Download Principles of MALDI

THE BASIC PRINCIPLES OF MASS SPECTROMETRY
At its core, mass spectrometry is a semiquantitative, analytic method traditionally used to
elucidate the composition or molecular structure of an unknown sample. This characterization is
performed entirely by the mass spectrometry instrument and is based on the acquisition and
analysis of mass and charge values from individual, ionized sample molecules. Mass
spectrometry instruments are composed of 3 basic modules: an ionization chamber, a mass
analyzer, and an ion detector (Figure 1). Once placed in the ionization chamber, the unknown
sample, which may be in a gas, liquid, or solid phase, is pulsed with an energy source. This
energy serves dual functions: ionization of individual molecules and desorption of solid or liquid
phase samples into the gas phase. The vaporized sample is next directed into and accelerated
through the mass analyzer, which separates ions based on their mass-to-charge ratio. Upon
emerging from the mass analyzer, ionized particles collide with the ion detector, which measures
both the mass and charge of each molecule as derived from their individual force and time to
impact. These signals are converted to an electrical output and ultimately depicted to the user in
a mass spectrum, which graphs the relative abundance of each detected ion on the y-axis versus
its mass-to-charge ratio on the x-axis (Figure 1). The composition or structure of the unknown
sample is subsequently derived from careful interpretation and analysis of the ion peaks.
Figure 1. Mass Spectrometry Instrument Design
Many different types of mass spectrometry instruments are available, primarily differentiated by
their method of sample ionization and the type of mass analyzer used. Selection among the
various instruments is largely dependent on the phase of the input sample, in addition to the
physical and chemical properties of the unknown molecules: their molecular weight, thermal
stability, side-chain modifications, etc. Each of these properties strongly influence the choice of
ionization method and the type of mass analyzer best suited to separate the ionized molecules.
Prior to the 1980s, mass spectrometry technology was largely restricted to the analysis of small,
thermostable compounds able to withstand the harsh electric ionization techniques available at
the time. Larger polypeptides and other biomolecules were found to rapidly degrade under these
conditions, which significantly impedes their characterization. With the onset of the proteomics
era, however, research into alternative mass spectrometry methods was accelerated and resulted
in the development of low energy or “soft ionization” techniques — the principle behind
MALDI-TOF MS. The major advantage of this method is its ability to ionize and desorb high
molecular weight biomolecules into the gas phase while preserving their intact state. Based on
these general considerations, MALDI-TOF MS has emerged as the premier method of choice for
the analysis and identification of large polypeptides and even whole microorganisms.
BASIC PRINCIPLES OF MALDI-TOF MASS SPECTROMETRY
Sample characterization by MALDI-TOF MS begins by spotting the sample (either solid or
liquid) into a defined indentation on a solid target support plate (Figure 2). The composition of
the input sample can vary greatly from purified protein to whole-cell microorganisms. Following
application onto the target plate, the sample may be further treated, depending on composition,
but is ultimately overlaid with a chemical matrix which must dry completely prior to analysis.
The matrix is essential for the “soft ionization” process and is chosen for both its efficient
desorption into the gas phase and for its ability to effectively absorb the majority of pulsed
ionizing energy, thereby protecting sample molecules from fragmentation. A number of matrix
compounds have been developed and are each composed of small (<1000 Dalton), acidic
molecules dissolved in an organic solvent. An approximately 10 to 1 ratio of matrix to sample is
used for MALDI-TOF MS preparation to ensure efficient dilution and protection of sample
molecules from fragmentation. Once dried, the prepared target plate is placed into the ionization
chamber where each sample is irradiated with 240 brief pulses of energy from an ultraviolet
nitrogen laser (337 nm). This process desorbs individual sample and matrix molecules from the
target plate into the gas phase, with the majority of energy absorbed by the matrix, which
becomes ionized with a single positive charge. This positive charge is subsequently transferred
from the matrix to native sample proteins through their random collision in the gas phase.
Figure 2. Matrix-Assisted Laser Desorption Ionization - Time of Flight Process
The cloud of ionized proteins is next funneled through a positively charged, electrostatic field
which accelerates the molecules into the time of flight (TOF) mass analyzer. The TOF chamber
is an empty, pressurized tube that allows ions to travel down a field-free region toward the ion
detector. The velocity at which individual ions fly through the TOF chamber is dependent on
their mass-to-charge ratio. Because each sample analyte has an identical, single positive charge,
ions are ultimately separated based on their difference in mass — heavier ions will travel through
the mass analyzer at a slower velocity, compared to lighter ions. As the ions emerge from the
TOF mass analyzer, they collide with the ion detector, which measures their charge and time to
impact (Figure 2). Based on standards of known mass, the time to impact for each unknown
analyte is converted into a mass-to-charge ratio, which is depicted on a mass spectrum.
