Mass spectrometry(Ionization Techniques) by Ashutosh Panke

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It's silde about ionization tech.in mass spectrometry.

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  • Remember the proteomics lab tour and the explanation of tryptic fragments for the peptide BSA. The trypsin enzyme was used to cut the peptide at specific amino acids. The calculated mass for each fragment is used to identify the protein based on the measured mass of the fragments. The unknown protein was compared to know masses from proteins digested with trypsin.
  • Mass spectrometry(Ionization Techniques) by Ashutosh Panke

    1. 1. Presented by:- Ashutosh Panke (M.Pharmacy,Pune.)
    2. 2. Ionization TechniquesIonization Techniques  Electron Impact (EI)Electron Impact (EI)  Chemical Ionization (CI)Chemical Ionization (CI)  Negative Ion Chemical Ionization (NICI)Negative Ion Chemical Ionization (NICI)  Spray Methods:Spray Methods:  Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Chemical Ionization (APCI)  Fast Atom Bombardment (FAB)Fast Atom Bombardment (FAB)  Field Ionization (FI)Field Ionization (FI)  Good for Gas Phase Samples (e.g. for GC Detector)Good for Gas Phase Samples (e.g. for GC Detector)  Continuous Ion Source, High degree ofContinuous Ion Source, High degree of FragmentationFragmentation
    3. 3. Ionization TechniquesIonization Techniques  Electro spray Ionization (ESI)Electro spray Ionization (ESI)  Matrix Assisted Laser Desorption IonizationMatrix Assisted Laser Desorption Ionization (MALDI)(MALDI)  Other Methods:Other Methods:  Secondary Ion Mass Spectrometry (SIMS)Secondary Ion Mass Spectrometry (SIMS)  Inductively Coupled Plasma (ICP)Inductively Coupled Plasma (ICP)
    4. 4. Ionization SourcesIonization Sources  The scope and utility of a mass spectrometric method is dictatedThe scope and utility of a mass spectrometric method is dictated by the ionization process.by the ionization process.  The appearance of mass spectra for a given molecular species isThe appearance of mass spectra for a given molecular species is highly dependent upon method used for ion formation.highly dependent upon method used for ion formation.  Ion sources are classified into two major categoriesIon sources are classified into two major categories Gas Phase:Gas Phase: - Applicable to thermally stable compounds with mol.Wt. less- Applicable to thermally stable compounds with mol.Wt. less than 10than 1033 D or Boiling point less than 500D or Boiling point less than 50000 C.C. - Sample is 1- Sample is 1stst vaporized and then ionised.vaporized and then ionised. Desorption:Desorption: - Applicable to analytes with mol. Wt. as large as 10- Applicable to analytes with mol. Wt. as large as 1055 D .D . - Sample in solid state or liquid state is directly converted into- Sample in solid state or liquid state is directly converted into gaseous ions.gaseous ions.
    5. 5. Fast Atom bombardment (FAB)Fast Atom bombardment (FAB)  Polar molecules, such as peptides with mol. wt. up to 10000Polar molecules, such as peptides with mol. wt. up to 10000 daltons can be analyzed by a ‘soft’ ionization technique calleddaltons can be analyzed by a ‘soft’ ionization technique called FAB.FAB.  The bombarding beam consists of xenon or argon atoms of highThe bombarding beam consists of xenon or argon atoms of high translational energy. This beam is produced by 1translational energy. This beam is produced by 1stst ionizing xenonionizing xenon atoms with electrons to give xenon radical cations.atoms with electrons to give xenon radical cations.  These radical cations are accelerated to 5-15 kev to give radicalThese radical cations are accelerated to 5-15 kev to give radical cations of high translational energy which are then passedcations of high translational energy which are then passed through xenon.through xenon.  During this passage, the charged high energy xenon obtainsDuring this passage, the charged high energy xenon obtains electron from the xenon atoms and the Xe.+ ions removed by anelectron from the xenon atoms and the Xe.+ ions removed by an electric field.electric field.
    6. 6. Fast atom bombardment ( FAB)Fast atom bombardment ( FAB) Softer than EI and CI. Ions are produced by bombardment withSofter than EI and CI. Ions are produced by bombardment with heavy atoms. Good for more polar compounds.heavy atoms. Good for more polar compounds. Xe + e XeXe + e Xe++ acceleration (5-15 KeV)acceleration (5-15 KeV) Xe+ + Xe Xe + XeXe+ + Xe Xe + Xe++ fast slowfast slow ++ 8 KeV8 KeV fast slowfast slow
    7. 7. AdvantagesAdvantages Parent IonParent Ion High Mass CompoundsHigh Mass Compounds (10,000 amu)(10,000 amu) Thermally LabileThermally Labile Compounds (R.T.)Compounds (R.T.) DisadvantagesDisadvantages No Fragment LibraryNo Fragment Library Solubility in MatrixSolubility in Matrix (MNBA, Glycerol)(MNBA, Glycerol) Quantitation DifficultQuantitation Difficult Needs Highly SkilledNeeds Highly Skilled OperatorOperator Relatively Low SensitivityRelatively Low Sensitivity Properties of FABProperties of FAB
    8. 8. Matrix Assisted Laser DesorptionMatrix Assisted Laser Desorption Ionization (MALDI)Ionization (MALDI)  MALDI is achieved in two steps. In the first step, the compound to be analyzed is dissolved in a solvent containing in solution small organic molecules, called the matrix. The second step occurs under vacuum conditions inside the source of the mass spectrometer. sample is co-crystallized with a matrix and then irradiated with laser.
