Transcript MALDI-MS

MALDI-MS
Matrix Assisted Desorption/Ionization
Mass Spectrometry
Phillip Mnirajd
Introduction
 Mass Spectrometry (MS)
 Vital tool used to characterize and analyze molecules
 Limitations
 Biomolecules and organic macromolecules are fragile
 Molecular ions or meaningful fragments were limited to only 5-
10 kDa at the time
 New technique
 In 1987, Michael Karas and Franz Hillenkamp successfully
demonstrated the use of a matrix to ionize high molecular
weight compounds [1].
MALDI
 Matrix Assisted Laser Desorption/Ionization (MALDI)
 Method where a laser is used to generate ions of high molecular
weight samples, such as proteins and polymers.
 Analyte is embedded in to crystal matrix
 The presence of an aromatic matrix causes the large molecules to
ionize instead of decomposing.
MALDI
 The mechanism remains
uncertain
 It may involve absorption of
light by the matrix
 Transfer of this energy to the
analyte
 which then ionizes into the gas
phase as a result of the relatively
large amount of energy
absorbed.
 To accelerate the resulting ions
into a flight-tube in the mass
spectrometer they are subjected
to a high electrical field [2].
MALDI
 The MALDI technique combined with a MS detector
(MALDI-MS) became an indispensable tool in analysis of
biomolecules and organic macromolecules.
 MALDI involves incorporation of the analyte into a matrix,
absorption/desorption of laser radiation, and then ionization
of the analyte.
MALDI
see reference 3
MALDI Matrix
 The analyte incorporation in to a suitable matrix is the first
step of the MALDI process, and is an important feature of
the MALDI method.
 A typical sample preparation involves using 10-6 M solution
of the analyte mixed with 0.1 M solution of the matrix.
 The solvents are then evaporated in a vacuum of the MS, and
the matrix crystallizes with the analyte incorporated [4].
MALDI Matrix
 According to Sigma Aldrich, the matrix must meet the
following properties and requirements [5]:
 Be able to embed and isolate analytes (e.g. by co-crystallization)
 Be soluble in solvents compatible with analyte
 Be vacuum stable
 Absorb the laser wavelength
 Cause co-desorption of the analyte upon laser irradiation
 Promote analyte ionization
MALDI Matrix
Reference 5
MALDI Matrix
Reference 6
MALDI Matrix
 For compounds that are not soluble in the standard solvents,
a solventless method was developed, in particular for
synthetic polymers.The method involves mixing the matrix
and analyte powders that were ground in a mortar. The
mixture is then applied to a MALDI target support and the
spectrum is obtained. However, this particular method leads
to increased fragmentation of ions and has a mass limit of 3055 kDa [4].
MALDI Laser
 Numerous gas and solid state lasers have been developed for use in
MALDI.
 Most MALDI devices use a pulsed UV laser
 N2 source at 337 nm
 neodymium-yttrium aluminum garnet (Nd:YAG)
 emits at 355 nm and gives a longer pulse time
 IR lasers are also used
 The most common IR laser is the erbium doped-yttrium aluminum
garnet (Er:YAG)
 Emits at 2.94 micrometer
 it is “softer” than the UV, which is useful for certain biomolecules
 matrices available for IR absorption are limited
MALDI Laser
Reference 5
MALDI Laser
 The MALDI method uses a pulse laser
 Laser fires in intervals
 Pulsed laser produces individual group of ions
 1st pulse=1st group of ions
 2nd pulse= 2nd group of ions, etc.
 Each group of ions generated are detected
 With continuous pulsing, the signal resolution increases
Time Mass Detectors
 The typical detector used with
MALDI is the time of flight mass
detector (TOF-MS)
 TOF is a method where the ions
are accelerated by an electric
field, resulting in ions of the same
strength to have the same kinetic
energy [7]
 The time it takes for each ion to
traverse the flight tube and arrive
at the detector is based on its
mass-to-charge ratio; therefore
the heavier ions have shorter
arrival times compared to lighter
ions

http://www.kore.co.uk/mtof.htm
Reflectron Design in TOF-MS
 The TOF detector is also equipped with a reflectron, or an ion mirror
 The reflectron deflects the ion using an electric field and increases the path
length, improving signal resolution [7].
