Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry (MALDI TOF MS)
MALDI TOF MS has matured into an indispensable tool in the field of modern
bioanalysis. It allows rapid analysis and identification of a wide scale of biomolecules
(proteins, peptides, nucleic acids, lipids, saccharides), as well as synthetic polymers, drugs,
small organic molecules and inorganic compounds.
Theoretical part
Together with the electrospray ionization, MALDI is a soft ionization technique that
allows analysis of various biomolecules, especially biopolymers, and synthetic polymers,
which tend to fragment when ionized by classical ionization techniques. As a pulsed
ionization technique MALDI it usually coupled with TOF mass analyzers.
A. Principle of MALDI
MALDI is a two-step process (Fig. 1). First, the sample, composed of an analyte (A)
mixed with an excess of MALDI matrix (M), is irradiated by short (~ ns) laser pulses. The
matrix molecules strongly absorb laser light leading to fast sample desorption. Second, the
protonized matrix species transfer protons to the analyte molecules and charge them. For
MALDI, a low fragmentation level and the formation of so called pseudomolecular ions
[A+H]+
are typical. .
Figure 1: Principle of the MALDI process
The requirements for a suitable matrix selection result from the MALDI mechanism:
- strong absorption at the wavelength of the employed laser, usually a nitrogen laser (337
nm) or frequency-tripled Nd:YAG lasers (355 nm)
- the formation of desirable „co-crystals“ with analyte molecules (usually based on
previous experience, by trial and error to some extent)
- acidic properties of matrix (efficient proton source for analyte ionization)
Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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The most commonly used MALDI matrices are small aromatic acids, such as 3,5-dimethoxy-
4-hydroxycinnamic acid (sinapinic acid, SA) or α-cyano-4-hydroxycinnamic acid (CHCA)
(Fig. 2).
OH
OH
O
N
OH
OH
O
O
O
CH3
CH3
Figure 2: Commonly used MALDI matrices:
sinapinic acid and α-cyano-4-hydroxycinnamic acid
B. Principle of Time-of-flight (TOF)
Ions generated by MALDI are usually analyzed in a time-of-flight mass analyzer (Fig.
3). Ions are given a defined kinetic energy by an accelerating electric field and are allowed to
drift through a field-free region. Their time of flight, t can be calculated as a function of the
m/z ratio of the ion according to the following equation:
2
2
2
L
t
eU
z
m

where m is mass, z is number of charges, e is the elemental charge, U is the acceleration
voltage and L is the length of the drift zone.
The advantage of the TOF mass analyzer is the high ion transmission (and thus high
sensitivity), short time of the analysis (~hundred s for a single-shot laser pulse) and
theoretically unlimited m/z range.
Figure 3: A schematic of the MALDI TOF mass spectrometer operating in the reflector mode
Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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In MALDI TOF MS, mass resolution can be improved in two ways, which correct the
negative influence of dispersion of the initial kinetic energy (Ekin) of the ions:
The use of delayed extraction (DE) refers to delaying the application of the extraction
potential by a certain time of 100 – 1000 ns after the laser pulse. Within the delay period, ions
with a given m/z that have high Ekin travel further away from the sample target (repeller) than
ions with lower Ekin. When the extraction voltage is applied, the slow ions, which are closer to
the repeller, are accelerated more, leading to the simultaneous arrival of ions of a given m/z on
the detector. The drawback of the DE is that the optimum delayed extraction time differs for
various m/z and therefore this method works well only for the selected interval of m/z.
Another method for the correction of the ion initial kinetic energy distribution is the
use of an ion mirror (reflector, reflectron). The reflector uses an electrostatic field to reflect
the ion beam toward the detector and it consists of a series of ring electrodes. The first
electrode is held at a potential of the same polarity but slightly higher magnitude compared to
the acceleration voltage, the voltage on the following electrodes drops gradually to zero on the
last electrode. More energetic ions penetrate deeper into the reflector, travel a slightly longer
path and spend a longer time in the reflector. Less energetic ions of the same m/z will
penetrate a shorter distance into the reflector, thus spending a shorter time in the reflector. The
detector is placed at the focal point, where ions of the same m/z, but with different energies
strike the detector at the same time. The disadvantage of using the reflector is a certain
decrease in the ion transmission and consequently sensitivity.
