Peptide mass fingerprinting
Abstract
Peptide mass fingerpring (PMF) is an analytical technique for protein identification. A key feature for PMF is that the protein sequence of the protein has to be known and available. In short, the unknown protein of interest is cut to peptides by an enzyme such as Trypsin, V8, Chymotrypsin and others to create peptides. The absolute mass of these (still unknown) peptides is accurately measured with a mass spectrometer such as MALDI-TOF, ESI-TOF and MALDI-TOF-TOF. These masses, from peptides coming from one protein, are then in silico compared to the genome. Software programs translate the known genome of the organism into proteins and then theoretically cut all these proteins into peptides with the same enzyme as used (for example trypsin). The absolute mass of all these peptides is then theoretically calculated. A comparison is made between the mass list of your measured peptide masses and all the masses from the calculated peptides. The results are statistically analyzed and eventually you get a positive identification of your protein of interest. The great advantage is that only the masses of the peptides have to be known (so no de novo sequencing); a disadvantage is that the genome of your organism has to be published. Additionally most PMF algorithms assume the peptides come from a single protein. The presence of a mixture can significantly complicate the analysis and potentially compromise the results.
Sample preparation
Protein samples can be derived from SDS-PAGE (1) and are then subject to some chemical modifications. Disulfidbridges in proteins are reduced and cystein aminoacids are carboxymethylated prior proteolytic cleavage. The protein sample is cut into several fragments using proteolytic enzymes such trypsin, chymotrypsin or V8 protease. Usually is the proteolysis carried out with a minor amount of enzyme and a sample to protease ratio of 50:1 is used. The proteolysis is typically carried out over night and the resulting peptides are extracted with acetonitrile and tried 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 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 entered the gas phase they become accelerated in the electric field of the mass spectrometer and fly towards an ion detector were there 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 weight 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 as used (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 result table.
- Peak list:
676.3718 825.4571 1017.5370 1026.5964 1040.6121 1105.4550 1196.7132 1218.5796 1243.5887 1255.6538 1260.5928 1266.6281 1276.5836 1288.6094 1305.6310 1388.6975 1398.6805 1413.7126 1420.6741 1482.8101 1576.8138 1932.7067 2188.0850
Protein Prospector Peptide Search Engine
Interpreting results
Literature
- 1) Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14440-5.
- 2) Clauser K. R., Baker P. R. and Burlingame A. L., Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Analytical Chemistry, Vol. 71, 14, 2871- (1999)