Pressure Chemical Ionization (APCI) Similar to ESI taking place in atmospheric pressure but only differ in mode of ionization In APCI, no voltage is applied to inlet capillary instead a separate corona discharge needle is used to emit a cloud of electrons that ionize compounds Because eluent molecules like water, methanol are present in excess relative to the analytes in the sample, they are predominantly ionized and then act as a reagent gas that reacts secondarily to ionize analyte molecules Relatively little fragmentation and mainly used in Tandem MS

25. 2. Ion Source e. Atmospheric Pressure Photoionization (APPI) ESI and APCI less effectively ionize nonpolar compounds APPI is similar to ESI and APCI but differ in photon flux used instead of corona discharge needle Better quantitative and a potential higher dynamic range is obtained by use of photon source Krypton discharge lamp with magnesium fluoride window is used

26. 2. Ion Source

27. 2. Ion Source f. Inductively Coupled Plasma ( ICP) It is atmospheric Pressure ionization method which can bring complete ionization Particularly useful for trace metal and heavy metal analysis in tissue or body fluids ICP is extremely sensitive and capable of extremely high dynamic range ICP-MS is comparatively free from most interferences

28. f. Inductively Coupled Plasma (ICP)

29. 2. Ion Source g . Fast Atom Bombardment (FAB) Ionization technique in which the analyte and non-volatile liquid matrix mixture is bombarded by a high energy beam of inert gas such as Argon or Xenon. Used for large compounds with low volatility ( eg peptides , proteins, carbohydrates) Solid or liquid sample is mixed with a non-volatile matrix ( eg Glycerol Monothioglycerol Carbowax 2,4 – dipentyl phenol 3 – nitrobenzyl alcohol (3 – NBA) Immobilised matrix is bombarded with a fast beam of Argon or Xenon atoms. Charged sample ions are ejected from the matrix and extracted into the mass analysers Gives M+H + or M-H + ions

30. 2. Ion Source g. Fast Atom Bombardment (FAB ) Advantages Best for fast heating of samples and slow down the sample fragmentation Good for rapid ionization . Used in conjunction with Tandem MS for diagnosis of short chain fatty acid acylcarnitine deficiencies from newborn blood spots Disadvantage Difficulty in tracing and distinguishing between low molecular weight compounds. Not good for multiply charged compounds Choosing correct matrix is difficult

31. 2. Ion Source h. Matrix-Assisted Laser Desorption Ionization (MALDI) Ionization is carried out by bombarding a laser beam on the sample dissolved in a matrix solution . Matrix is used in MALDI to Absorb the laser energy. Prevent analyte agglomeration. Protect analyte from being destroyed by direct laser beam . Matrix consists of a crystallized molecules of which the most commonly used are :- 3,5 – dimethoxy – 4 – hydroxy cinnamic acid ( sinapinic acid). α – cyano – 4 – cinnamic acid ( α – cyano or α – matrix). 2,5 – dihydroxy benzoic acid (DHB)

32. h. Matrix-Assisted Laser Desorption Ionization (MALDI ) Solution of the matrix is made in a mixture of highly purified water and another organic compound (acetonitrile or ethanol ). Triofluoro acetic acid (TFA) is also added. If sinapinic acid is used as a matrix the solution is prepared by adding: 20 mg/ml of sinapinic acid. Water: acetonitrile: TFA (50:50:0.1 ). Matrix solution is then mixed with the analyte to be investigated . The organic compound acetonitrile dissolves hydrophobic proteins present in the sample while water dissolves hydrophilic proteins . The solution is then spotted in a air tight chamber on the tip of the sample probe . With a vacuum pump the air is removed and vacuum is created which leads to evaporation of the solvent leaving behind a layer of recrystalized matrix containing analyte molecules.

33. h. Matrix-Assisted Laser Desorption Ionization (MALDI ) Now the laser beam (EMR) is shooted to the sample, the range of uv radiation used is 360-390nm.due to the absorbing substance is present in matrix ,it absorbs radiation or energy and thus it transfers some of its energy to sample molecule where by the molecular ions are formed and then accelerate to analysers .

