1 Bruker Biflex III MALDI- TOF User Training Jonathan A. Karty, Ph.D. July 29, 2008 Topics Covered Sample Requirements Instrument Overview (technical) General Instrument Use Instructions Calibration Discussion Tips and Tricks Software reference at end of PowerPoint file will not be covered in this session
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Bruker Biflex III MALDI- TOF User Trainingmsf.chem.indiana.edu/pdfs/MALDI training talk.pdf · 1 Bruker Biflex III MALDI-TOF User Training Jonathan A. Karty, Ph.D. July 29, 2008 Topics
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Bruker Biflex III MALDI-TOF User Training
Jonathan A. Karty, Ph.D.July 29, 2008
Topics Covered
� Sample Requirements� Instrument Overview (technical)� General Instrument Use Instructions
� Calibration Discussion
� Tips and Tricks
� Software reference at end of PowerPoint file will not be covered in this session
2
What is the Bruker Biflex 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
� Capable of limited MS-MS� Instruments in Proteomics R&D Facility are MUCH better for
MS-MS and MSn
� MALDI/LDI source� 384 position target plate (~1 µL spot size)� 337 nm N2 laser (can cause photolysis)� Can analyze positive or negative ions (same spot)
What Samples Can It Run?� Biopolymers
� Peptides, proteins, DNA, RNA, oligosaccharides
� Organometallic complexes� Organometallic salts work great
� Some synthetic polymers� Polypropylene glycol, PAMAM dendrimers� Polycyclic aromatic hydrocarbons with TCNQ
� Molecules that photoionize upon irradiation by 337 nm laser� Porphyrins� Organometallic complexes
3
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 ionize in source� Lack charge acceptor/donor site
� Cannot photoionize with N2 laser
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
� Acidic conditions required for proper crystallization of many matrices� Lack of acidic conditions can be overcome in some
cases
� Need at least 2 µL
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Biflex III Picture
Target vacuum lock
Rough Pumps
Target Camera Monitor
Camera light switch
Source Compartment
Camera
Laser
5
Instrument Diagram
ReflectorDetector
LinearDetectorLens
Target
ExtractionPlate
FlightTube
Entrance
Reflectron
337 nm Nitrogen laser
Linear Mode
ReflectorDetector
LinearDetectorLens
Target
ExtractionPlate
FlightTube
Entrance
Reflectron
337 nm Nitrogen 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 (1000 RP @ 12 kDa)
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Reflectron Mode
ReflectorDetector
LinearDetectorLens
Target
ExtractionPlate
FlightTube
Entrance
Reflectron
337 nm Nitrogen laser
Reflectron mode is used for small species (<3.5 kDa) and is capable of 11,000 resolving
power @ 3.2 kDa.
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
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Some Common MALDI Matrices
CHCH
C
CHCH
C
CH
CC
C N
O
OH
OH
CCH
C
CHCH
C
CH
CH
C O
OH
OH
OCH3C
CH
C
CHC
C
CH
CH
C O
OH
OH
OCH3
O
CH3
C
CHCH
C
CCH
OH
OH C
O
OH
CH
CHCH
C
CN
OH
C
O
OH C NC
C C NN
N
a-cyano-4-hydroxycinnamic acid = CCAMolecular Formula =C10H7NO3 ferulic acid = FA
Molecular Formula =C10H10O4
sinapinic acid = SAMolecular Formula =C11H12O5
2,5 dihydroxybenozoic acid = DHBMolecular Formula =C7H6O4
3-hydroxypicolinic acid = HPAMolecular Formula =C6H5NO3
tetracyanoquinodimethane = TCNQMolecular Formula =C12H4N4
CCA Matrix
� Good matrix for compounds <10 kDa� Makes relatively homogenous flat spots
� Mix 10 g/L in 45% CH3CN, 5% (CH3)2CO, 0.1% TFA in H2O (instructions on balance)
� Combine 1 part analyte with 5 parts matrix� Ratio can be adjusted depending on sample
� Deposit 1 µL on target and let air dry
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FA and SA Matrices� Matrices for compounds >10 kDA
� Spots are not homogenous or flat� Crystallization often must be assisted (tap spot)
