Mass spectrometry

introduction to mass spectrometry

1. MASS SPECTROMETRY Dipesh Tamrakar M.Sc. Clinical Biochemistry 1

2. Mass spectrometry powerful qualitative & quantitative analytical technique Use to measure wide range of clinically relevant analytes Universal detector & detects everything (theoretically) Coupled with either gas or liquid chromatography results in expanded analytical capabilities with widespread clinical applications Key tool in emerging field of proteomics A mass spectrometer is a device that measures the mass-to-charge (m/z) ratio of ions. 2

3. The concept of mass spectrometry was put forth by Sir J.J Thomson, English physicist who discovered the electron in 1887; NP-1906 Thomson II Experiment The beam of electron get deflected in magnetic field The degree of deflection depends on mass and charge 3 Video 1

4. Basic concepts Requires an ionization of ions from neutral atom/molecules Useful for determining the elemental composition & structure of both inorganic & organic compounds In MS, ions are formed continuously in the ion source of mass spectrometer and accelerated toward the detector by an electrical potential or accelerating voltage V The mass spectrum can be compared with spectra in various instruments Computer-based libraries of spectra are available to assist in identification of the analytes 4

5. PRINCIPLE 5 Video 2

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8. 1. Inlet 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 8

9. Ionization In 2002, the Nobel Prize was shared by John Fenn & Koichi Tanaka for their development of electrospray and laser desorption ionization respectively If low energy or soft ionization technique are used, the mass of the target molecule can be determined Advances in soft ionization techniques have extended the use of MS to the direct measurement of peptide & protein mass Ionization at higher energy results in more extensive fragmentation of target molecules 9

10. 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+ 10

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 11

12. 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 p.d . 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 2. Ion source 12

13. a. Electron Ionization (EI) 13

14. b. Chemical ionization Soft ionization technique (proton transfer) 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 This is major 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 2. Ion source 14

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16. c. Electrospray Ionization (ESI) A technique in which a sample is ionized at atm. 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 2. Ion source 16

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18. d. Sonic spray ionization (SSI) 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 2. Ion source 18

19. e. Atmospheric Pressure Chemical Ionization (APCI) Similar to ESI taking place in atm. 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 2. Ion source 19

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21. f. 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 2. Ion source 21

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23. g. Inductively Coupled Plasma (ICP) It is atm. 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 ( e.g parts per trillion) and capable of extremely high dynamic range ICP-MS is comparatively free from most interferences 2. Ion source 23

24. h. Matrix-Assisted Laser Desorption Ionization (MALDI) Originally described in 1987 consisting purely of laser desorption/ionization Due to limitation on size and stability of analyte, addition of matrix to assist the process under vacuum Currently , analyte is dissolved in solution of matrix (small mol. Wt. UV-absorbing compound) Generally matrix to analyte ratio is 1000:1 As volatile solvents evaporate, the matrix compound crystallizes and incorporates analyte molecules MALDI is mostly coupled with Time of Flight-MS 2. Ion source 24

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26. 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 2. Ion source 26

27. 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 2. Ion source 27

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29. k. Fast Atom Bombardment (FAB) FAB produces ions when a high-velocity beam of atoms impacts the surface of liquid containing analytes Potentization is thought to occur when analytes on the surface of vaporized droplets are transferred to the gas state Since the bombardment occurs in high vacuum, the liquid used must have a high boiling point Used to ionize proteins and small molecules Used in conjunction with Tandem MS for diagnosis of short chain fatty acid acylcarnitine deficiencies from newborn blood spots 2. Ion source 29

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31. 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 atm 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 31

32. 3. Mass analyzer Beam type instruments Quadrupole Magnetic sectors Time of flight (TOF) II. Trapping mass spectrometers Quadrupole ion trap Linear ion trap Ion cyclotron resonance General classes of MS In beam type instruments , the ions make one pass through the instrument and then strike the detector 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 32

33. 1. Beam Type Designs Quadrupole Currently most widely used MS Easy to use, flexibility, adequate performance for most applications, relatively low cost, non critical site requirements & highly developed software systems It consists of four parallel electrically conductive rods arranged in a square array forming long channel through which ion beam pass 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 33

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35. Quadrupole MS rely on superposition of Radio Frequency & DC potential applied Both RF & DC are fixed in SIM 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 1. Beam Type Designs 35

