Matrix Assisted Desorption/Ionization
Mass Spectrometry
Phillip Mnirajd

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Mass Spectrometry (MS)
 Vital tool used to characterize and analyze molecules
Limitations
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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].

 Matrix Assisted Laser Desorption/Ionization (MALDI)
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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.

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The mechanism remains uncertain
It may involve absorption of light by the matrix
Transfer of this energy to the analyte
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 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].

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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.

see reference 3

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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].

 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)
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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

Reference 5

Reference 6

 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 30-
55 kDa [4].

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Numerous gas and solid state lasers have been developed for use in
MALDI.
Most MALDI devices use a pulsed UV laser
 N
2 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
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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

Reference 5

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The MALDI method uses a pulse laser
 Laser fires in intervals
Pulsed laser produces individual group of ions
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1 st pulse=1 st group of ions
2 nd pulse= 2 nd group of ions, etc.
Each group of ions generated are detected
With continuous pulsing, the signal resolution increases

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The typical detector used with
MALDI is the time of flight mass detector (TOF-MS)
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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

 The TOF detector is also equipped with a reflectron, or an ion mirror
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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.

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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

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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

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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

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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

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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

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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

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Very simple to implement
Low cost (<$100k)
Moderate vacuum required (~10 -5 torr)
Small size
Most common MS in use
Reference 8

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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

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Applications of MALDI mass spectrometry [9]
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Peptides and proteins
Synthetic polymers
Oligonucleotides
Oligosaccharides
Lipids
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Inorganics
Bacterial identification
Used especially
 Proteomics

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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

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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

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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

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US Patent #6177266 B1 [10]
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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

 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.

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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-hydroxyα -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).

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Protein extracts
 1 μ l of a protein extract was mixed with 9 μ l 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 μ l of aqueous buffer, typically
0.1% trifluoroacetic acid, vortexed for 30 seconds, and 1 μ l of the resulting suspension was either frozen for later use and thawed and combined with 9 μ l of a matrix solution or used immediately.

 Mass spectral analysis of protein extracts
 Distinguishes among 4 strains of Bacillus

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Mass spectral data of whole, intact cells
Capable of detecting virulent and non virulent strains
 Bacillus REV-1 and
Abortus

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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

see reference 11

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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)

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M. Karas, et al and F. Hillenkamp; International Journal of Mass Spectrometry and Ion Processes, 78; 1987, p53.
“Matrix Assisted Laser Desorption Ionization (MALDI).” http://www.tau.ac.il/lifesci/units/proteomics/voyager.html
(6/18/2009).
“MALDI-TOF Mass Analysis.” http://www.protein.iastate.edu/maldi.html
(6/18/2009).
Jasna Peter-Katalinic; Franz Hillenkamp (2007). “MALDI MS: A Practical Guide to Instrumentation, Methods and
Applications.”Weinheim: Wiley-VCH.
“Maldi Mass Spectrometry.” http://www.sigmaaldrich.com/img/assets/4242/fl_analytix6_2001_new.pdf
(6/17/09).
“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).
Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A. J. Chem. Educ. 1997, 74, 1289.
Karty, Johnathan A.” Introduction to Walk-Up Mass Spectrometry.” msf.chem.indiana.edu/.../Introduction%20to%20Mass%20Spectrometry%20july2008.ppt (6/21/09).
“MALDI Mass.” http://www.sigmaaldrich.com/analytical-chromatography/spectroscopy/maldi-mass.html
(6/22/09).
Krishnamurthy, T. U.S. Patent 6,177,266, 2001.
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)