MASS SPECTROPHOTOMETRY

1. MASS SPECTROMETRY M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

2. INTRODUCTION • Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. • It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds

3. PRINCIPLE • The sample is ionised to generate parent molecular ions • Further fragmentation will generate fragment ions • The process of ionisation has to be controlled to generate similar ions from all the molecules in the mixture

4. The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields • The ions are detected, usually by a quantitative method • The ion signal is processed into mass spectra

7. INSTRUMENTATION • The analyser, detector and ionisation source are under high vacuum to allow unhindered movement of ions • Operation is under complete data system control

8. How does a Mass Spectrometer work? • Samples easier to manipulate if ionised • Separation in analyser according to mass-to-charge ratios (m/z) • Detection of separated ions and their relative abundance • Signals sent to data system and formatted in a m/z spectrum

9. Sample introduction • Prior to sample introduction, 2 things must be achieved: • Sample must be introduced into vacuum • Sample must be vaporized • The sample is introduced by placing it on the probe, which is then inserted through a vacuum lock into ionisation region of mass spectrometer

10. Ionization • Ionization means placing a charge on a neutral molecule • Methods: • 1) Electron ionization • 2) Electrospray • 3) Matrix-assisted laser desorption/ ionization(MALDI)

11. Electron ionization • Also known as electron bombardment / electron impact method • The sample is heated to vaporize it • The sample in the gas phase is now delivered into electron ionization region • Here a beam of electrons with energy of 70EV is made to interact with the sample • This interaction causes electron ejection in the sample molecules leading to ionization

12. Electrospray • Generates ions directly from aqueous or aqueous/organic solutions • The solution is forced through a narrow needle which is kept at a high potential (3.5 kV) • The voltage on the needle causes the spray to be charged as it is nebulized • Thus, very small droplets are created and they are charged on their surfaces

13. The electric charge density on the surface of the droplet is a function of its size- smaller the droplet, larger is the electric charge density • Thus, as the droplets decrease in size, there is repulsion between mutually charged droplets • At this point, ions begin to leave the droplet • Ions are led into mass analyzer

14. Matrix-assisted laser desorption/ionization(MALDI) • It nondestructively vaporizes & ionizes both big and small molecules • The analyte is first co-crystallized with an excess of a matrix compound • Matrix compounds are organic acids, which absorb in the UV range • After the co-crystallization, a pulse UV laser beam is focused on the surface of the crystal

15. The matrix absorbs the radiation & is vaporized • The analyte is also vaporized and carried along with the matrix • The matrix doubles up as a proton acceptor or donor & thus also ionizes the analyte • Different matrices: • 2,5 dihydroxy benzoic acid-proteins,peptides & oligonucleotides • Sinapinic acid – proteins & peptides

17. Mass analyzers • After ionization, ions that are in the gas phase enter the mass analyzer • It separates ions within a selected range of mass-to-charge (m/z) ratios • To separate ions,different mass analyzers use magnetic fields,electric fields or the time taken by an ion to move over a fixed distance

18. Magnetic sector mass analyzer • J.J Thompson,who built the 1st mass spectrometer, used a magnet to measure the m/z value of electrons • It separates ions in a magnetic field according to the momentum & charge of ion • A 1 to 10kV electric field accelerates ions from the source region into the magnetic sector • Once it reaches the magnetic field,the ion beam is bent in an arc by the magnetic field

19. The radius of the arc(r) depends on: • Momentum of the ion • Charge of the ion • Magnetic field strength • The greater the momentum of the ions, the larger is their arc radius • The separation of ions is thus based upon their momentum • Hence, magnetic analyzers are also called momentum analyzers

21. The quadrapole analyzer • In the mass spectrometer, an electric field accelerates ions out of the source region and into the quadrupole analyzer • The quadrapole analyzer consists of 4 rods/electrodes arranged across from each other • The ions are made to travel through the quadrupole • Here, they get filtered according to their m/z ratio

22. Only one of the separated ion beams is allowed to strike the detector • The separation according to m/z ratio is based upon the radio frequency & direct current voltages applied to these electrodes • These voltages produce an oscillating electric field that transmits ions according to their m/z value by alternatively focusing them in different planes

24. Time of flight Analyzer • Used mostly with MALDI • The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through the same potential, and then measures the time they take to reach the detector • The smaller ions will reach the detector first because they will acheive great velocities • The larger ions will have lesser velocities & reach the detector late

25. DETECTOR • The final element of the mass spectrometer is the detector • The detector generates a signal current from incident ions by generating secondary electrons which are further amplified • Types: • Faradey Cup • Electron Multiplier • Photomultiplier Conversion Dynode

26. Faradey Cup • Concept: A change in charge on a metal plate results in a flow of electrons • The flow creates a current • When a single ion strikes the surface of a dynode in faradey cup, it results in ejection of several electrons • This ejection induces a current in the cup

