Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI TOF)

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Matrix-Assisted Laser Desorption Ionization Time
of Flight (MALDI TOF)

MALDI is an efficient process for generating
gas-phase ions of peptides and proteins for mass
spectrometric detection. It is widely used in
proteomics research as a high-throughput
technique to identify proteins and their post
translational modifications.

• Sandipan Ray
• Suruchi Rao
• Harini Chandra

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Master Layout (Part 1)
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Part 1 Fundamentals of MALDI-TOF MSPart 2
Sample preparation and spotting Part 3
Ionization and detection
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Matrix-Assisted Laser Desorption Ionization
Time-of-flight (MALDI TOF)
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Flight tube
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Ion source
Detector
1.Sample preparation and spotting 2. Ionization
3. Separation on the basis of time of flight 4.
Detection
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Definitions of the componentsPart 1
Fundamentals of MALDI-TOF MS
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1. Ion source One of the major components of any
MS instrumentation which fragments the sample
into an ionic form for further detection. MALDI
and ESI are most commonly used for proteins
samples 2. Matrix Assisted Laser Desorption
Ionization (MALDI) MALDI is an efficient
ionization source for generating gas-phase ions
of peptides and proteins for mass spectrometric
detection. Target analyte embedded in dried
matrix-sample is exposed to short, intense pulses
from a UV laser. 3. Mass analyzer The mass
analyzer resolves the ions produced by the
ionization source on the basis of their
mass-to-charge ratios. Various characteristics
such as resolving power, accuracy, mass range and
speed determine the efficiency of these
analyzers. Commonly used mass analyzers include
Time of Flight (TOF), Quadrupole (Q) and ion
trap. 4. Time-of-Flight (TOF) This is a mass
analyzer in which the flight time of the ion from
the source to the detector is correlated to the
m/z of the ion. 5. Flight tube Connecting tube
between the ion source and detector within which
the ions of different size and charge migrate to
reach the detector.
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Definitions of the componentsPart 1
Fundamentals of MALDI-TOF MS
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• 6. Reflectron The reflectron acts as an ion
  mirror, and extends the flight length without
  increasing the instrument size. The reflectron
  compensates for the initial energy spread of ions
  having the same mass.
• 7. Reflectron detector Detects the ions
  reflected by ion mirror. This over all setup
  improves the resolution.
• 8. Detector The ion detector determines the mass
  of ions that are resolved by the mass analyzer
  and generates data which is then analyzed. The
  electron multiplier is the most commonly used
  detection technique.

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Part 1, Step 1
Time-of-Flight Mass Analyzer
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Linear Mode
Flight Tube
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Detector
Ion Source
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The lighter ions travel faster and strike the
detector before the heavier ions. This time of
flight (TOF) can be correlated with mass of the
ion
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Action
Audio Narration
Description of the action
First show the rectangular tube with the
detector on the right end the ion source on
the left end. Next, show the appearance of the
colored circles which must then move towards the
detector such that the smallest circle moves
the fastest the largest one moves slowly. Once
they reach the detector, the text below must be
shown.
As shown in animation.
The time-of-flight analyzer resolves ions
produced by the ionization source on the basis of
their mass-to-charge ratio. The TOF tube can be
operated in the linear mode or the reflectron
mode depending on the sample to be detected. In
case of small molecules, this mode usually
provides sufficient resolution. The generated
ions are accelerated towards the detector with
the lighter ions travelling through the TOF tube
faster than the heavier ions. The flight time of
the ions is correlated with the m/z ratio.
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Part 1, Step 2
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Reflectron mode
Flight Tube
Reflectron detector
Reflectron (Ion Mirror)
Sample target
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3
Ion Source
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Action
Audio Narration
Description of the action
As shown in animation.
The TOF analyzer can also be operated in the
reflectron mode, which is more commonly used for
proteomics studies. A reflectron, which acts as
an ion mirror, is incorporated at one end of the
TOF tube. This helps in extending the path length
and in turn the flight time of the ion without
having to increase the actual size of the
instrument. This helps to even out any kinetic
energy differences between ions having the same
mass and thereby improves the resolution.
First show the rectangular tube with the
reflectron, detector the ion source on
the left end. Next, show the appearance of the
colored circles which must then move towards the
reflectron where they get deflected and
ultimately reach the reflectron detector. The
smallest circle must move fastest while the
largest must move slowly as shown in the
animation.
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Part 1, Step 3
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Time-of-Flight Equation
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Where t time-of-flight (s) m mass of the ion
(kg) q charge on ion (C) V0 accelerating
potential (V) L length of flight tube (m)
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Action
Audio Narration
Description of the action
As shown in animation.
The time of flight of a charged ion can be
calculated by means of the equation shown. The
flight time is directly proportional to the
square root of mass of the ion.
Show appearance of the equation and meaning of
each of its terms.
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Master Layout (Part 2)
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Part 1 Fundamentals of MALDI-TOF MSPart 2
Sample preparation and spotting Part 3
Ionization and detection
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Sample Preparation
Matrix selection
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Matrix preparation
Sample purification
Sample deposition
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Matrix deposition
Drying target
Analysis
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Definitions of the componentsPart 2 Sample
preparation and spotting
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• 1. In-gel digestion The in-gel digestion is part
  of the sample preparation process for the mass
  spectrometric identification of proteins during
  the course of proteomic analysis. Protein spots
  or bands excised from the gels are digested using
  trypsin.
• 2. Trypsin Trypsin is a serine protease found in
  the digestive system of many vertebrates, where
  it hydrolyses proteins. It cleaves at the
  C-terminal of lysine (K) and Arginine (R)
  residues with the exception of K-proline and
  R-proline sites.
• 3. ZipTip Very small tip like device for removal
  of salts and other interfering agents from the
  protein samples before analysis. ZipTips can be
  incorporated into high throughput robotic devices
  for automated sample clean up.
• 4. Sample/Target plate Multiple well plates on
  which the samples are spotted.
• 5. Analytes The samples that are under study.
  Analytes may be proteins, peptides or
  carbohydrates and are ionized prior to mass
  spectrometric detection.
• 6. Matrix Solution containing high concentration
  of a UV absorbing molecule deposited on sample
  plate along with samples. It is essential to
  select a matrix appropriate for the type of
  sample to be analysed.
• 7. Ion desorption A process in which atomic and
  molecular species residing on the surface of a
  solid leave the surface and enter the surrounding
  gas or vacuum.