� Waters premix SA (directions on balance) � spots are made by mixing 5:1 matrix:analyte� Deposit 1 µL on target (no tapping required)
� Mix 0.15 M matrix in EtOH (alternate method)� 32.4 g/L for FA, 33.6 g/L for SA� Mix 7 parts analyte + 3 parts matrix; put 1 µL onto target� Wait 30 sec� Tap spot with pipette tip until tiny crystals form� Stir crystals around so entire spot is covered with crystals
� SA and FA spots require more laser power than CCA
HPA Matrix
� Used for oligonucleotides� 7 mg HPA in 50 µL SCX:NH4+ resin
A Word About Isotopes� Instrument can resolve the isotopic pattern of
compounds < 4 kDa in reflectron mode� Many molecular weight calculators compute the
isotopically averaged mass� Assume 1.1% of C is 13C, not 12C
� Monoisotopic masses are what we label in reflectron mode data� All 12C, 1H, 14N, 32S, 16O
� Monoisotopic masses are NOT usually observed in linear mode for species > 2.5 kDa
� Be aware of which mass your computer program predicted
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Monoisotopic vs. Average Mass
3650 3660 3670
Inte
nsi
tym/z (Th)
Linear3660.18
Reflectron3657.96
1560 1570 1580
Inte
nsity
m/z (Th)
Linear1570.70
Reflectron1570.81
13C0
13C1
13C2
13C3
13C4
13C0
13C1
13C2
13C0
13C1
13C2
Interpreting Data: Mass Defect� Atomic weights are not integers (except 12C)
�14N = 14.0031 Da; 11B = 11.0093 Da; 1H = 1.0078 Da
�16O = 15.9949 Da; 19F = 18.9984 Da; 56Fe = 55.9349 Da
� Difference from integer mass is called “mass defect” or “fractional mass”� Related to nuclear binding energy
� Sum of the mass defects depends on composition� H, N increase mass defect
� Hydrogen-rich molecules have high mass defects � Eicosane (C20H42)= 282.3286
� O, Cl, F, Na decrease it� Hydrogen deficient species have low mass defects� Morphine, (C17H19NO3) = 285.1365
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Peptide Mass Defect Figure
Dependence of the fractional masses of predicted Caulobacter tryptic peptides on their nominal masses. The red squares represent the locations of the matrix-alkali
cluster masses [Figure from Karty et al in J. Chrom. B. v782 pp363-383 (2002)].
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
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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
IsoPro on Your PC� IsoPro 3.0 can be used in the MSF or downloaded� Type in formula by selecting Formula from the Edit
menu (use proper capitalization)� Select Distribution from Calculate (text table)� Select Display from Calculate (picture)� Use Parameters to alter charge state or theoretical
resolution� Recalculate distribution/display after making changes
� Protein “sequences” can be entered by entering amino acid composition on Amino Acids page
� Remember to account for ionization cations� Charge state function on Parameters page automatically
adds 1 H per charge
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IsoPro 3.0 ScreensAverage mass
Monoisotopicmasses
Target Loading Issues� Common target introduction error:
� Target inserted in wrong orientation� Take target out of lock, put it back in, but let the door
push the target in
� Any other error, contact Jon Karty or Angie Hansen� Call Jon at home if you have to
� $3,300 minimum service fee if we need to call a Bruker engineer onsite
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Biflex III Software Package� XAcq 4.0: acquisition of data files
� All instrument functions are controlled by XAcq� Calibration and smoothing can be performed here
� XMass 5.1: process saved data� Smooth and recalibrate raw data� Label peaks and print data� Compare different spectra in the same window
� Both programs run on UNIX workstation� ASCII formatted data can be exported and
retrieved by SFTP
Hands-on Training
� 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