34. 2. Ion source i . Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization ( AP-MALDI) Works as MALDI at atmospheric pressure Major advantage: ability to switch sources easily while coupling the inherent speed & multiple sample wells of traditional MALDI with MS Major drawback as compared with ESI is poor fragmentation of the slightly charged ions produced in MALDI

35. 2. Ion Source J. Surface- Enhanced Laser Desorption/Ionization ( SELDI) Combines affinity purification & MALDI on the target The most common setup involves producing a MALDI target surface modified with some type of affinity capture property (hydrophobic, ionic, immobilized metal affinity chromatography (IMAC), DNA, antibody, etc ) Sample of interest is exposed to one or more of these affinity surfaces where certain analyte will bind preferentially, then a matrix is added to enhance desorption/ionization & analyzed by TOF Major advantage is low sample loss as purification & analysis occur on the same surface

36. Surface- Enhanced Laser Desorption/Ionization (SELDI)

37. Ionization Method Typical Analytes Sample I nt r oduct i o n Mass Ra ng e I on iz at i o n [mass] Electron Impact (EI) relatively small, volatile GC or li q u i d /so lid probe up to 1000 Daltons hard, [M] •+ if observed Chemical Ionization (CI) relatively small, volatile GC or li q u i d /so lid probe up to 1000 Daltons hard to soft; varies with carrier [M+H] + Fast Atom Bombardment (FAB ) carbohydrates, organometallics, peptides, nonvolatile sample mixed in viscous matrix up to 6000 Daltons soft, but harder than MALDI, ESI [M+Na] +, [M+H] + Matrix Assisted Laser Desporption (MALDI) peptides, proteins, nucleotides sample mixed in solid matrix up to 500 , 000 Daltons soft [ M+ H ] + Electrospray (ESI) peptides. proteins, nonvolatile HPLC or syringe up to 6000 Daltons soft [M+Na] +, [M+H] + Comparison of different ion source

38. Vacuum system To prevent collision of ions in magnetic or electrical field Vacuum of 10 -3 torr to 10 -9 torr is applied depending on MA type Mass analyzer is maintained at elevated temp (150 – 250  C ) to avoid absorbing of molecules inside the vacuum chamber Efficient high vacuum pumps generally don’t operate well near atmospheric pressure so it should have mechanical vacuum pump to evacuate system pressure Diffusion pump, Turbomolecular Pump, Cryopump can be used. Higher pump capacities are associated with lower detection limits because noise arising from the gas background is reduced

39. 3. Mass analyzer General classes of MS In beam type instruments , the ions make one pass through the instrument and then strike the detector Beam type instruments Quadrupole Magnetic sectors Time of flight (TOF ) In trapping type analyzer , ions are held in a spatially confined region of space by a combination of magnetic or electrostatic or radio frequency electrical field Trapping mass spectrometers Quadrupole ion trap Linear ion trap Ion cyclotron resonance

40. I . Beam Type Designs Quadrupole Ion beam entering the quadrupole have various m/z values but with only narrow range will transport Ions outside the narrow range are ejected radially

41. Quadrupole Quadrupole MS rely on superposition of Radio Frequency & DC potential applied Both RF & DC are fixed in Selected Ion Monitoring mode In scanning mode of operation, the RF &/or DC voltages are continuously varied to scan a range of m/z values The effective force with pseudopotential points inward towards the quadrupole axis & is proportional to the distance from axis Therefore it acts as confining force preventing ions from being ejected radially from quadrupole assembly

42. Quadrupole

43. I . Beam Type Designs Quadrupole Currently most widely used MS Advantages Easy to use Flexibility adequate performance for most applications highly developed software systems

44. I . Beam Type Designs B. Magnetic source Magnetic field is used to deflect ions around a curved path Radius of curvature of an ion depends on m/z ratio and strength of magnetic field Ions with correct m/z pass through the detector Ions too heavy or too light do not make it through Can vary strength of magnetic field so that all ions can be detected Can increase resolution further by subjecting ions to an additional electric field double focusing mass analyzer double focusing mass analyzer

45. I . Beam Type Designs B. Magnetic source Double- focussing magnetic sector mass spectrometer . combination of a large electromagnetic and some kind of electrostatic focusing device.

