| Peptide Mass Fingerprinting |
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SAMPLE PREPARATION Protein samples can be derived from and dried under vacuum. The peptides are then dissolved in a small amount of distilled water and are ready for the mass spectrometric analysis. MASS SPECTROMETRIC ANALYSIS The digested protein can be analyzed with different types of mass spectrometers such as ESI-TOF or MALDI-TOF . MALDI-TOF is often the preferred instrument because it allows a higher sample throughput and several proteins can be analyzed in a single experiment. A small fraction of the peptide (usually 1 microliter or less) is pipetted onto a MALDI target and a chemical called Matrix is added to the peptide mix. The matrix molecules are required for the desorption of the peptide molecules. Matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed. The target is inserted into the vacuum chamber of the mass spectrometer and the analysis of peptide masses is initiated by a pulsed laser beam which transfers high amounts of energy into the matrix molecules. The energy transfer is sufficient to promote the transition from the solid state into the gas state of matrix molecules and peptides. Once the peptides have entered the gas phase they become accelerated in the electric field of the mass spectrometer and fly towards an ion detector where their arrival is detected as an electric signal. The peptide mass is proportional to the Time Of Flight (TOF) in the drift tube and can be calculated accordingly. COMPUTATIONAL ANALYSIS The result of the mass spectrometrical analysis is a list of molecular weights which is often called peak list. The peptide masses are now compared to huge databases such as Swissprot, Genbank which contain protein sequence information. Software programs (2) cut all these proteins into peptides with the same enzyme used in the chemical cleavage (for example trypsin). The absolute mass of all these peptides is then theoretically calculated. A comparison is made between the peak list of measured peptide masses and all the masses from the calculated peptides. The results are statistically analyzed and possible matches are returned in a results table. LITERATURE
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