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Part 2, Step 1
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In-gel digestion
Trypsin digestion
Cut out spot
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Arginine or Lysine
Completed gel
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C-terminal
N-terminal
Trypsin
Digested fragments
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Action
Audio Narration
Description of the action
The protein sample must be prepared suitably
before it can be analyzed by MS. The purified
protein of interest is excised from the gel on
which it has been electrophoresed and dissolved
in a suitable buffer. Trypsin is then added to
this in order to carry out digestion of the
protein. This enzyme cleaves the protein at the
C-terminal of the its arginine lysine residues
unless there is a proline present immediately
after. The protein is thus digested into smaller
fragments of manageable size.
As shown in animation.
First show appearance of the grey image on top
left with all the spots. Next show the red
circle. That black spot must be excised from
there and must enter the tube as depicted. Next
show a hand adding a solution into the tube with
the micropipette which must turn green in color.
The tube must then be zoomed into and the brown
rectangle interspersed with green region must be
shown. The orange object must then be shown which
must cleave the rectangle exactly at the green
regions. Once it is cleaved, the fragments must
separate out as shown below the arrow mark.
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Part 2, Step 2
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Removal of salt, buffers and detergents from the
sample
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Digested protein sample
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Action
Audio Narration
Description of the action
Once the protein sample has been digested, all
the salt, buffers and any detergents must be
removed from this sample. This can be efficiently
done with the help of filters (e.g. ZipTip). It
offers several advantages such as quick
purification, sample enrichment and ensuring
there is no contamination. However, it can purify
only limited volume of the sample and also
adsorbs some amount of the protein sample thereby
leading to losses.
As shown in animation.
First show the tube with the grey solution in it.
Next show the hand with the micropipette in it.
This must move into the tube, be held there very
briefly and then be removed out again. This must
be repeated at least 5 times. Once the hand is
outside again, the tip of the micropipette must
be zoomed in and the figure in the circle must be
shown. Again the white region must be zoomed into
and the figure on the right must be shown.
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Part 2, Step 3
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Addition of matrix
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Mixing
Sample
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Action
Audio Narration
Description of the action
The purified protein sample is then mixed with an
aromatic matrix compound like a-cyano-4-hydroxycin
namic acid, sinapinic acid etc. in the presence
of an organic solvent. The components are then
mixed thoroughly.
As shown in animation.
First show the tube with a grey solution in it.
Next show the hand with the micropipette entering
the tube. Once it enters, the grey solution must
turn blue and the hand must then be removed. Then
show this tube being placed in one of the holes
in the instrument on the right.
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Part 2, Step 4
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Sample spotting
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196 well MALDI Plate
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Action
Audio Narration
Description of the action
The solution containing the organic matrix with
the embedded analyte is then spotted on to a
metallic MALDI sample plate.
As shown in animation.
First show the grey surface with white spots.
Next show the hand with pipette moving down and a
liquid being dispensed into one of the spots.
This spot must then be zoomed into and the figure
on top left must be shown.
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Master Layout (Part 3)
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Part 1 Fundamentals of MALDI-TOF MSPart 2
Sample preparation and spotting Part 3
Ionization and detection
Laser
Matrix analyte
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Detector
Flight tube
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MALDI
Target plate
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Peptide spectrum
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Definitions of the componentsPart 3
Ionization and detection

• 1. Laser Light amplification by stimulated
  emission of radiation (LASER or laser) is a
  mechanism for emitting electromagnetic radiation.
• 2. Matrix analyte Solution containing high
  concentration of UV absorbing molecules embedded
  with the analyte of interest, deposited on the
  sample plate. It is essential to select a matrix
  that is appropriate for the type of sample being
  analysed. Commonly used matrices are sinapinic
  acid and a-cyano-4-hydroxycinnamic acid
• 3. Sample plate Plate onto which the
  matrix-analyte solution is spotted.
• 4. Matrix Assisted Laser Desorption Ionization
  (MALDI) MALDI is an efficient ionization source
  for generating gas-phase ion of peptides and
  proteins for mass spectrometric detection. Target
  analyte embedded in dried matrix-sample is
  exposed to short, intense pulses from a UV laser.
• 5. Time-of-Flight (TOF)- This is a mass analyzer
  in which the flight time of the ion from the
  source to the detector is correlated to the m/z
  of the ion.

