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    Matrix Assisted Laser Desorption / Ionization: Ronak Lazarus 2021PHXP0015H Supervisor: Dr. Amit Nag

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    RONAK LAZARUS
    Matrix Assisted Laser Desorption/Ionization (MALDI) is a technique used to ionize large biomolecules like proteins for mass spectrometry analysis. In MALDI, the sample is mixed with an aromatic organic compound called the matrix and deposited on a target plate. A pulsed laser is used to excite the matrix which transfers energy to the analyte molecules, causing them to ionize. The ions are then accelerated into a time-of-flight mass spectrometer for separation and detection based on their mass-to-charge ratios. Commonly used matrices include sinapinic acid for proteins and 2,5-dihydroxybenzoic acid for glycans. MALDI is often coupled with

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    Matrix Assisted Laser Desorption / Ionization: Ronak Lazarus 2021PHXP0015H Supervisor: Dr. Amit Nag

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    Matrix Assisted Laser Desorption/Ionization (MALDI) is a technique used to ionize large biomolecules like proteins for mass spectrometry analysis. In MALDI, the sample is mixed with an aromatic organic compound called the matrix and deposited on a target plate. A pulsed laser is used to excite the matrix which transfers energy to the analyte molecules, causing them to ionize. The ions are then accelerated into a time-of-flight mass spectrometer for separation and detection based on their mass-to-charge ratios. Commonly used matrices include sinapinic acid for proteins and 2,5-dihydroxybenzoic acid for glycans. MALDI is often coupled with

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    Maldi Presentation Ronak

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    Matrix Assisted Laser Desorption/Ionization (MALDI) is a technique used to ionize large biomolecules like proteins for mass spectrometry analysis. In MALDI, the sample is mixed with an aromatic organic compound called the matrix and deposited on a target plate. A pulsed laser is used to excite the matrix which transfers energy to the analyte molecules, causing them to ionize. The ions are then accelerated into a time-of-flight mass spectrometer for separation and detection based on their mass-to-charge ratios. Commonly used matrices include sinapinic acid for proteins and 2,5-dihydroxybenzoic acid for glycans. MALDI is often coupled with

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    44 views19 pages

    Matrix Assisted Laser Desorption / Ionization: Ronak Lazarus 2021PHXP0015H Supervisor: Dr. Amit Nag

    Uploaded by

    RONAK LAZARUS
    Matrix Assisted Laser Desorption/Ionization (MALDI) is a technique used to ionize large biomolecules like proteins for mass spectrometry analysis. In MALDI, the sample is mixed with an aromatic organic compound called the matrix and deposited on a target plate. A pulsed laser is used to excite the matrix which transfers energy to the analyte molecules, causing them to ionize. The ions are then accelerated into a time-of-flight mass spectrometer for separation and detection based on their mass-to-charge ratios. Commonly used matrices include sinapinic acid for proteins and 2,5-dihydroxybenzoic acid for glycans. MALDI is often coupled with

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    Matrix Assisted Laser Desorption / Ionization

    Ronak Lazarus
    2021PHXP0015H
    BITS Pilani Supervisor : Dr. Amit Nag
    Hyderabad Campus
    Introduction

     It is a method where a laser is used to generate ions of high molecular weight


    samples such as proteins and polymers.
     Analyte is embedded into crystal matrix.
     The presence of an aromatic matrix causes the large molecules to ionize
    instead of decomposing.

    BITS Pilani, Pilani I Goa I Hyderabad


    How does MALDI work

    Required items : - a sample to be analyzed


    - a matrix substance
    - a laser

    MALDI preparation :
    1) Mix sample and matrix solutions on suitable ratio.
    2) Put an aliquot of this mixture on the target plate.
    3) Let the mixture co-crystallize.
    4) Insert the target plate in the MALDI mass spec
    (running under high vacuum)
    5) Shoot with the laser on the MALDI preparation
    to generate ions.
    6) Collect , analyze and detect the resulting ions.