46. I . Beam Type Designs B. Magnetic source Advantages : Versatile Reliable highly sensitive double focusing variation are capable of very high m/z resolution & mass accuracy Demerits : typically expensive, large & heavy, difficult to use

47. I . Beam Type Designs C. Time of Flight (TOF ) Based on the fact that a lighter ion travels faster than a heavier ion, provided they both have same kinetic energy Ions are accelerated by a potential of several kilovolts Ion travel down the flight tube and strike the detector at the far end of t he flight tube. The time it takes to traverse the tube is known as the flight time; this is related to the mass-to-charge ratio of the ion m=ion of mass E= kinetic energy L = distance to travel in a region free of electric fields

48. C. Time of Flight (TOF) TOF is coupled readily to pulse ionization method, MALDI (most commonly)

49. C. Time of Flight (TOF) Use of Reflectron : to improve resolution by compensating for kinetic energy variations

50. I . Beam Type Designs C. Time of Flight (TOF) Non-scanning technique Advantages : unlimited m/z range high acquisition speed, high mass accuracy, moderately high resolution, high sensitivity, reasonable cost. Significant advantage of modern TOF-MS produce exact mass measurements, typically with low parts per million(ppm) accuracy Practical use in routine chromatography & clinical analysis

51. II. Trapping Mass Spectrometers A. Quadrupole Ion Trap (QIT): Primarily used as GC or HPLC detectors Relatively compact, inexpensive & versatile instrument Similar physical principle as Quadrupole MS however RF field of an ion trap is designed to trap ions in 3D Known for high sensitivity

52. A . Quadrupole Ion Trap (QIT):

53. II. Trapping Mass Spectrometers B. Linear Ion Trap (LIT): Based on modified linear Quadrupole Mass Filter Electrostatic fields are applied to the ends to prevent ions from exiting out of the ends of device An advantage is the trapping field can be turned off at will & the device operated as a normal QMF

54. II. Trapping Mass Spectrometers C. Ion Cyclotron Resonance (ICR): ICR is a trapping technique with high sensitivity Based on principle that ion immersed in a magnetic field undergo circular motion (cyclotron motion) A typical ICR-MS uses a high field superconducting magnet Ions are suspended inside the cell and undergo cyclotron motion which keeps ions from being lost radially . A low potential is applied to the end caps to keep ions from leaving the trap axially.

55. C. Ion Cyclotron Resonance (ICR ): Each m/z is associated with a specific cyclotron frequency. Ions circulating in the ICR cell induce an electrical current in detection electrodes. The time-dependent image current is Fourier transformed to obtain the component frequencies of the different ions, which correspond to their m/z

56. When the ions pass into the magnetic field they are bent into a circular motion in a plane perpendicular to the field by the Lorentz Force (see figure 1 and equation 1) The frequency of rotation of the ions is dependent on their m/z ratio (equation 2). Deconvolution of this signal by FT methods results in the deconvoluted frequency vs. intensity spectrum which is then converted to the mass vs. intensity spectrum (the mass spectrum) by equation 3 (the mass conversion). It is also usual to correct for mass errors at this stage by applying a calibration.

57. II. Trapping Mass Spectrometers C. Ion Cyclotron Resonance (ICR ): Advantages High mass accuracy Ultrahigh resolution The ability to perform multiple stages of MS/MS( MS n ) Disadvantages : High instrument cost Very demanding site requirements: space & access restriction Uses high field superconducting magnet: erase of credit cards and magnetically encoded strips Cost of operation, care & maintenance is high A highly skilled operator level

58. Tandem Mass Spectrometers Physical principle: 2 MS (mass filter)are arranged sequentially with a collision cell placed between 2 instruments 1 st filter : used to select ions of particular m/z called either parent ion or precursor ion directed into collision cells, precursor ions collide with background gas molecules & broken to form product ion 2 nd second : acquires the mass spectrum of the product ions Possible scan function involving 1 st MS to select a given m/z and full scanning through mass spectrum of product

