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Mass Spectrometry: Methods & Theory

Mass Spectrometry: Methods & Theory

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Mass Spectrometry: Methods & Theory

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  1. Mass Spectrometry: Methods & Theory

  2. Proteomics Tools • Molecular Biology Tools • Separation & Display Tools • Protein Identification Tools • Protein Structure Tools

  3. Mass Spectrometry Needs • Ionization-how the protein is injected in to the MS machine • Separation-Mass and Charge is determined • Activation-protein are broken into smaller fragments (peptides/AAs) • Mass Determination-m/z ratios are determined for the ionized protein fragments/peptides

  4. Protein Identification • 2D-GE + MALDI-MS • Peptide Mass Fingerprinting (PMF) • 2D-GE + MS-MS • MS Peptide Sequencing/Fragment Ion Searching • Multidimensional LC + MS-MS • ICAT Methods (isotope labelling) • MudPIT (Multidimensional Protein Ident. Tech.) • 1D-GE + LC + MS-MS • De Novo Peptide Sequencing

  5. Mass Spectrometry (MS) • Introduce sample to the instrument • Generate ions in the gas phase • Separate ions on the basis of differences in m/z with a mass analyzer • Detect ions

  6. Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif. How does a mass spectrometer work? Create ions Separate ions Detect ions • Mass spectrum • Database analysis

  7. Artificially trypsinated Fragmented using trypsin Artificial spectra built Spot removed from gel Generalized Protein Identification by MS Spectrum of fragments generated MATCH Library Database of sequences (i.e. SwissProt)

  8. Methods for protein identification

  9. MS Principles • Different elements can be uniquely identified by their mass

  10. N -CH2- OH COOH HO -CH2CH-NH2 HO HO MS Principles • Different compounds can be uniquely identified by their mass Butorphanol L-dopa Ethanol CH3CH2OH MW = 327.1 MW = 197.2 MW = 46.1

  11. Mass Spectrometry • Analytical method to measure the molecular or atomic weight of samples

  12. Weighing proteins A mass spectrometer creates charged particles (ions) from molecules. Common way is to add or take away an ions: NaCl+ e- NaCl- NaCl NaCl+ + e- It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure.

  13. Mass Spectrometry • For small organic molecules the MW can be determined to within 5 ppm or 0.0005% which is sufficiently accurate to confirm the molecular formula from mass alone • For large biomolecules the MW can be determined within an accuracy of 0.01% (i.e. within 5 Da for a 50 kD protein) • Recall 1 dalton = 1 atomic mass unit (1 amu)

  14. MS History • JJ Thomson built MS prototype to measure m/z of electron, awarded Nobel Prize in 1906 • MS concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919 • Designed to measure mass of elements • Aston Awarded Nobel Prize in 1922

  15. MS History • 1948-52 - Time of Flight (TOF) mass analyzers introduced • 1955 - Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize) • 1968 - Tandem mass spectrometer appears • Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

  16. MS Principles • Find a way to “charge” an atom or molecule (ionization) • Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) • Detect ions using microchannel plate or photomultiplier tube

  17. Mass Spec Principles Sample + _ Detector Ionizer Mass Analyzer

  18. Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif. How does a mass spectrometer work? Create ions Separate ions Detect ions • Mass spectrum • Database analysis

  19. Mass spectrometers • Time of flight (TOF) (MALDI) • Measures the time required for ions to fly down the length of a chamber. • Often combined with MALDI (MALDI-TOF) Detections from multiple laser bursts are averaged. Multiple laser • Tandem MS- MS/MS -separation and identification of compounds in complex mixtures - induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon • Different MS-MS configurations • Quadrupole-quadrupole (low energy) • Magnetic sector-quadrupole (high) • Quadrupole-time-of-flight (low energy) • Time-of-flight-time-of-flight (low energy)

  20. Typical Mass Spectrometer

  21. LC/LC-MS/MS-Tandem LC, Tandem MS

  22. Typical Mass Spectrum • Characterized by sharp, narrow peaks • X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion) • Height of peak indicates the relative abundance of a given ion (not reliable for quantitation) • Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others)

  23. All proteins are sorted based on a mass to charge ratio (m/z) m/z ratio: Molecular weight divided by the charge on this protein

