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

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

3. What is the Bruker Autoflex III? Time-of-flight mass spectrometer Time-of-flight mass spectrometer Ions of given same kinetic energy, heavy ions travel slower than lighter ones Ions of given same kinetic energy, heavy ions travel slower than lighter ones Two modes of operation Two modes of operation Linear Linear Reflectron Reflectron MALDI/LDI source MALDI/LDI source 384 position target plate (~1 µL spot size) 384 position target plate (~1 µL spot size) 355 nm Nd:YAG laser 355 nm Nd:YAG laser Can analyze positive or negative ions (same spot) 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 Analyte is mixed with UV-absorbing matrix ~10,000:1 matrix:analyte ratio ~10,000:1 matrix:analyte ratio Analyte does not need to absorb laser Analyte does not need to absorb laser A drop of this liquid is dried on a target A drop of this liquid is dried on a target Analyte incorporated into matrix crystals Analyte incorporated into matrix crystals Spot is irradiated by a laser pulse Spot is irradiated by a laser pulse Irradiated region sublimes, taking analyte with it Irradiated region sublimes, taking analyte with it Matrix is often promoted to the excited state Matrix is often promoted to the excited state Charges exchange between matrix and analyte in the plume (very fast <100 nsec) Charges exchange between matrix and analyte in the plume (very fast <100 nsec) Ions are accelerated toward the detector 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 Technique is relatively simple Volatilize and ionize labile molecules Volatilize and ionize labile molecules Imagine electron ionization on a protein Imagine electron ionization on a protein MALDI creates very simple mass spectra MALDI creates very simple mass spectra Ions are usually (M+nH) n+ or (M-nH) n- Ions are usually (M+nH) n+ or (M-nH) n- Only 1-3 charge states are observed Only 1-3 charge states are observed Usually 1 charge state for peptides < 3.5 kDa Usually 1 charge state for peptides < 3.5 kDa MALDI ideal for time-of-flight analyzers MALDI ideal for time-of-flight analyzers Theoretically unlimited mass range (100 kDa done here) Theoretically unlimited mass range (100 kDa done here) MALDI is very rapid (<1 min/spot) MALDI is very rapid (<1 min/spot) Low sample consumption (1 µL) Low sample consumption (1 µL) Wide array of matrices available for different analytes Wide array of matrices available for different analytes

8. Some Common MALDI Matrices

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

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

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

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

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

14. Linear Mode Reflector Detector Linear Detector Lens Target Extraction Plate Flight Tube Entrance Reflectron 355 nm Nd:YAG laser 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)

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

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

17. MALDI Example I Continued

18. MALDI-TOF Example 2

19. General Sample Guidelines Purify analyte if possible Purify analyte if possible Analyte should be 5 – 100 µM in concentration Analyte should be 5 – 100 µM in concentration ZipTips can help purify dirty samples (C 4 and C 18 available in MSF) ZipTips can help purify dirty samples (C 4 and C 18 available in MSF) Use only volatile solvents/buffers Use only volatile solvents/buffers MeOH, H 2 O, acetone, CH 3 CN, THF, CH 2 Cl 2, C 6 H 6 MeOH, H 2 O, acetone, CH 3 CN, THF, CH 2 Cl 2, C 6 H 6 TFA, HOAc, formic acid, NH 3, etc. TFA, HOAc, formic acid, NH 3, etc. Ionic strength < 20 mM (e.g. 0.1% v/v HOAc) Ionic strength < 20 mM (e.g. 0.1% v/v HOAc) If you need a detergent, 20 mM n- octylglucopyranoside can work If you need a detergent, 20 mM n- octylglucopyranoside can work No SDS, TWEEN, CHAPS, etc No SDS, TWEEN, CHAPS, etc Need at least 2 µL Need at least 2 µL

20. Sample Prep Tricks Ziptip to clean up dirty samples Ziptip to clean up dirty samples C 18 for peptides < 3 kDa C 18 for peptides < 3 kDa C 4 for peptides/proteins > 3kDa C 4 for peptides/proteins > 3kDa Elute directly into matrix for added sensitivity Elute directly into matrix for added sensitivity ZipTip instructions on MSF website ZipTip instructions on MSF website If CCA liquid turns yellow, pH is too high If CCA liquid turns yellow, pH is too high Spots from non-acidic CCA do not crystallize correctly Spots from non-acidic CCA do not crystallize correctly Add a little 1% v/v or 10% v/v TFA to lower pH Add a little 1% v/v or 10% v/v TFA to lower pH If sample needs base for solubility, try over-layer method If sample needs base for solubility, try over-layer method Dissolve sample in NH 3 or other volatile base Dissolve sample in NH 3 or other volatile base Place 1 uL of sample on target, let dry completely Place 1 uL of sample on target, let dry completely Deposit 1 uL matrix over top of dried sample Deposit 1 uL matrix over top of dried sample

21. Sample Prep Tricks 2 Non-aqueous over-layer Non-aqueous over-layer Make 1 uL spot of matrix on plate, let dry Make 1 uL spot of matrix on plate, let dry Deposit small amount of sample in volatile solvent (e.g. CHCl 3, acetone, CH 2 Cl 2 ) Deposit small amount of sample in volatile solvent (e.g. CHCl 3, acetone, CH 2 Cl 2 ) You can even do internal calibration this way You can even do internal calibration this way Put calibrants in matrix spot Put calibrants in matrix spot For better mass accuracy, let voltages stabilize 10-30 minutes before recording data For better mass accuracy, let voltages stabilize 10-30 minutes before recording data

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