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Peptide mass fingerprinting (PMF) is an analytical technique for protein identification that was developed by John Yates and colleagues (3). In short, the unknown protein of interest is cleaved into peptides by a protease such as Trypsin. The collection of peptides resulting from this cleavage comprise a unique identifier of the unknown protein. The absolute masses of the (still unknown) peptides are accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. In a process called de novo sequencing, peptide sequences are derived from the masses of their fragments as shown on a tandem mass spectrum. When performing de novo sequencing, no protein sequence database is used for reference.www.bioinform.com These masses are then in silico compared to the genome. Computer programs translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides with the same protease (for example trypsin), and calculate the absolute masses of the peptides from each protein. They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. The results are statistically analyzed to find the best match.
The great advantage is that only the masses of the peptides have to be known (so de novo sequencing is not necessary). A disadvantage is that the protein sequence has to be present in the database of interest. 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. Disulfide bridges in proteins are reduced and cysteine amino acids are carboxymethylated.
Then the proteins are cut into several fragments using proteolytic enzymes such trypsin, chymotrypsin or V8 protease. A typical sample:protease ratio is 50:1. The proteolysis is typically carried out overnight and the resulting peptides are extracted with acetonitrile and dried under vacuum. The peptides are then dissolved in a small amount of distilled water and are ready for 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 a 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 of matrix molecules and peptides from the solid state into the gas state. Then the molecules 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. Their mass is proportional to their time of flight (TOF) in the drift tube and can be calculated accordingly.
Computational analysis
The mass spectrometrical analysis produces 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
- Bin Ma, 2003. University of Waterloo Bioinformatics Papers: PEAKS: Powerful Software for Peptide De Novo Sequencing by MS/MS.
- Clauser KR, Baker PR, Burlingame AL. Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal Chem. 1999 Jul 15;71(14):2871-82.
- Griffin PR, MacCoss MJ, Eng JK, Blevins RA, Aaronson JS, Yates JR 3rd. Direct database searching with MALDI-PSD spectra of peptides. Rapid Commun Mass Spectrom. 1995;9(15):1546-51.
- Mohd Nazri Ismail. 2005. Doping Control Centre. University Science Malaysia.
- 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.
External links
- PMF Tutorial
- Mascot Peptide Fingerprint Search Engine
- PEAKS de novo sequencing and protein ID
- Protein Prospector Peptide Search Engine
Protein methods | biochemistry | Laboratory techniques | mass spectrometry
"Peptide mass fingerprinting"