    9. 9. MALDI ( Matrix assisted laser desorption/MALDI ( Matrix assisted laser desorption/ ionization)ionization)  In this technique anIn this technique an aqueous/alcoholic solution ofaqueous/alcoholic solution of sample was mixed with a largesample was mixed with a large excess of a radiation absorbingexcess of a radiation absorbing matrix material.matrix material.  The resulting solution wasThe resulting solution was evaporated on the surface of aevaporated on the surface of a metallic probe that was used formetallic probe that was used for introduction of the sample intointroduction of the sample into the mass spectrometer.the mass spectrometer.  The solid mixture is exposed to aThe solid mixture is exposed to a pulsed laser beam, which resultedpulsed laser beam, which resulted in sublimation of the analyte asin sublimation of the analyte as ions.ions.
    10. 10. 1111 SELDI-TOF MS
    11. 11. 1212 SELDI – Surface-Enhanced Laser Desorption and Ionization For SELDI :- • Proteins are cocrystallized with UV-absorbing compounds. • Vaporized by a pulsed-UV laser . • Ionized proteins are accelerated in an electric field • Mass to charge ratios of the different protein ion species are measured. Also called SELDI-TOF-MS • TOF – Time of flight
    12. 12. 1313 Differences between SELDI and MALDI SELDI • Wider mass range than MALDI • Biomarker discovery MALDI • High accuracy in the lower peptide range • Protein identification
    13. 13. Electrospray IonizationElectrospray Ionization  Most important technique for analyzing biomolecules such as polypeptides,Most important technique for analyzing biomolecules such as polypeptides, proteins and oligonucleotides having mol. Wt. of 10000 D or more.proteins and oligonucleotides having mol. Wt. of 10000 D or more.  Electrospray ionization takes place under atmospheric pressure andElectrospray ionization takes place under atmospheric pressure and temperature.temperature.  A solution of the sample is pumped through a stainless steel capillary needleA solution of the sample is pumped through a stainless steel capillary needle at a rate of few microliter/min.at a rate of few microliter/min.  The needle is maintained at several kilo-volts with respect to a cylindricalThe needle is maintained at several kilo-volts with respect to a cylindrical electrode that surrounds the needle.electrode that surrounds the needle.  The resulting charged spray of fine droplets then passes through a desolvatingThe resulting charged spray of fine droplets then passes through a desolvating capillary, where evaporation of the solvent and attachment of a charge to thecapillary, where evaporation of the solvent and attachment of a charge to the analyte molecules takes place.analyte molecules takes place.  As the droplet becomes smaller as a consequence of evaporation of theAs the droplet becomes smaller as a consequence of evaporation of the solvent, their charge density becomes greater and desorption of ions into thesolvent, their charge density becomes greater and desorption of ions into the ambient gas occurs.ambient gas occurs.
    14. 14. AdvantagesAdvantages Electrospray Ionization can be easily interfaced to LC. Absolute signals from Electrospray are more easily reproduced, therefore, better quantitation. Mass Accuracy is considered better. Multiple charging is more common then MALDI. DisadvantagesDisadvantages No Fragmentation Need Polar Sample Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best) Sensitive to Salts Suppression Properties of ESIProperties of ESI
    15. 15. MASS ANALYZERS:MASS ANALYZERS:  Function:Function: To separate the ions produced inTo separate the ions produced in the ion source acc. to their mass/charge ratio.the ion source acc. to their mass/charge ratio.  Ideally mass analyzer should be capable ofIdeally mass analyzer should be capable of distinguishing small mass differences.distinguishing small mass differences.  It should also allow passage of a sufficientIt should also allow passage of a sufficient number of ions to yield radially measurablenumber of ions to yield radially measurable ion current.ion current.
    16. 16. Classification of Mass Analyzer:Classification of Mass Analyzer: 1)1) SECTOR ANALYZERSSECTOR ANALYZERS a)a) Magnetic field only - Single Focusing Mass AnalyzerMagnetic field only - Single Focusing Mass Analyzer b)b) Magnetic and electrostatic field - Double FocusingMassMagnetic and electrostatic field - Double FocusingMass AnalyzerAnalyzer 2)2) TIME OF FLIGHT ANALYZER ( TOF )TIME OF FLIGHT ANALYZER ( TOF ) 3)3) QUADRAPOLE ANALYZERSQUADRAPOLE ANALYZERS a)a) Quadrapole filterQuadrapole filter b)b) Ion trap analyzerIon trap analyzer 4) FT – MS ( Ion cyclotion Resonance – Mass Spectrometer )4) FT – MS ( Ion cyclotion Resonance – Mass Spectrometer ) ( ICR-MS)( ICR-MS) 5) MS – MS ( Tandem Mass Spectroscopy )5) MS – MS ( Tandem Mass Spectroscopy )
    17. 17. Time of Flight Mass AnalyzerTime of Flight Mass Analyzer:: • “Pulse” of ions are accelerated into analyzer – Very small range of kinetic energies (ideally all have same KE) – Since masses vary, velocity must also vary • Ions enter a field-free region, the drift tube, where they are separated on the basis of their velocities – Lighter ions (smaller m/z) arrive at the detector first, heavier ions (larger m/z) arrive later
    18. 18. REFERANCEREFERANCE  Organic Spectroscopy byOrganic Spectroscopy by Pavia,Lampman,Kriz page no:402-415.Pavia,Lampman,Kriz page no:402-415.  Instrumental method of chemical analysis byInstrumental method of chemical analysis by G.R. Chatwal & S.K. Anand.G.R. Chatwal & S.K. Anand. Page no: 2.275-2.282.Page no: 2.275-2.282.  Website:-Website:-  www.freepdf.comwww.freepdf.com  www.bioinformatics.ualr.eduwww.bioinformatics.ualr.edu  www.sildeshare.netwww.sildeshare.net
    19. 19. THANK YOU… THANK YOU…

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