 Figure from: Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A. J. Chem. Educ.
1997, 74, 1289.
Quadrupole Mass Filter (QMF)
 QMF involves the generation of radio frequency (RF) and
DC field between opposite pairs of 4 rods.
 Rods can be cylindrical or hyperbolic
 A narrow range of m/z’s have stable trajectories through the
quadrupole
 Ion motions governed by set of Mathieu equations
 Scanning the quadrupole generates the mass spectrum
see reference 8
Quadrupole Mass Filter (QMF)
TOF vs QMF
 TOF and QMF are both used in MALDI
 QMF detectors are used more in teaching application
 Cheaper than TOF
 High accuracy and resolution not imperative
 TOF is the most typical detector used in research
 High mass limit
MALDI Advantages
 Gentle Ionization technique
 High molecular weight analyte can be ionized
 Molecule need not be volatile
 Sub-picomole sensitivity easy to obtain
 Wide array of matrices
see reference 8
MALDI Disadvantages
 MALDI matrix cluster ions obscure low m/z species (<600)
 Analyte must have very low vapor pressure
 Pulsed nature of source limits compatibility with many mass
analyzers
 Coupling MALDI with chromatography can be difficult
 Analytes that absorb the laser can be problematic
 Fluorescein-labeled peptides
see reference 8
TOF Advantages
 All ions detected at once
 High mass accuracy and resolving power possible
 Reasonable performance for cost
 <5 ppm mass accuracy and >20,000 resolving power
commercially available
 High mass, low charge ions not a problem
 Theoretically unlimited mass range
Reference 8
TOF Disadvantages
 High vacuum required for resolution and accuracy (<10-7
torr)
 Complex vacuum system necessary
 Must be recalibrated often
 Temperature and voltage fluctuations alter flight times
 Fast detectors prone to saturation
 Long flight tubes for high resolving power can make
instruments large
Reference 8
QMF Advantages
 Very simple to implement
 Low cost (<$100k)
 Moderate vacuum required (~10-5 torr)
 Small size
 Most common MS in use
Reference 8
QMF Disadvantages
 Limited mass range (up to m/z 4,000)
 Limited resolving power and mass accuracy
 Scanning limits sensitivity and speed
 Quad can rapidly jump between select m/z ratios for increased
speed & sensitivity
Refrence 8
Applications of MALDI
 Applications of MALDI mass spectrometry [9]
 Peptides and proteins
 Synthetic polymers
 Oligonucleotides
 Oligosaccharides
 Lipids
 Inorganics
 Bacterial identification
 Used especially
 Proteomics
Synthetic Polymer Analysis
 Using MALDI-TOF-MS
 MS spectrum of
polybutylene adipate [7]
 In trans-3-indoleacrylic
acid matrix
 Oligomer distribution is
resolved
 Avg mol mass=4525 Da
 All ions are singly
charged
 Distance between
oligomers is mass of the
repeating unit
Bacterial Identification
 Rapid bacterial identification is useful in diagnosing disease,
monitoring contamination, etc.
 Important to identify related species
 Also identify strains in complex matrices
 Identified by:
 Biomarkers
 Cellular protein content
 MALDI-TOF-MS
Bacterial Identification
 MALDI-TOF-MS uses crude protein extract requiring
minimal sample preparation
 Masses obtained of unknown is compared to experimentally
determined signals
 Ions are specific to genus, species, or strain of bacteria
 MALDI-TOF-MS can determine mass of proteins of 1-40
kDa [10]
 Accuracy of  0.1%
 Due to the variability in percent composition of the isotopes
Bacterial Identification
 US Patent #6177266 B1 [10]
 January 23, 2001
 United States of America as represented by the Secretary of the
Army
 “Rapid Identification of Bacteria By Mass Spectrometry”
 Provides method to identify bacteria
 Genus, species, strain
 Bacteria identification on whole cells
 Provide library of biomarkers
Bacterial Identification
 The present invention provides a method for generating
unique mass spectral profiles for bacteria protein extracts or
whole bacteria cells. These profiles contain proteinaceous
biomarkers which distinguish between bacteria of different
genera, species and strains. Comparable profiles are
generated when the method is performed using different
MALDI-TOF instruments from different manufactures.