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Definitions of the componentsPart 3
Ionization and detection

• 6. Flight tube- Connecter between the ion
  source and detector within which the ions of
  different size and charge fly to reach the
  detector
• 7. Detector- The ion detector determines the
  mass of ions that are resolved by the mass
  analyzer and generates data which is then
  analyzed. The electron multiplier is the most
  commonly used detection technique.
• 8. Peptide spectrum The picks corresponding to
  individual peptides which are separated on the
  basis of their m/z ratio.

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Part 3, Step 1
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Ionization
Laser
Matrix analyte
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Detector
Flight tube
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MALDI
Target plate
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Action
Audio Narration
Description of the action
The target plate containing the spotted matrix
and analyte is placed in a vacuum chamber with
high voltage and short laser pulses are applied.
The laser energy gets absorbed by the matrix and
is transferred to the analyte molecules which
undergo rapid sublimation resulting in gas phase
ions.
As shown in animation.
First show the entire grey apparatus with all the
labels and the violet rectangle laser source.
Next show a beam emerging from the rectangular
box and falling on the white semicircular region.
Once this happens, the colored ions must emerge
from the white surface as shown in animation.
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Part 3, Step 2
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Resolution detection
Detector
Flight tube
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3
Peptide spectrum
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Action
Audio Narration
Description of the action
The gas phase ions generated are accelerated and
travel through the flight tube at different
rates. The lighter ions move rapidly and reach
the detector first while the heavier ions migrate
slowly. The ions are resolved and detected on the
basis of their m/z ratios and a mass spectrum is
generated. Parameters such as geometric design,
power supply quality, calibration method, sample
morphology, ion beam velocity etc. all affect the
accuracy of mass detection.
As shown in animation.
Once the colored circles have appeared, they must
move towards the detector. The smallest blue
circles must move the fastest followed by the
orange circles and then the green circles. Once
all the circles reach the detector, the computer
with the spectrum in it must be shown.
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Interactivity option 1Step No 1
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MALDI can also be used for tandem mass
spectrometry studies in combination with two
consecutive TOF tubes that are separated by a
collision cell. The ion fragment selected from
the first TOF tube is further fragmented in the
collision cell by bombarding against an inert gas
like argon. This technique can be used for
protein sequencing studies. Click on the laser to
view the working of MALDI-TOF-TOF-MS.
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Laser
Detector
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TOF 2
TOF 1
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Reflector
Collision cell
Results
Boundary/limits
Interacativity Type Options
When the user clicks on the red box marked as
laser the animation shown must take place. The
three circles (blue, red yellow) must appear
which must move towards the violet box. In this
only the yellow circle must be selected move
ahead. It must then get fragmented three
smaller circles (green, pink yellow) must
appear which must then travel towards the
detector, with the smallest circle moving
fastest.
User must click on the red box marked as laser
after which the animation shown above must appear.
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Click on the red box marked laser.
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Questionnaire
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• 1. Which are the two types of ionization sources
  used for the Mass Spectrometric analysis of
  biological samples?
• a) Fast Atom Bombardment and Chemical
  Ionization
• b) Electron Transfer Dissociation and
  Collision Induced Dissociation
• c) Matrix Associated Laser Desorption
  Ionization and Electrospray Ionization
• d) Electron Transfer Dissociation and Matrix
  Associated Laser Desorption Ionization
• 2. Which of the following is not a mass analyzer?
• a) Time-of-Flight (TOF) b) Quadrupole
  (Q) c) Ion traps d) MALDI
• MALDI is a
• a) Soft ionization technique b) Hard
  ionization technique c) Both of them
• d) None of them

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Questionnaire
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4. Which of the following matrix is most
suitable of carbohydrate samples? a) CHCA
b) Sinapinic acid c) Dithranol d) DHB 5. Which
of the following statement is right a)
MALDI is more tolerant to salts than ESI b)
MALDI is less tolerant to salts than ESI c)
Both of them are equally more tolerant to salts
d) There is no effect of salts on MALDI and
ESI
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Links for further reading
Books Link, A. J., LaBaer J., Proteomics A
cold spring harbour laboratory manual cold
spring harbour laboratory press.
Research papers 1. Karas, M., Hillenkamp, F.
"Laser desorption ionization of proteins with
molecular masses exceeding 10,000 daltons". Anal.
Chem. 1988, 60, 22992301. 2. Karas, M.,
Glückmann, M., Schäfer, J. Ionization in
matrix-assisted laser desorption/ionization
singly charged molecular ions are the lucky
survivors. J Mass Spectrom. 2000 ,35,1-12.