C. Selected applications of MALDI TOF MS
1. Molecular mass determination
Pseudo-molecular ions generated by MALDI in positive mode are usually single-charged
as [A+H]+
. Other types of ions, such as double-, triple- or even multi-charged ions [A+2H]2+
,
[A+3H]3+
might also be observed. Analyte dimers, such as [A2+H]+
as well as various adducts
of analyte/matrix/alkali metals [A+Na]+
, [A+K]+
, [A+MH]+
, [A+MNa]+
may occur in the
spectra. MALDI TOF MS is not usually applied for low molecular mass determination (below
500 Da) due to possible interferences of ions of matrix fragments, adducts and clusters.
MALDI TOF MS is mostly used for determination of molecular mass of peptides,
proteins, nucleic acids and saccharides. It can also be powerful for determining molecular
weight distribution and structures of synthetic polymers.
However, the molecular mass information itself is generally not sufficient for
unambiguous identification of a compound.
2. Peptide Mass Fingerprinting (PMF)
One of the most significant applications of MALDI TOF MS is protein
identification. Sequences of known proteins are stored in DNA and protein
databases. As mere determination of protein mass is not sufficient for protein
identification, other techniques based on analysis of peptides generated by
specific cleavage of the analyzed protein are employed. Proteins can be
identified based on more detailed information about a group of peptides
(PMF) or using tandem mass spectrometry (MS/MS) of a single peptide.
Peptide Mass Fingerprinting (PMF) is a technique used to identify
proteins by matching their constituent fragment masses (peptide masses) to the theoretical
Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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peptide masses generated from a protein or DNA databases. The first step in PMF is that an
intact, unknown protein is cleaved with a proteolytic enzyme to generate peptides. Trypsin is
most commonly used (it cleaves peptide bonds X-K or X-R if X ≠ P). Masses of the peptides
obtained from MALDI TOF mass analysis are compared with theoretical fingerprints
generated from protein databases using programs, such as Protein Prospector, Mascot,
Proteomics etc. The result of the database search is a list of proteins that might provide the
measured peptides. The probability of protein identification is given by parameters, such as
the ratio matched/entered peptides, so called expectation factor, or sequence coverage.
Presumption of successful identification is high accuracy of m/z determination and low
number of proteins in the original sample. Should more proteins be present in the sample, the
protein of interest has to be isolated using a suitable separation technique prior to the
enzymatic cleavage.
Gel electrophoresis (i.e. 1D or 2D SDS-PAGE) and liquid chromatography are used
almost exclusively for protein separation, nowadays. Ideally, the output of separation is a
single protein.
D. Solid Phase Extraction (SPE)– ZipTip™
ZipTip™ (Millipore, MA) is a 10 µl pipette tip with a 0.6 or 0.2 µl bed of
chromatography media fixed at its end with no dead volume. It is ideal for concentrating and
purifying samples for sensitive analyses by mass spectrometry, liquid chromatography,
capillary electrophoresis and other analytical methods (Fig. 4). The chromatographic medium
is placed in the very end of the tip assuring virtually zero dead volume.
Two types of resins are available in ZipTip™ pipette tips: C4 resin with 30 nm pores
for larger peptides and proteins and C18 resin with 20 nm pores for peptides.
Figure 4: Principle of the ZipTip™ pipette tips. Application details are explained in the following chapter
Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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Experimental part
Task: Identify two unknown proteins by MALDI TOF MS and peptide mass fingerprinting.
Use SPE ZipTip™ pipette tips for purification of protein digests.