59. Tandem Mass Spectrometers As with single-stage mass spectrometers, tandem mass spectrometers are categorized as: Beam-type instruments: Tandem in space ( Triple Quadrupole ) Trapping instruments : Tandem in time 2 magnetic sector instruments have been operated in Tandem with collision cell placed between 2 instruments: permit high resolution, rarely used, expensive, cumbersome to operate Double focusing MS also used in Tandem known as: Linked scanning technique QIT & ICR can be used in tandem MS and are capable of multiple stage of MS

60. Tandem Mass Spectrometers MS/MS mainly used for quantitative analysis of routine samples Excellent for structural characterization & compound identification The most important feature: very high selectivity together with good sensitivity Very low interference when coupled with HPLC, low consumable cost, high sample throughput rate

61. 4. Detectors All MS use detectors for electron multiplication except ICR MS(ion cyclotron resonance ) 3 classes of electron Multipliers: (similar principle) Discrete Dynode Multiplier Continuous Dynode Electron Multipliers Micro channel Plate Electron Multipliers Additional detectors Faraday cup and image current detection .

62. Discrete Dynode Multipliers (DDM) Cascade process that electron amplifies on striking dynode One electron can produce pulse of 10 4 – 10 8 electrons Duration of pulse is as low as <10 Nano seconds

63. 2. Continuous dynode electron multiplier (CDEM) Same as DDM, only differ in physical construction: set of dynode is replaced by a single continuous resistive surface That acts both as a (continuous) voltage divider to establish the potential gradient and as the secondary electron generating surface 3. Microchannel Plate Election Multipliers Microchannel Plate in a disk of glass that contains pores extending from the upper surface to the lower surface Channels are 3 -30 um diameter, length 200-1000 um

64. Faraday Cup: The Faraday Cup is a simple electrode that intercepts the ion beam directly This induces current is then amplified using electronic amplifier Provides absolute measure of ion current Some instruments use both electron multiplier & Faraday Cup to provide extended dynamic range of detection : useful for elemental analysis of trace metals in samples

65. 5. Computer & Software MS instruments generate enormous amounts of raw data In toxicology lab one important function of the data system is library searching to assist in compound identification Several commercial libraries with quality & quantity of available spectra are available Data systems exist that aid in characterization of spectral data to identify proteins Fragmentation information can also be compared with peptide databases to identify structural mutations that may be present Software programs are also available to locate & identify components in complex chromatographic separations

66. Application of MS Biotechnology :  the analysis of proteins, peptides, oligonucleotides Pharmaceutical :  drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism Clinical :  neonatal screening, haemoglobin analysis, drug testing Environmental :  W ater quality, food contamination Geological :  oil composition

67. Mass spectrometry help biochemists Accurate molecular weight measurements: sample confirmation, to determine the purity of a sample, to verify amino acid substitutions, to detect post-translational modifications, to calculate the number of disulphide bridges Reaction monitoring: to monitor enzyme reactions, chemical modification, protein digestion Amino acid sequencing: sequence confirmation,  de novo  characterisation of peptides, identification of proteins by database searching with a sequence "tag" from a proteolytic fragment Oligonucleotide sequencing: the characterization or quality control of oligonucleotides Protein structure: protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions, macromolecular structure determination

68. References C. A. Burtis , E. R. Ashwood : Tietz's Textbook of Clinical Chemistry and Molecular Diagnosis , 4 th Edition, Elsevier Saunders M. L. Bishop, E. P. Fody : Clinical Chemistry Principles, Techniques, Correlations , 7 th Edition, Lippincott Williams and Wilkins K. Wilson and J. Walker: Principles and Techniques of Biochemistry and Molecular Biology , 7 th Edition, Cambridge University Press Edmond de Hoffmann, Vincent Stroobant Mass Spectrometry-Principles and Applications , Third Edition http :// premierbiosoft.com/tech_notes/mass-spectrometry.html http:// www.chm.bris.ac.uk/ms/ionisation.xhtml http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm http://www.chem.ox.ac.uk/spectroscopy/mass- spec/Lecture/oxmain_lectureCI.html http:// science.widener.edu/svanbram