27. Ejection of several electronscurrent in the cup

28. Electron multiplier • Uses a series of dynodes maintained at successively higher potentials • Thus,electrons released by the 1st dynode (when ion impinges on it) are dragged to 2nd dynode because it has a higher potential • Highly sensitive

30. Photomultiplier Conversion Dynode • Ions strike a dynode resulting in emission of electrons • These electrons are made to strike a phosphorous screen • The screen releases photons • Photons detected by a photomultipier

31. TANDEM MS • Complex mixtures are now analyzed without prior purification by tandem MS • It employs the equivalent of 2 mass spectrometers linked in series • The 1st spectrometer separates individual peptides upon their differences in mass • By adjusting the field strength of 1st magnet, a single peptide can be directed into 2nd mass spectrometer ,where fragments are generated and their mass determined

33. Applications: • 1) Identification & quantification of proteins • 2) Drug screening • 3) Pesticides & pollutants screening • 4) Used to screen blood samples from new borns for the presence & conc of proteins,F.A,other metabolites • 5) Screening of inborn errors of metabolism (phenyl ketonuria, ethylmalonic encephalopathy,glutaric acidemia type 1)

34. Gas chromatography-mass spectrometry In this technique, a gas chromatograph is used to separate different compounds. This stream of separated compounds is fed online into the ion source, a metallicfilament to which voltage is applied. This filament emits electrons which ionize the compounds. The ions can then further fragment, yielding predictable patterns. Intact ions and fragments pass into the mass spectrometer's analyzer and are eventually detected

35. Liquid chromatography-mass spectrometry • Separates compounds chromatographically before they are introduced to the ion source and mass spectrometer. • It differs from GC/MS in that the mobile phase is liquid, usually a mixture of water and organic solvents • Most commonly, an electrospray ionization source is used in LC/MS. There are also some newly developed ionization techniques like laser spray

36. APPLICATIONS • PROTEIN CHARACTERIZATION: • Proteins are 1st digested into smaller peptides using different proteases • A collection of these smaller peptides is then introduced into the mass analyzer • ANALYSIS OF BIOLOGICAL NONCOVALENT COMPLEXES • Electrospray ionization gets these noncovalent complexes into gaseous phase & MS can be used to observe these complexes • Eg: Hb complex , DNA duplex , cell surface carbohydrates, whole viruses

37. CHARACTERIZAION OF SMALL BIOMOLECULES ASSOCIATED WITH DIFFERENT BIOLOGICAL STATES • MS successfully discovered that cis-9,10-octadecenoamide was present in the sleep state & was absent during the wake state • APPLICATIONS IN VIROLOGY: • Identification of a virus in a given sample by analyzing the mass of the capsid proteins or DNA/RNA through MS • SEQUENCING PEPTIDES & OLIGONUCLEOTIDES • MALDI has been used recently to sequence proteins & oligonucleotides

38. Isotope ratio MS: isotope dating and tracking • Mass spectrometry is also used to determine the isotopic composition of elements within a sample

39. Trace gas analysis • selected ion flow tube (SIFT-MS), andproton transfer reaction (PTR-MS), are variants of chemical ionization dedicated for trace gas analysis of air, breath or liquid headspace • Use well defined reaction time allowing calculations of analyte concentrations from the known reaction kinetics without the need for internal standard or calibration.

40. Atom probe • An atom probe is an instrument that combines time-of-flight mass spectrometry and field ion microscopy (FIM) to map the location of individual atoms. • Pharmacokinetics • Pharmacokinetics is often studied using MS because of the complex nature of the matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time point data. • The most common instrumentation used in this application is LC-MS with a triple quadrupole MS

41. Glycan analysis • Mass spectrometry (MS), with its low sample requirement and high sensitivity, has been the predominantly used in glycobiology for characterization and elucidation of glycan structures. • Mass spectrometry provides a complementary method to HPLC for the analysis of glycans

42. Space exploration • As a standard method for analysis, mass spectrometers have reached other planets and moons. • Two were taken to Mars by the Viking program. • In early 2005 the Cassini-Huygens mission delivered a specialized GC-MS instrument aboard the Huygens probe through the atmosphere of Titan, the largest moon of the planet Saturn. • This instrument analyzed atmospheric samples and was able to vaporize and analyze samples of Titan's frozen, hydrocarbon covered surface once the probe had landed.

43. Advantages • Provides molecular weights of peptides and proteins with high accuracy (0.1-0.01%) • Highly sensitive; requires fmol-pmol quantities of sample • Sample purity not important • Can be coupled with on-line separation methods such as HPLC and capillary electrophoresis for the analysis of mixtures

44. Disadvantages • Noncovalent complexes are often disrupted • Cannot distinguish stereoisomers • Expensive instrumentation

45. Thank you