    BITS Pilani, Pilani I Goa I Hyderabad


    Role of matrix in MALDI

    The matrix transfers the energy needed for ionization from the laser light to the
    sample molecules.
    Positive ionization mode:

    BITS Pilani, Pilani I Goa I Hyderabad


    Negative ionization mode:

    BITS Pilani, Pilani I Goa I Hyderabad


    Type of laser commonly used in MALDI

    Most MALDI devices use a pulsed UV laser


    • N2 laser source emits at 337 nm
    Neodymium – yttrium aluminum garnet ( Nd: YAG)
    • Emits at 355nm 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)
    • It is softer than the UV, which is useful for certain biomolecules.

    BITS Pilani, Pilani I Goa I Hyderabad


    Commonly used in MALDI matrix substances

    Peptides : 4-Hydroxy -α- cyanocinamic acid (HCCA)


    Proteins: 2,5- Dihydroxyacetophenone (DHAP)
    Sinapinic acid (SA)
    2,5- dihydroxybenzoic acid (DHB)
    Glycans: 2,5- dihydroxybenzoic acid (DHB)
    Nucleic Acids: 3- Hydroxypicolinic acid (HPA)
    2,4,6- Trihydroxyacetophenone (THAP)

    Why different matrices for different sample?


    1) The amount of energy needed to ionize a particular sample compound ( specific energy threshold)
    2) The stability of a particular sample compound ( for desired fragmentation)

    BITS Pilani, Pilani I Goa I Hyderabad


    Time of flight mass detectors
    The typical detector used with MALDI is time of flight mass detector (TOF)
    • TOF is a method where ions are accelerated by electric field, resulting ions
    of same strength to have same kinetic energy.
    • The time it takes for each ion to transverse the flight tube to arrive at detector
    is based on its mass-to-charge ratio.
    • The larger its m/z , the slower an ion will fly, the longer the measured flight
    time will be.

    BITS Pilani, Pilani I Goa I Hyderabad


    MALDI-TOF: Resolution
    Resolution R defines, how well two peaks are separated from each other:
    R=m / Δm
    Δm is usually derived from a mass peak´s full width at half maximum (FWHM), as shown below for
    the analysis of a mixture of two compounds X and Y:

    BITS Pilani, Pilani I Goa I Hyderabad


    MALDI-TOF: Resolution
    Example spectrum containing 3 singly charged [M+H]+ signals at m/z 922Da, 923Da and 924Da:

    Resolution = m/Δm = 922.00852/0.07264 = 12693


    The resolution here is obviously more than sufficient to achieve baseline separation of the three compounds
    contained in the mixture.

    BITS Pilani, Pilani I Goa I Hyderabad


    MALDI-TOF : Linear mode

    BITS Pilani, Pilani I Goa I Hyderabad


    MALDI-TOF : Linear mode
    Observation:
    Ions of identical mass arrive at the detector at slightly different time points, causing peak
    broadening (i.e. limiting the resolution)

    BITS Pilani, Pilani I Goa I Hyderabad


    How to improve resolution…

    • Optimized matrix preparation homogeneity


    (minimizing spatial spread)

    • Pulsed ion extraction for efficient ion focusing


    (minimizing initial energy spread)

    • Further ion focusing by means of a reflector TOF setup


    (minimizing remaining energy spread)

    BITS Pilani, Pilani I Goa I Hyderabad


    How to improve resolution…

    • Optimized matrix preparation homogeneity (minimizing spatial spread)

    BITS Pilani, Pilani I Goa I Hyderabad


    How to improve resolution…

    • Pulsed ion extraction for efficient ion focusing (minimizing initial energy spread)

    BITS Pilani, Pilani I Goa I Hyderabad


    How to improve resolution…

    • Further ion focusing by means of a reflector TOF setup. (minimizing remaining energy spread)

    BITS Pilani, Pilani I Goa I Hyderabad


    How to improve resolution…

    BITS Pilani, Pilani I Goa I Hyderabad


    Applications
    MALDI-TOFMS use in profiling of microorganisms :

    BITS Pilani, Pilani I Goa I Hyderabad


    Thank YOU

    BITS Pilani, Pilani I Goa I Hyderabad

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