In the late nineties of the 20Th century Ciphergen developed The Surface Enhanced Laser Desorption/Ionisation Time of Flight (SELDI-TOF) mass spectrometry platform. Prinicipally the system include the three main elements for proteomics; e.g. a separation module, an ionization device and a mass spectrometer.
Separation
Figure 2: Two proteinChip arrays with 8 spots.
The most sophisticated and powerful tool of the SELDI-TOF platform is the separation module. The SELDI-TOF platform uses “ProteinChips” with affinity chromatographic properties. Different ProteinChip arrays (one array contains 8 or 16 spots) are available with different chromatographic properties. The choice of the ProteinChip depends on the type of experiment. Two different kinds of chips are available for use in practice. The chemically loaded ProteinChip contain hydrophobic, hydrophilic, anion exchange, cation exchange, anion exchange and immobilized –metal affinity surfaces. The pre-activated ProteinChip arrays can be used to covalently link ‘bait’ molecules.
Crude biological samples can be applied directly to the ProteinChips arrays using the bioprocessor. After a short incubation period, unbound and aspecific proteins are washed off by using different wash procedures. Finally, the ProteinChip arrays are prepared for reading by the incubation with the energy absorbing molecules.
Figure 3: Bioprocessor for liquid handling procedures. The arrays are put into the processor (in this examples lane 1 and 2) and processing occurs via a 96 well format.
Ionization and Time of Flight detection
Ionization immediately starts when a laser desorbs the bound molecules from the array surface. The ionized molecules will be accelerated through a high voltage electric field and detected by a time of flight analyzer. Time of flight analysis is based on acceleration of ionized molecules to a detector. Because the ionized molecules have the same energy the time of flight is directed related to the mass of the molecules. The smaller the molecules the greater the velocity of the molecules. Altogether the time of flight depends on the mass, charge and kinetic energy of the ionized molecule.
Figure 4 (click to enlarge): Principle of Surface Enhanced Laser Desorption Ionization and Time of Flight Mass Spectroscopy: SELDI-TOF-MS. 4A: Protein extracts are bound to various chemical surfaces (cationic, anionic, hydrophobic, metal ion etc, provided in an array format by the manufacturer) under varying pH and salt strength conditions. 4B: Proteins are eluted from the different surfaces using laser desorption and ionization and the molecular mass of the released protein is measured by time of flight (TOF) mass spectroscopy. Results can visualised in various ways, including a "false image" gel view.