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Lecture 9. MALDI/TOF

Lecture 9. MALDI/TOF. Introduction.

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Lecture 9. MALDI/TOF

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  1. Lecture 9.MALDI/TOF

  2. Introduction • Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. It is most similar in character to electrospray ionization both in relative softness and the ions produced (although it causes many fewer multiply charged ions). • The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization.

  3. Matrix • crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is made, often in a mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroacetic acid (TFA) may also be added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0.1).

  4. Laser • typically employ the use of UV lasers such as nitrogen lasers (337 nm) and frequency-tripled and quadrupled Nd:YAG lasers (355 nm and 266 nm respectively). • infrared lasers are used due to their softer mode of ionization. IR-MALDI also has the advantage of greater material removal (useful for biological samples), less low-mass interferences, and compatibility with other matrix-free laser desorption mass spectrometry methods.

  5. Ionization • The laser is fired at the crystals in the MALDI spot. The matrix absorbs the laser energy and it is thought that primarily the matrix is ionized by this event. • The matrix is then thought to transfer part of its charge to the analyte molecules (e.g. protein), thus ionizing them while still protecting them from the disruptive energy of the laser • Ions observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecular ion, for example [M+H]+

  6. Mass spectrometer (TOF) • The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range. • The TOF measurement procedure is also ideally suited to the MALDI ionization process since the pulsed laser takes individual 'shots' rather than working in continuous operation.

  7. MALDI-TOF instruments are typically equipped with an "ion mirror", deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution. • resolving power m/Δm of well above 20'000 FWHM (full-width half-maximum, Δm defined as the peak width at 50% of peak height).

  8. Capability • determine the molar mass distribution.[29] Polymers with polydispersity greater than 1.2 are difficult to characterize with MALDI due to the signal intensity discrimination against higher mass oligomers. • allow chemists to analyze the results of as catenanes and rotaxanes, dendrimers and hyperbranched polymers

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