Sample Preparation
 Bacteria
 supplied as γ-irradiated and lyophilized samples by the U.S.
Army Laboratories at Dugway Proving Ground, Utah.
 Nonpathogenic bacteria cells of different strains were grown inhouse by incubating for 24 hrs. at 37° C. on trypticase soy agar
or nutrient agar plates, harvested and lyophilized
 Matrix
 10 mg/ml of either 4-hydroxy-α-cyano-cinnamic acid (4
CHCA; 10 mg/ml) or 3,5-dimethoxy-4-hydroxy cinnamic acid
(sinapinic acid) in an aqueous solvent solution comprising 0.1%
aqueous trifluoroacetic acid (TFA) and acetronitrile in a ratio of
70/30 (v/v).
Sample Preparation
 Protein extracts
 1 μl of a protein extract was mixed with 9 μl of matrix
solution.
 For analysis of whole cells
 Small quantity (0.1-0.2 mg) of intact, whole cells are
suspended were added to 20 μl of aqueous buffer, typically
0.1% trifluoroacetic acid, vortexed for 30 seconds, and 1 μl
of the resulting suspension was either frozen for later use and
thawed and combined with 9 μl of a matrix solution or used
immediately.
Bacterial Identification
 Mass spectral analysis of
protein extracts
 Distinguishes among 4 strains
of Bacillus
Bacterial Identification
 Mass spectral data of
whole, intact cells
 Capable of detecting
virulent and non virulent
strains
 Bacillus REV-1 and
Abortus
Bacterial Identification
 Comparison of two tables
show common biomarkers
and unique biomarkers in
Bacillus species
 Different strains of a
bacteria species can also be
MALDI-TOF-MS analysis of
protein and of mass spectral
analysis of intact, whole
cells by the above procedure
also produced biomarkers
which distinguished
between bacteria at the
genus, species and strain
levels
Bacterial Identification
see reference 11
Conclusion
 MALDI-MS is a vital tool in mass analysis of biomolecules
and organic macromolecules
 Detection limits of femtomole to attomole [7]
 Reproducibility is relative
 Complimentary technique to ESI (electrospray ionization)
References
1.
M. Karas, et al and F. Hillenkamp; International Journal of Mass Spectrometry and Ion Processes, 78; 1987, p53.
2.
“Matrix Assisted Laser Desorption Ionization (MALDI).”
http://www.tau.ac.il/lifesci/units/proteomics/voyager.html (6/18/2009).
3.
“MALDI-TOF Mass Analysis.” http://www.protein.iastate.edu/maldi.html (6/18/2009).
4.
Jasna Peter-Katalinic; Franz Hillenkamp (2007). “MALDI MS: A Practical Guide to Instrumentation, Methods and
Applications.”Weinheim: Wiley-VCH.
5.
“Maldi Mass Spectrometry.” http://www.sigmaaldrich.com/img/assets/4242/fl_analytix6_2001_new.pdf
(6/17/09).
6.
“Lecture 2: Basic Maldi and Electrospray Theory.”
http://www.hopkinsmedicine.org/mams/mams/middleframe_files/teaching_files/me330.884/2005/ms20
05-lecture-2-basic-maldi-esi.pdf (6/20/2009).
7.
Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A. J. Chem. Educ. 1997, 74, 1289.
8.
Karty, Johnathan A.” Introduction toWalk-Up Mass Spectrometry.”
msf.chem.indiana.edu/.../Introduction%20to%20Mass%20Spectrometry%20july2008.ppt (6/21/09).
9.
“MALDI Mass.” http://www.sigmaaldrich.com/analytical-chromatography/spectroscopy/maldi-mass.html
(6/22/09).
10.
Krishnamurthy, T. U.S. Patent 6,177,266, 2001.
11.
Lee,Y. “Highly Efficient Classification and Identification of Human Pathogenic Bacteria By MALDI-TOF-MS”;
http://www.mcponline.org/cgi/reprint/7/2/448 (6/19/09)