  24. Typical Mass Spectrum Relative Abundance aspirin 120 m/z-for singly charged ion this is the mass

  25. DM M Resolution & Resolving Power • Width of peak indicates the resolution of the MS instrument • The better the resolution or resolving power, the better the instrument and the better the mass accuracy • Resolving power is defined as: M is the mass number of the observed mass (DM) is the difference between two masses that can be separated

  26. Resolution in MS

  27. Resolution in MS 783.455 QTOF 784.465 785.475 783.6

  28. Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Ion Source Mass Filter Inlet Data System Detector Sample Plate Target HPLC GC Solids probe TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. PC’s UNIX Mac MALDI ESI IonSpray FAB LSIMS EI/CI Mass Spectrometer Schematic

  29. Different Ionization Methods • Electron Impact (EI - Hard method) • small molecules, 1-1000 Daltons, structure • Fast Atom Bombardment (FAB – Semi-hard) • peptides, sugars, up to 6000 Daltons • Electrospray Ionization (ESI - Soft) • peptides, proteins, up to 200,000 Daltons • Matrix Assisted Laser Desorption (MALDI-Soft) • peptides, proteins, DNA, up to 500 kD

  30. Electron Impact Ionization • Sample introduced into instrument by heating it until it evaporates • Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV) • Molecule is “shattered” into fragments (70 eV >> 5 eV bonds) • Fragments sent to mass analyzer

  31. EI Fragmentation of CH3OH CH3OH CH3OH+ CH3OH CH2O=H+ + H CH3OH + CH3 + OH CH2O=H+ + H CHO=H+ Why wouldn’t Electron Impact be suitable for analyzing proteins?

  32. Why You Can’t Use EI For Analyzing Proteins • EI shatters chemical bonds • Any given protein contains 20 different amino acids • EI would shatter the protein into not only into amino acids but also amino acid sub-fragments and even peptides of 2,3,4… amino acids • Result is 10,000’s of different signals from a single protein -- too complex to analyze

  33. Soft Ionization Methods 337 nm UV laser Fluid (no salt) + _ Gold tip needle cyano-hydroxy cinnamic acid MALDI ESI

  34. Soft Ionization • Soft ionization techniques keep the molecule of interest fully intact • Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale) • MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) • Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF

  35. Ionization methods • Electrospray mass spectrometry (ESI-MS) • Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid. • Matrix-assisted laser desorption ionization (MALDI) • Analyte (protein) is mixed with large excess of matrix (small organic molecule) • Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix.

  36. Electrospray Ionization • Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 mm) at a rate of 10-100 mL/min • Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to “nebulize” or aerosolize • Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)

  37. Electrospray (Detail)

  38. Electrospray Ionization • Can be modified to “nanospray” system with flow < 1 mL/min • Very sensitive technique, requires less than a picomole of material • Strongly affected by salts & detergents • Positive ion mode measures (M + H)+ (add formic acid to solvent) • Negative ion mode measures (M - H)- (add ammonia to solvent)

  39. Positive or Negative Ion Mode? • If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS • If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA

  40. Matrix-Assisted Laser Desorption Ionization 337 nm UV laser cyano-hydroxy cinnamic acid MALDI

  41. MALDI • Sample is ionized by bombarding sample with laser light • Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4-hydroxycinnaminic acid for peptides) • Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)

  42. HT Spotting on a MALDI Plate

  43. MALDI Ionization Matrix + + + - • Absorption of UV radiation by chromophoric matrix and ionization of matrix • Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule • Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/”popping”) Laser - - + Analyte + + + - + + - - + - + + + + + +

  44. MALDI • Unlike ESI, MALDI generates spectra that have just a singly charged ion • Positive mode generates ions of M + H • Negative mode generates ions of M - H • Generally more robust that ESI (tolerates salts and nonvolatile components) • Easier to use and maintain, capable of higher throughput • Requires 10 mL of 1 pmol/mL sample

  45. Principal for MALDI-TOF MASS

  46. Principal for MALDI-TOF MASS

  47. MALDI =SELDI 337 nm UV laser cyano-hydroxy cinnaminic acid MALDI

  48. MALDI/SELDI Spectra Normal Tumor