A. Solutions and materials
MALDI matrices
- solution TA30 (30% acetonitrile, 0.1% TFA): mix 300 l ACN, 600 l H2O
and 100 l 1% TFA
- CHCA matrix solution: dissolve ~ 2 mg of CHCA in 200 l TA30, sonicate cca 1
min in an ultrasonic bath and centrifuge undissolved crystals
- SA matrix solutions:
- solution I: dissolve cca 2 mg of SA in 200 l 100% EtOH, sonicate cca 1 min in an
ultrasonic bath and centrifuge undissolved crystals
- solution II: dissolve cca 2 mg of SA in 200 l TA30, sonicate cca 1 min in an
ultrasonic bath and centrifuge undissolved crystals
ZipTip™ purification
- ZipTip™ C18 pipette tips
- 0.1% TFA: mix 50 l 1% TFA with 450 l of H2O
- 50% acetonitrile (ACN): mix 200 l of ACN with 200 l of H2O
Proteins – unknown samples
- Protein solutions: 0.1 mg.ml-1
in 50 mmol.l-1
NH4HCO3
- Tryptic digests of unknown proteins (0.1 mg.ml-1
)
Peptide calibration mixture
- Mixture of peptides designed for mass spectrometer calibration. Composition of the
mixture is given in the following table:
peptide
concentration
(µM)
Relative molecular
weight, M
m/z ([M+H]
+
)
monoisotopic dominant
Bradykinin 1 1059.5610 1060.5692 1060.5692
Angiotensin I 1 1295.6775 1296.6853 1296.6853
Renin 2 1758.9332 1759.9396 1760.9474
ACTH 5 2464.1911 2465.1989 2466.2067
Insulin 10 5733.6 5734.7 – average
Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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Protein calibration mixture
- Mixture of proteins designed for mass spectrometer calibration. Composition of the
mixture is determined in the following table:
Protein M Ion m/z (average)
Trypsinogen 23981 [M+H]
+
23982 Da
Protein A 44612 [M+H]
+
44613 Da
Protein A [M+2H]
2+
22307 Da
Albumin-bovine (BSA) 66463 [M+H]
+
approx. 66.5 kDa
Albumin-bovine (BSA) [M+2H]
2+
approx. 33.3 kDa
Protein digest desalinating with ZipTip™ (see Fig. 4)
1. Prepare 3n aliquots of 0.1% TFA (50 l), where n is the number of the unknown
samples. For each sample, 1 aliquote will be used for equilibration and 2 aliquotes
will be used for washing.
2. Add 1 l 1% TFA to the samples (10 l in each).
3. Dispense 5 l of 50% ACN into an empty microvial using a standard pipette tip
and close it. This microvial will be used to elute the peptides in step 7.
4. Equilibrate a ZipTip™ pipette tip:
a. Pre-wet the tip with 10 l 50% ACN: aspirate the solution into the tip,
dispense to waste. Repeat.
b. Equilibrate the tip by aspirating and dispensing 10 l of 0.1% TFA from the
prepared aliquot. Repeat.
5. Bind the peptides to ZipTip™: Aspirate and dispense the sample (10 l)
carefully and slowly for 10 times, avoid introducing air.
6. Wash the peptides: Aspirate 10 lf0.1% TFA from a new aliquot and
dispense to waste. Repeat with another aliquot.
7. Elute the peptides: Insert the tip into the microvial (from step 3) and dispense 5
l of 50% ACN. Slowly aspirate and dispense the solution at least three times
without introducing air bubbles.
8. The peptides are in 5 l of 50% ACN.
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B. Sample preparation for MALDI TOF MS
Deposit your samples on the MALDI target as described below. Choose two free
columns and deposit each sample twice onto two neighbor wells (write down the exact well
positions for all samples).
row analyte matrix
1 H2O SA (2)
2 H2O SA (1+2)
3 Protein calibration mixture SA (1+2)
4 unknown protein 1 SA (1+2)
5 unknown protein 2 SA (1+2)
6 - -
7 H2O CHCA
8 Protein digest 1 CHCA
9 Peptide calibration mixture CHCA
10 Protein digest 2 CHCA
11 Protein digest 1 (desalted) CHCA
12 Peptide calibration mixture CHCA
13 Protein digest 2 (desalted) CHCA
Sample preparation of proteins – „double layer“ method
Deposit ~ 0.1 l of the SA matrix solution I (in 100% EtOH) on each spot in the row
2-5. A thin layer of matrix crystals will be formed. Premix 2 l of SA matrix solution
(solution II) with 2 l of water/protein calibration mixture/unknown protein in a cap of a
small microvial. Apply 0.5 l of this solution on the top of the thin layer of matrix in row 2-5.
In the first row, apply 0.5 l of the solution of the SA matrix with water directly on the
MALDI target. Write down all sample well positions.
Sample preparation of peptides – „quick and dirty“ method
For the samples in row 7-13, apply 0.5 l of the sample solution and 0.5 l of the
CHCA matrix solution and mix carefully with the pipette tip directly on the MALDI target.
Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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C. MALDI TOF MS of peptides and proteins
Mass spectra acquisition is performed using a MALDI TOF/TOF mass spectrometer
AutoflexSpeed (Bruker). This instrument is equipped with a 1 kHz Nd:YAG laser (355 nm).
Except for the insertion of the MALDI target into the instrument, the whole system is
controlled by the software FlexControl (Fig. 5). Other programs, such as FlexAnalysis and
BioTools are available for spectra processing and analysis.