Each generated mass spectrum can be thought of as a unique protein “fingerprint” or a protein
profile of the unknown sample. Specifically for analysis of microorganisms, MALDI-TOF MS
will detect the most abundant proteins over a predefined mass range (typically 2 to 20 kDa).
These are mostly intracellular, hydrophilic proteins and are primarily ribosomal components or
other noncatalytic, structural complexes. Based on this protein profile, identification of the
unknown microorganism is performed by computerized comparison of the acquired spectra to a
database of reference spectra composed of previously well-characterized isolates.
Principles of MALDI
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The sample is dispersed in a large excess of matrix material which will strongly absorb the
incident light.
The matrix contains chromophore for the laser light and since the matrix is in a large molar
excess it will absorb essentially all of the laser radiation
The matrix isolates sample molecules in a chemical environment which enhances the probability
of ionization without fragmentation
Short pulses of laser light focused on to the sample spot cause the sample and matrix to volatilize
The ions formed are accelerated by a high voltage supply and then allowed to drift down a flight
tube where they separate according to mass
Arrival at the end of the flight tube is detected and recorded by a high speed recording device
The time-of-flight of the ion is converted to mass using the following principles:
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An accelerating potential (V) will give an ion of charge z an energy of zV. This can be equated
to the kinetic energy of motion and the mass (m) and the velocity (v) of the ion
zV = 1/2mv2
Since velocity is length (L) divided by time (t) then
m/Z = [2Vt2]/L2
V and L cannot be measured with sufficient accuracy but the equation can be rewritten
m/Z = B(t-A)2
where A and B are calibration constants that can be determined by calibrating to a known m/Z
Mass of an ion is determined by the following method:
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1. Measure time-of-flight (t) of the ion
2. External or internal calibration is used to determine the constants A and B so the time-of-flight
can be converted to mass
m/Z = B(t-A)2
3. Store B/V so changes in the 20 kV voltage supply does not effect calibration
It is assumed that all ions have the same kinetic energy.
Samples are loaded onto metal plates for analysis on the instrument. A sample concentration of 1
mg/mL is ideal and usually from one to ten picomoles of sample is required for analysis. This is
spotted onto the sample position on the metal strip and then 0.5 uL of matrix (usually 10 mg/mL)
is applied to the sample position as well. There are many different matrices that can be used for
MALDI-TOF. Some of the most common include Sinapinnic Acid (SA) for protein samples,
alpha-Cyano-4-hydroxycinnamic acid (ACH) for peptide samples, and a 9:1 mixture of 2,5Dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (sDHB) for carbohydrate and
sometimes protein samples. DNA can also be analyzed using MALDI-TOF by employing
different matrices. New matrix solutions are now in development which will yield greater
sensitivity
and
resolution.
MALDI technology has many applications in the biochemical field. It can be used to easily
monitor and optimize enzymatic digests, characterize proteins, or can be used for quality control
for peptide synthesis. MALDI has also been used as a method of N-terminal and C-terminal
protein/peptide sequencing. There are also applications in the rapid conformation of post
translational modifications and the quantitation of drugs and chelators conjugated to monoclonal
antibodies.
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MALDI Sample Preparation
MALDI samples should be in liquid form (or lyophilized powder). It should free of SDS, buffers,
etc. if possible and should not be radioactive. We will usually need ~5-10 µL. It is best to
remove buffer salts and detergents (e.g. by dialysis) prior to analysis and to dissolve the sample
in a suitable solvent (e.g. 0.1% TFA/water) which will not degrade the spectrum. If there is too
much salt in a sample, the salt signal intensity is so large that it effectively suppresses out the
sample signal, giving no sample spectrum. In cases where it is not possible to remove these
contaminants the sample should be in a higher concentration. It may then be possible to dilute
the sample to the point where the contaminants will have little effect on the spectrum.
Levels of buffers and detergents which exceed the following limits will probably cause
noticeable degradation of the spectrum:
Phosphate buffer
>50mM
Ammonium bicarbonate
>30mM
Tris buffer
>100mM
Guanidine
>1M
Detergents(e.g. Triton-X)
>0.1%
SDS
>0.01%
Alkali metal salts
>1M
Glycerol
>1%
Sodium Azide
>1mM