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)