36. B. Magnetic source Nowadays rarely used in MS It is versatile, reliable, highly sensitive & in their double focusing variation are capable of very high m/z resolution & mass accuracy Demerits: typically expensive, large & heavy, difficult to use 1. Beam Type Designs 36

37. C. Time of Flight (TOF) Non-scanning technique Advantages: unlimited m/z range, high acquisition, high sensitivity & reasonable cost Significant advantage of modern TOF-MS produce exact mass measurements, typically with low ppm accuracy Practical use in routine chromatography & clinical analysis Simply based on the fact that a lighter ion travels faster than a heavier ion, provided they both have same kinetic energy 1. Beam Type Designs 37

38. The flight time for an ion of mass m & kinetic energy E to travel a distance L in a region free of electric fields is given by: T = L(m/2E)1/2 TOF is coupled readily to pulse ionization method, MALDI (most commonly) TOF-MS is extremely fast, 5000 spectra/s or greater 1. Beam Type Designs 38

39. 2. Trapping Mass Spectrometers 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 39

40. B. Linear Ion Trap (LIT): Based on modified linear Quadruple 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 2. Trapping Mass Spectrometers 40

41. 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 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 2. Trapping Mass Spectrometers 41 Video 3

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43. D. 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 rates Physical principle: 2 MS are arranged sequentially with a collision cell placed between 2 instruments 2. Trapping Mass Spectrometers 43

44. D. Tandem Mass Spectrometers 1 st : used to select ions of particular m/z called either parent ion or precursor ion 2 nd : directed into collision cells, precursor ions collide with background gas molecules & broken to form product ion Possible scan function involving 1 st MS to select a given m/z and full scanning through mass spectrum of product ion (structural characterization) In constant neutral loss scan, 2MS are scanned synchronously with m/z offset between parent and product ion Another scan function is multiple reaction monitoring (MRM) 44

45. D. Tandem Mass Spectrometers MRM primarily used for quantitative analysis of few selected target compounds & is a closer analogue of SIM monitoring used in GC-MS Classification: as with single stage MS Beam type instrument: Tandem in space (Triple Quadrupole) Trapping instrument: Tandem in time Q1 as MS1, Q3 as MS3 and Q2 as function cell in triple quadrupole 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: linked scanning technique; a product ion scan by linked scanning involves low resolution for MS1 & high resolution for MS2 45

46. D. Tandem Mass Spectrometers 2 TOF-MS gives excellent sensitivity & throughput Hybrid MS: combination of 2 different types of MS more popular quadrupole-MS1 , TOF-MS2; used in proteomics & clinical lab applications Trapping mass spectrometers: ions are held in one region of space QIT & ICR can be used in tandem MS and are capable of multiple stage of MS Extremely versatile but unable to perform true precursor ion scans or constant neutral loss scans 46

47. 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 1. 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 47

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49. 2. Continuous dynode electron multiplier (CDEM) Same as DDM, only differ in physical construction: set of dynode is replaced by single continuous surface The surface of the tube contain an electrically resistive layer The resistive layer also serves as the secondary electron emitter CDEM is generally fabricated from a specialized glass 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 4. Detectors 49

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51. Faraday Cup: The Faraday Cup is a simple electrode that intercepts the ion beam directly This 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 4. Detectors 51

52. 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 52 Video 4

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55. Applications of MS: GC-MS Gas chromatography- mass spectrometry (GC-MS) is a method that combines the features of gas liquid chromatography and mass spectrometry to identify different substances within a test sample Application of GC_MS Drug detection Fire investigation Environmental investigation Explosives investigation , and Identification of unknown samples. 55

56. Applications of Tandem MS Biotechnology and Pharmaceutical To determine chemical structure of drugs and drug metabolites Detection/quantification of impurities, drugs and their metabolites in biological fluids and tissues Analysis of liquid mixtures Fingerprinting Nutraceuticals/ herbal drugs/ tracing source of natural products or drugs Clinical testing and Toxicology Inborn errors of metabolism, cancer, diabetes, various poisons, drugs of abuse, etc. 56

57. Pharmaceutical analysis Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets Biomolecule characterization Protein and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis 57

58. Advantages of MS/MS as a screening tool in NBS Sensitive Specific Accurate Quantitation Internal standards: gold standard for accuracy High impact Multiple Metabolite, Multiple Disease Screening cost effective High throughput 58