Maldi

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Maldi

  1. 1. Biological Mass Spectrometry A mini course
  2. 2. Biological Mass SpectrometryGeneral Mass Spectrometry The General mass spectrometer Analyzers types Calibrations and mass Accuracy DetectionMatrix Assisted Laser Desorption (MALDI) TOFElectrospray Ionization (ESI) Quadrupoles, Ion Traps, FTMS, and O-TOFFragmentation Post source dissociation (PSD) (with MALDI) In source dissociation (with ESI) Collision induced dissociation CID IRMPD and ESI
  3. 3. The General Mass Spectrometry M a s s S p e c tro m e te r Io n S o u rc e A n a ly z e r D e te c to r M ALDI TO FS p ra y S o u rc e s Q u a d r u p o le s T r a p p in g in s t r u m e n t s
  4. 4. Analyzers• Time of Flights mass spectrometer• Quadrupole mass spectrometer• Quadrupole Ion traps• FT-MS, FT-ICR
  5. 5. Time-of-flight Mass Spectrometers• Basically a tube that ions travel through with lower mass ions traveling quicker through the tube.• Reflectron increases resolution by compensating for energy and increasing path length.• Delayed extraction increases resolution.
  6. 6. O-TOF PulserIon Beam Grid T T o OF
  7. 7. Quadrupole Mass Spectrometer - Rods + + - - + + Ions -
  8. 8. Quadrupole Ion Traps - End Caps ++ - - +Ring Ions + -
  9. 9. FT-MS, FT-ICR + + B
  10. 10. Calibration• All Mass Spectrometers MUST BE CALIBRATED. This adjust parameters in the mass assignment of the ions.• In general, the closer in space and time the calibration is the more accurate the calibration.• Default calibrations are usually set up on instruments for nominal mass accuracy.• Calibrations can be done with any compound with a known elemental formula. The best calibration compounds are similar type of compounds to the type you are going to measure accurately.• It is best to have calibration compounds above and below the compound of interest
  11. 11. Mass Accuracy• Higher resolution allows for higher mass accuracy in two ways – Resolves stardards and sample from background – Better centroiding of peaks• Use of internal standards with a high resolution (>10,000) is the “Gold standard” for mass accuracy.• Mass Accuracy needed is dependent on use and available information.
  12. 12. Ion Detec tion R Volta ge ac ross resistor
  13. 13. WHY MALDI?• Less Sensitive to Salts• Lower PRACTICAL detection limits• Easier to interpret spectra (less multiple charges)• Quick and easy• Higher mass detection• Higher Throughput (>1000 samples per hour)
  14. 14. Matrix• Needs to be involatile (most are solids at room temperature)• Needs to absorb the laser wavelength that you are using. (most cases 337 nm)• Preferably dissolves in same solvent as the sample• Typically, the matrices are acidic.
  15. 15. HOT and Cold Matrices• DHB is a cold matrix, the samples are not as likely to be fragmented, may not ionize some molecules.• Alpha-cyano dihydroxybenzoic acid is considered a hot matrix. More likely to fragment the molecules. Can produce multiply charged proteins.
  16. 16. Sample preparation• Dried Droplet- mix sample with matrix and drop on plate at the same time. –Easy to do.• Layered method- put matrix on plate then dry before adding sample. Good for low concentrations, more difficult.• Want high Matrix-Analyte ratio.
  17. 17. The MALDI Instrument T im e - o f F lig h t M a s s S p e c tro m e te r S o u rc e A n a ly z e r D e te c to rD e la y e d E x t r a c t io n R e fle c t r o n W h y d e fle c t t h e io n s ? PSD
  18. 18. Basic Physics• Kinetic Energy= ½ mv2• Potential Energy of charged particle=qV• Potential Energy +Kinetic Energy=Constant
  19. 19. Delayed Extraction• Kinetic Energy of ions leaving the surface is not constant. Also, their could be a time delay, in addition.• Delayed Extraction is a method used to compensate for the spread of kinetic energy of the ions leaving the surface.• Delayed Extraction is mass dependent.• Delayed Extraction will also compensate for some time delay of ions coming off the surface.
  20. 20. M1 kE < M2 kE
  21. 21. T t4 =Sam ple plate M1 kE< M2 kE E= kE + PE Potential Voltage M1 kE > M2 kE Detector M1 M2
  22. 22. Reflectron• The reflectron is another method used to compensate for the spread in Kinetic energies in the time of flight.• Reflectron does not compensate for any time delay coming off the surface.• Three types of reflectrons ( single stage, two stage, and curved field)
  23. 23. KE M1 > KE M2M1M2
  24. 24. KE M1 > KE M2 T t2 = M1 M2
  25. 25. KE M2= KE M1= 0KE M1 > KE M2 M1 T t3 = M2
  26. 26. T 5 =
  27. 27. Why Deflect the ions?• Detector is made up of multiple detectors similar to the electron multiplier called “channels”.• When an ion strikes the detector the “channel” is dead for milliseconds.• When to many matrix ions hit the detector, it kills the signal.• Linear detector is specially design to compensate for larger number of ions, but is not perfect.
  28. 28. Why Electrospray Ionization?• 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.
  29. 29. Detection of PeptidesMALDIDetec tedPeptid es
  30. 30. Advantages of Multiple Charging• Can use instruments with lower maximum m/z (i.e., Quadrupoles, ion traps, FTMS)• For FTMS, the resolution is better at lower m/z values, therefore, ESI helps one obtain better resolution at higher m/z values.• Multiply charge ions tend to fragment easier then singly charge ions.
  31. 31. Basic ESI Source - 3kV T Pum ping o
  32. 32. Needle options• High flow needles (for connection to regular LC columns(up to 1ml/min))• Nanospray needles (high sensitivity work)• Microspray needles (connected to micro LC columns)• Atmospheric Pressure Chemical Ionization (for non-polar molecules)• Multiple needles setups
