Analyzing Biological and Organic Polymers by MALDI-TOF

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 NdYAG 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
• 10,0001 matrixanalyte 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 lt100 nsec)
• Ions are accelerated toward the detector

6
MALDI Diagram
Image from http//www.noble.org/Plantbio/MS/iontec
h.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 (MnH)n or (M-nH)n-
• Only 1-3 charge states are observed
• Usually 1 charge state for peptides lt 3.5 kDa
• MALDI ideal for time-of-flight analyzers
• Theoretically unlimited mass range (100 kDa done
  here)
• MALDI is very rapid (lt1 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 Cant 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 NdYAG 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
  (lt600) 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 NdYAG laser
Target
Reflectron
Linear Detector
Lens
Extraction Plate
Reflector Detector
Flight Tube Entrance
14
Linear Mode
355 nm NdYAG laser
Target
Reflectron
Linear Detector
Lens
Linear mode is used for large (gt3.5 kDa)
molecules or exceedingly fragile species
(oligosaccharides). It is capable of 4,000
resolving power _at_ 3.2 kDa (1,000 RP _at_ 12 kDa)
Extraction Plate
Reflector Detector
Flight Tube Entrance
15
Reflectron Mode
355 nm NdYAG laser
Target
Reflectron
Linear Detector
Lens
Extraction Plate
Reflectron mode is used for small species (lt4
kDa) and is capable of 11,000 resolving power _at_
3.2 kDa.
Reflector Detector
Flight Tube Entrance
16
MALDI Example
(ACTH 7-38H)
(ACTH 18-37H)
(Ubiq2H)2
(UbiqH)
(InsH)
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 lt 20 mM (e.g. 0.1 v/v HOAc)
• If you need a detergent, 20 mM n-octylglucopyranos
  ide 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 lt 3 kDa
• C4 for peptides/proteins gt 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