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

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 Organometallic complexes
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

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 ~10-7 torr
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 3.2 kDa (1, kDa)
Extraction
Plate
Reflector
Detector
Flight
Tube
Entrance

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