  33. 33. Why Fragment the ions?• Molecular weight is only one piece of data the instrument can provide• Data base searches on tryptic peptides do not always reveal the protein.• Sequence and modification information can be obtained by fragmentation
  34. 34. Ways to Induce Fragmentation• Post Source Decay-MALDI• In source Dissociation-ESI• Low Energy –High Energy Collision induced dissociation• Inferred multi-photon dissociation (in a trap)• Electron Capture dissociation (in a trap)
  35. 35. Tandem Mass Spectrometers• Traps• Methods using single MS’s• Triple Quadrupoles• Q-Tofs• TOF-TOF
  36. 36. PSD• Easily done on simple MALDI with reflectron• Might not produce enough fragments• Relatively low precursor ion selection
  37. 37. Physics Reminder• Conservation of Energy assures that the kinetic energy of fragments is determined by the relative masses of the products.• KE=1/2mv2• KEprecursor=KEproduct ion+KEproduct neutral• vprecursor=vproduct ion• KEproduct ion=KEprecursor*(mproduct ion/mprecursor)
  38. 38. M > M2K > K2 1
  39. 39. M > M2K > K2 1
  40. 40. M > M2 K > K2 1Velocity in Field FreeRegion on the samefor both fragments
  41. 41. Types of Reflectrons• Two stage- Give best resolution on single MS mode, requires doing PSD in Stages.• Single Stage-Large Reflector takes a significant amount of space. PSD can be done in single stage, however, low mass product ions have poor resolution.• Curved field-Another Large Reflector that takes up space. Allows PSD to be obtained in single stage with good resolution throughout PSD, but single MS mode does not have as good a resolution.
  42. 42. In Source Dissociation• This is done by increasing the voltage between the entrance to the sample orifice and the first skimmer.• Can be done on simple electrospray instrument.• Produces results similar to low energy CID• No mass selection, therefore, requires sample to be extremely pure.• Can be used to produce multiple MS on triple and hybrid instruments.
  43. 43. CID• CID is divided into low energy (<100 eV) and high energy(>1000 eV) based on the collision energy.• High energy CID produces more fragments, but is more complicated to interpret.• Low energy CID has a limit on the m/z it can dissociate of approximately 1000.• High energy CID produces charge remote fragmentation that can be used to distinguish leucine and isoleucine.
  44. 44. IRMPD and ECD• Complimentary techniques• Can only be performed in trapping instruments-Mostly FTMS.• ECD requires a multiply charged ion• Used for dissociating high molecular weight fragments
  45. 45. Interpretation of Tandem Mass Spectra of Peptides• Known Sequence- Calculate expected fragments and compare to tandem spectra to see match• Modified Sequence-Calculate unmodified sequence compare to tandem spectra to see difference where modification occurs.• Unknown Sequence- Check Database to see if it is a match• Unknown Sequence not in Database- Manual Interpretation (Practice!Practice!Practice!)
  46. 46. Peptide fragmentation a b c ONterm -RCH-C-NH-CHR-Cterm x y z
  47. 47. Manual Interpretation• Goal-Assign as many abundant fragments as possible to a spectra• Remember Cysteine modifications• Know type of fragments that are typically observed by dissociation method. – Low Energy (b and y, loss of neutrals from these fragments) – High Energy (x,y,z, a,b,c, v, d,w) Reference: Ioannis A Papayannopoulos, Mass Spec. Rev.,1995, vol. 14, 49-73.
  48. 48. Steps in Manual Interpretation• Choose a large peak• Look for ions that are different in mass from the fragment by a specific amino acid mass. If there is look for an other fragment that differs by a specific amino acid mass to build a partial sequence.• Decide the type of fragments that corresponds to the above sequence by either mass difference to the precursor or complimentary fragments.• Look for other complimentary fragments and low mass ions to confirm the partial sequence.• Choose another unassigned peak and repeat procedures until sequence is determined.
  49. 49. Peptide modifications to help Interpretation• O18 water digestion- all C terminal fragments will have unique Isotope pattern• N-terminal or C-terminal modifications- results in distinct fragmentation patterns.
  50. 50. LC Interfaces• Direct LC with Electrospray. – Direct connection to mass spectrometer – Real Time monitoring• Vacuum deposition for MALDI – Indirect conection to mass spectrometer – Not Real Time, allows one to make decisions later. Multiple MS/MS on single LC peaks.
  51. 51. LC detection methods• Simple acquiring of spectra.• Precursor and product ion scans• Data Dependent scans• Selective Ion Monitoring• Selective Reaction monitoring
  52. 52. Applications• Protein Identifications• Protein Modifications• Bacteria Identifications• SNP’s
  53. 53. Protein Identification• Identify Protein mass• Digest Protein• Analyze by MALDI• Data Base Search• If negative results, go to Tandem Mass Spectrometry• Data Base Search on Tandem Mass Spectrum
  54. 54. Protein Modifications• Tandem Mass Spectrometry is very important• Can obtain full mass of proteins to help determine mass and number of modifications• Digest proteins and run mass spectrometry.• Use Tandem to determine the exact position• Use of unmodified protein or peptide can be useful in the interpretation
  55. 55. Bacteria Identification• Uses MALDI of Bacteria to Identify Proteins produced by bacteria• Data base search of MALDI spectra identifies bacteria• At this point, no large consistent Data Base is available.

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