http://www.tau.ac.il/lifesci/units/proteomics/voyager.html

Matrix assisted laser desorption ionization mass spectrometry is carried out on an AppliedBiosystems Voyager DE-STR instrument equipped with delayed extraction and reflector. A total sample volume of up to 2 µl is loaded onto MALDI sample plate in volatile solvents. In reflector mode, masses of peptides (low molecular weight, 750-4,500) can be determined on low fmol quantities with an average mass accuracy lower than 10 ppm. Under optimum conditions, the limit of sensitivity of tryptic peptides (below 4,000) is in the high amol range. Masses can potentially be obtained on numerous biopolymers including oligosaccharides, nucleotides and proteins that range from ~600 to 750,000 Daltons.

Some MALDI-TOF theory

This MS approach uses a nitrogen UV laser (337 nm) to generate ions from high mass, non-volatile samples such as peptides and proteins. The key to this technique is that in the presence of an aromatic matrix large molecules like peptides ionize instead of decomposing. Although the mechanism remains uncertain, it may involve absorption of UV light by the matrix followed by transfer of this energy to the peptide - which then ionizes into the gas phase as a result of the relatively large amount of energy absorbed. To accelerate the resulting ions into a flight-tube in the mass spectrometer they are subjected to a high electrical field.

Three different models have been proposed to explain desorption of the matrix-sample material from the crystal surface: (1) quasithermal evaporation as a result of increased molecular motion, (2) expulsion of upper lattice layers, and (3) an increase in the hydrodynamic pressure due to the rapidly expanding molecules in the crystal lattice. However, there is no consensus yet as to how the sample molecules are ionized. The widely accepted view is that, following there desorption as neutrals, the sample molecules are ionized by acid-base proton transfer reactions with the protonated matrix ions in a dense phase just above the surface of the matrix. The protonated matrix molecules are generated by a series of photochemical reactions.

The matrix performs two important functions: (1) it absorbs photon energy from the laser beam and transfers it into excitation energy of the solid system, and (2) it serves as a solvent for the analyte, so that the intermolecular forces are reduced and aggregation of the analyte molecules is held to a minimum. Some desirable characteristics of a typical MALDI matrix are:

• A strong light absorption property at the wavelength of the laser flux.
• The ability to form micro-crystals with the sample.
• A low sublimation temperature, which facilitates the formation of an instantaneous high-pressure plume of matrix-sample material during the laser pulse duration.
• The participation in some kind of a photochemical reaction so that the sample molecules can be ionized with high yields.


Several matrix-laser combinations have been tested successfully. For peptides and small molecular mass proteins (<10,000 Da), good results are obtained with a-cyno-4-hydroxycinnamic acid (CHCA), whereas high-mass proteins are analyzed with sinapinic acid. The use of 3-amino-4 hydroxybenzoic acid and 2,5-dihydroxybenzoic acid (DHB) has been recommended for the analysis of oligosaccharides.


Our MALDI instrument can be used in either linear or reflector mode. In linear mode the ions travel down a linear flight path and their mass/charge (m/z) ratio (see below for an explanation of the difference between mass and mass/charge ratio) is determined by the time it takes for them to reach the detector. Hence, this instrument is called a time of flight (TOF) instrument. The relationship that allows the m/z ratio to be determined is E = ½ (m/z)v². In this equation, E is the energy imparted on the charged ions as a result of the voltage that is applied by the instrument and v is the velocity of the ions down the flight path. Because all of the ions are exposed to the same electric field, all similarly charged ions will have similar energies. Therefore, based on the above equation, ions that have larger mass must have lower velocities and hence will require longer times to reach the detector, thus forming the basis for m/z determination by a mass spectrometer equipped with a time of flight detector.


A reflector MALDI has an ion mirror at its end which reflects the ions back (at a slight angle) to a detector. Reflector mode have several major advantages: (1) it permits limited mass spectrometric sequencing to be carried out via a process called post source decay (PSD); (2) it permits higher mass accuracy.


During high voltage extraction of the peptide ions produced by exposure to UV light, there are slight differences in the amount of energy that is actually acquired by similarly charged ions. In a linear instrument these differences result in slight differences in times of flight which results in broader peaks and lower mass accuracy. In terms of resolving fragment ions, a reflector also compensates for similarly charged ions having slightly different overall energies (the more energetic ions that have slightly faster velocities will penetrate further into the ion mirror and hence be slightly delayed relative to less energetic ions - thus both will tend to reach the detector at the same time). As a result, the reflector improves both resolution and mass accuracy. Although there is always the possibility of observing fragmentation ions when using the reflector (and mistaking these for contaminating peptide ions), by adjusting the settings on the instrument it is possible to minimize the possibility of seeing peptide fragmentation in the reflector mode.

 

Relevant literature

• Chapman, J. R., Mass Spectrometry of Proteins and Peptides, 2001,Humana Press
• Dass, C., Principles and practice of biological mass spectrometry, 2001, John Wiley & Sons
• James, P., Proteome research: mass spectrometry, 2001, Springer
• Kellner, R., F. Lottspeich, and H. E. Meyer, Microcharacterization of Proteins, 2nd Ed, 1999, Wiley-VCH.
• Kinter, M., and N. E. Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry, 2000, Wiley Interscience
• Siuzdak, G., Mass Spectrometry for Biotechnology, 1996, Academic Press