Transcript PowerPoint 2007

Analyzing Biological and
Organic Polymers by
MALDI-TOF
Jonathan A. Karty, Ph.D.
Topics Covered
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Sample Requirements
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Instrument Overview
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General Instrument Use Instructions
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Tips and Tricks
What is the Bruker Autoflex III?
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Time-of-flight mass spectrometer
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Ions of given same kinetic energy, heavy ions travel
slower than lighter ones
Two modes of operation
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Linear
Reflectron
MALDI/LDI source
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384 position target plate (~1 µL spot size)
355 nm Nd:YAG laser
Can analyze positive or negative ions (same spot)
Autoflex III Picture
Matrix-Assisted Laser Desorption/Ionization
(MALDI)
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Analyte is mixed with UV-absorbing matrix
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A drop of this liquid is dried on a target
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Analyte incorporated into matrix crystals
Spot is irradiated by a laser pulse
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~10,000:1 matrix:analyte ratio
Analyte does not need to absorb laser
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
MALDI Diagram
Image from http://www.noble.org/Plantbio/MS/iontech.maldi.html
MALDI Advantages
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Technique is relatively simple
Volatilize and ionize labile molecules
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Imagine electron ionization on a protein
MALDI creates very simple mass spectra
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Ions are usually (M+nH)n+ or (M-nH)nOnly 1-3 charge states are observed
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MALDI ideal for time-of-flight analyzers
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Usually 1 charge state for peptides < 3.5 kDa
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
Some Common MALDI Matrices
What Samples Can It Run?
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Biopolymers
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Organometallic complexes
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Organometallic salts work great
Synthetic polymers
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Peptides, proteins, DNA, RNA, oligosaccharides
Polymer need not be soluble in same solvent as
matrix
Molecules that photoionize upon irradiation by
355 nm laser
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Porphyrins
Organometallic complexes
What Samples Can’t It Run?
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“Dirty” samples
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Significant concentration of involatiles
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Alkali metal salts can be quite problematic
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RNA/DNA analyses require extensive desalting
Molecules with significant vapor pressures
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Glycerol, urea, most buffers, many detergents
Instrument is held at ~10-7 torr
Molecules that do not make stable ions in source
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Lack charge acceptor/donor site
Cannot photoionize with Nd:YAG laser
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
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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
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Fluorescein-labeled peptides
Instrument Diagram
355 nm Nd:YAG laser
Target
Reflectron
Linear
Detector
Lens
Extraction
Plate
Flight
Tube
Entrance
Reflector
Detector
Linear Mode
355 nm Nd:YAG laser
Target
Reflectron
Linear
Detector
Lens
Extraction
Plate
Flight
Tube
Entrance
Reflector
Detector
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)
Reflectron Mode
355 nm Nd:YAG laser
Target
Reflectron
Linear
Detector
Lens
Extraction
Plate
Flight
Tube
Entrance
Reflector
Detector
Reflectron mode is used for
small species (<4 kDa) and is
capable of 11,000 resolving
power @ 3.2 kDa.
(ACTH 7-38+H)+
(Ubiq+2H)2+
(ACTH 18-37+H)+
MALDI Example
(Ins+H)+
(Ubiq+H)+
MALDI Example I Continued
Intens. [a.u.]
Intens. [a.u.]
MALDI-TOF Example 2
8476.61
16952.53
4000
6000
3000
16952.53
4000
2000
1000
2000
17056.51 17131.54
0
4802.98
16600
16800
17000
17200
1740
0
5000
7500
10000
12500
15000
17500
20000
22500
25000
27500
m/z
General Sample Guidelines
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Purify analyte if possible
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Use only volatile solvents/buffers
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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 noctylglucopyranoside can work
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Analyte should be 5 – 100 µM in concentration
ZipTips can help purify dirty samples (C4 and C18
available in MSF)
No SDS, TWEEN, CHAPS, etc
Need at least 2 µL
Sample Prep Tricks
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Ziptip to clean up dirty samples
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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
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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
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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
Sample Prep Tricks 2
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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
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 Put
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calibrants in matrix spot
For better mass accuracy, let voltages
stabilize 10-30 minutes before recording
data
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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