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Analyzing Biological and Organic Polymers by MALDI-TOF

Analyzing Biological and Organic Polymers by MALDI-TOF. Jonathan A. Karty, Ph.D. Topics Covered. Sample Requirements Instrument Overview General Instrument Use Instructions Tips and Tricks. What is the Bruker Autoflex III?. Time-of-flight mass spectrometer

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Analyzing Biological and Organic Polymers by MALDI-TOF

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  1. Analyzing Biological and Organic Polymers by MALDI-TOF Jonathan A. Karty, Ph.D.

  2. Topics Covered • Sample Requirements • Instrument Overview • General Instrument Use Instructions • Tips and Tricks

  3. What is the BrukerAutoflex III? • Time-of-flight mass spectrometer • Ions of given same kinetic energy, heavy ions travel slower than lighter ones • Two modes of operation • Linear • Reflectron • MALDI/LDI source • 384 position target plate (~1 µL spot size) • 355 nm Nd:YAG laser • Can analyze positive or negative ions (same spot)

  4. Autoflex III Picture

  5. Matrix-Assisted Laser Desorption/Ionization (MALDI) • Analyte is mixed with UV-absorbing matrix • ~10,000:1 matrix:analyte ratio • Analyte does not need to absorb laser • A drop of this liquid is dried on a target • Analyte incorporated into matrix crystals • Spot is irradiated by a laser pulse • Irradiated region sublimes, taking analyte with it • Matrix is often promoted to the excited state • Charges exchange between matrix and analyte in the plume (very fast <100 nsec) • Ions are accelerated toward the detector

  6. MALDI Diagram Image from http://www.noble.org/Plantbio/MS/iontech.maldi.html

  7. MALDI Advantages • Technique is relatively simple • Volatilize and ionize labile molecules • Imagine electron ionization on a protein • MALDI creates very simple mass spectra • Ions are usually (M+nH)n+ or (M-nH)n- • Only 1-3 charge states are observed • Usually 1 charge state for peptides < 3.5 kDa • MALDI ideal for time-of-flight analyzers • Theoretically unlimited mass range (100 kDa done here) • MALDI is very rapid (<1 min/spot) • Low sample consumption (1 µL) • Wide array of matrices available for different analytes

  8. Some Common MALDI Matrices

  9. What Samples Can It Run? • Biopolymers • Peptides, proteins, DNA, RNA, oligosaccharides • Organometallic complexes • Organometallic salts work great • Synthetic polymers • Polymer need not be soluble in same solvent as matrix • Molecules that photoionize upon irradiation by 355 nm laser • Porphyrins • Organometallic complexes

  10. What Samples Can’t It Run? • “Dirty” samples • Significant concentration of involatiles • Glycerol, urea, most buffers, many detergents • Alkali metal salts can be quite problematic • RNA/DNA analyses require extensive desalting • Molecules with significant vapor pressures • Instrument is held at ~10-7torr • Molecules that do not make stable ions in source • Lack charge acceptor/donor site • Cannot photoionize with Nd:YAG laser

  11. MALDI Advantages • Relatively gentle ionization technique • Very high MW species can be ionized • Molecule need not be volatile • Very easy to get sub-picomole sensitivity • Spectra are easy to interpret • Positive or negative ions from same spot • Wide array of matrices available

  12. MALDI Disadvantages • MALDI matrix cluster ions can obscure low m/z (<600) range • Analyte must have very low vapor pressure • Coupling MALDI with chromatography can be difficult • Analytes that absorb the laser can be problematic • Fluorescein-labeled peptides

  13. Instrument Diagram 355 nm Nd:YAG laser Target Reflectron Linear Detector Lens Extraction Plate Reflector Detector Flight Tube Entrance

  14. Linear Mode 355 nm Nd:YAG laser Target Reflectron Linear Detector Lens Linear mode is used for large (>3.5 kDa) molecules or exceedingly fragile species (oligosaccharides). It is capable of 4,000 resolving power @ 3.2 kDa (1,000 RP @ 12 kDa) Extraction Plate Reflector Detector Flight Tube Entrance

  15. Reflectron Mode 355 nm Nd:YAG laser Target Reflectron Linear Detector Lens Extraction Plate Reflectron mode is used for small species (<4 kDa) and is capable of 11,000 resolving power @ 3.2 kDa. Reflector Detector Flight Tube Entrance

  16. MALDI Example (ACTH 7-38+H)+ (ACTH 18-37+H)+ (Ubiq+2H)2+ (Ubiq+H)+ (Ins+H)+

  17. MALDI Example I Continued

  18. MALDI-TOF Example 2

  19. General Sample Guidelines • Purify analyte if possible • Analyte should be 5 – 100 µM in concentration • ZipTips can help purify dirty samples (C4 and C18 available in MSF) • Use only volatile solvents/buffers • MeOH, H2O, acetone, CH3CN, THF, CH2Cl2, C6H6 • TFA, HOAc, formic acid, NH3, etc. • Ionic strength < 20 mM (e.g. 0.1% v/v HOAc) • If you need a detergent, 20 mM n-octylglucopyranoside can work • No SDS, TWEEN, CHAPS, etc • Need at least 2 µL

  20. Sample Prep Tricks • Ziptip to clean up dirty samples • C18 for peptides < 3 kDa • C4 for peptides/proteins > 3kDa • Elute directly into matrix for added sensitivity • ZipTip instructions on MSF website • If CCA liquid turns yellow, pH is too high • Spots from non-acidic CCA do not crystallize correctly • Add a little 1% v/v or 10% v/v TFA to lower pH • If sample needs base for solubility, try over-layer method • Dissolve sample in NH3 or other volatile base • Place 1 uL of sample on target, let dry completely • Deposit 1 uL matrix over top of dried sample

  21. Sample Prep Tricks 2 • Non-aqueous over-layer • Make 1 uL spot of matrix on plate, let dry • Deposit small amount of sample in volatile solvent (e.g. CHCl3, acetone, CH2Cl2) • You can even do internal calibration this way • Put calibrants in matrix spot • For better mass accuracy, let voltages stabilize 10-30 minutes before recording data

  22. Hands-on Training • Starts AFTER 11/7 • Groups of no more than three • One hour or so to complete • No charge for first session • After training, students must demonstrate competency by running their own samples prior to being granted after-hours access

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