Figure 5: Screen of the FlexControl software
1. Determination of the molecular weight of unknown proteins
- Insert the MALDI target into the instrument and wait until the system is pumped down
completely (p < 3x10-6
mbar).
- Load the method for measurement of proteins (Select Method -> file vyuka ->
method linear_proteins.par). Parameters (extraction voltage, delay time etc.) of this method
were set in advance for measurement of proteins with size of 10 – 100 kDa in the linear positive mode.
- Find the position with protein calibration mixture on the MALDI target.
- Start the spectra acquisition by clicking the Start button; the final spectrum is an
average spectrum from 1000 accumulated mass spectra.
Manual to the Advanced Laboratory Course on Analytical Chemistry: MALDI TOF MS
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Optimal laser energy adjustment
Increase the laser energy gradually until the first analyte peaks appear (this energy is
called threshold energy and it differs for various matrices, analytes and sample
preparation), increase the energy by 10 – 30 % over the threshold energy; record this
value and use it in further experiments.
Calibration:
In TOF mass spectrometry, only the x-axis is usually calibrated. The y-axis is often normalized, i.e. the
most intense peak is assigned 100% intensity. Calibration is performed in a defined range of m/z, which a
calibration mixture of selected compounds is prepared for. In the case of low m/z (<500) peaks of matrix,
matrix clusters and fragments can be used; for m/z in the range of 500 - 5000 Da a mixture of peptides can
be applied. Synthetic polymers or a mixture of selected proteins can be used for heavy molecules (<6000
Da).
After the spectra acquisition of the protein calibration mixture, open the bookmark
Calibration and assign successively the m/z values to the relevant peaks. When all peaks
are assigned, confirm the calibration with the Apply button. In the next measurement, the
instrument will work with this calibration.
Save the spectrum of the protein calibration mixture (Save as).
Measurement of the mass spectra of the unknown proteins
Acquire the mass spectra of the unknown proteins in the same way as the spectra of
the protein calibration mixture. Acquire also the spectra of the matrix. Save all spectra.
Questions:
1) Compare the morphology of the sinapinic acid crystals using the „double layer“ and the
„dry droplet“(the spot on row 1) sample preparation. Try to explain what are advantages
of the „double layer“ sample preparation for analysis of proteins.
2) Explain what you observed in the mass spectra – number of peaks, their shape and
resolution.
3) Calculate the approximate amount of substance (mol) and the number of molecules of the
unknown proteins which were deposited on the MALDI target.
1. Measurement of peptides/protein digests
Acquire spectra of the protein digests in the same way as in the case of proteins. Measure
in the linear mode (method: linear_peptides.par) first and then in the reflector mode
(reflector_peptides.par). In both cases, calibrate the instrument using the peptide calibration
mixture before the measurement of the digests of the unknown proteins and blank samples
(matrix only). Note the calibration error and save the spectra for further database search.
While saving the spectra, choose the methods PMF.FAMS in the section Processing and
check the box Open in FlexAnalysis.
Questions:
4) Explain the peak isotope distribution. Characterize the monoisotopic peak.
5) Explain the presence of signals in the blank sample.
6) Explain the difference between the spectra acquired in linear and reflector mode
(resolution, peak intensity). Summarize advantages and disadvantages of both modes.
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D. Protein Mass Fingerprinting (PMF)
Open the mass spectrum of the digest of an unknown protein in the FlexAnalysis. Send
this spectrum (list of monoisotopic peaks) to BioTools by clicking on the traffic lights icon
. This program will be used for data analysis. In BioTools, click on MS icon , which opens
the dialog (Fig. 6) for the database search. Enter your name, email address, the database name
(Swissprot), the digesting enzyme (trypsin), the maximal number of uncleaved sites (partials;
1), variable modification (oxidation (M)), mass tolerance (50 ppm) and start the search with
the Start button.
The results of the search will appear in a new window; more details of the results can be
displayed in the BioTools (button Get Hits).
Discuss the results with your supervisor.
Figure 6: Dialog for the PMF database search
E. Laboratory report
The report has to contain the date, all names of participants, and titles of the finished
tasks. One report for the entire group is sufficient, preferably in electronic format. Please,
enclose major experimental results, such as spectra and tables, answers to the questions and a
brief discussion of the results. Save the results to the computer and turn them in to your
supervisor.