To use all functions of this page, please activate cookies in your browser.
With an accout for my.chemeurope.com you can always see everything at a glance – and you can configure your own website and individual newsletter.
- My watch list
- My saved searches
- My saved topics
- My newsletter
Peptide mass fingerprinting
Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification that was developed 1993 by several groups independently.     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. These masses are then in silico compared to either a database containing known protein sequences or even 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. Typical for the PMF based protein identification is the requirement for an isolated protein. Mixtures exceeding a number of 2-3 proteins typically require the additional use of MS/MS based protein identification to achieve sufficient specificity of identification (6). Therefore, the typical PMF samples are isolated proteins from Two-dimensional gel electrophoresis (2D gels) or isolated SDS-PAGE bands. Additional analyses by MS/MS can either be direct, e.g., MALDI-TOF/TOF analysis or downstream nanoLC-ESI-MS/MS analysis of gel spot eluates.
Additional recommended knowledge
Protein samples can be derived from SDS-PAGE and are then subject to some chemical modifications. Disulfide bridges in proteins are reduced and cysteine amino acids are carboxymethylated chemically or acrylamidated during the gel electrophoresis.
Then the proteins are cut into several fragments using proteolytic enzymes such as trypsin, chymotrypsin or Glu-C. 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 high sample throughput and several proteins can be analyzed in a single experiment - if complemented by MS/MS analysis.
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.
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 (see web resources and references in Hufnagel, 2006) 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.
Web resources for protein ID based on PMFs
Categories: Protein methods | Laboratory techniques | Mass spectrometry
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Peptide_mass_fingerprinting". A list of authors is available in Wikipedia.|