![Peptide mass fig](https://upload.wikimedia.org/wikipedia/commons/c/c6/Peptide_mass_fig.jpg)
Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
identification in which the unknown protein of interest is first cleaved into smaller
peptides
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
A p ...
, whose absolute masses can be accurately measured with a
mass spectrometer
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used ...
such as
MALDI-TOF
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of ...
or
ESI-TOF.
The method was developed in 1993 by several groups independently.
The peptide masses are compared to either a database containing known protein sequences or even the genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, 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 advantage of this method is that only the masses of the peptides have to be known. Time-consuming
de novo peptide sequencing In mass spectrometry, de novo peptide sequencing is the method in which a peptide amino acid sequence is determined from tandem mass spectrometry.
Knowing the amino acid sequence of peptides from a protein digest is essential for studying the biolo ...
is then unnecessary. A disadvantage is that the protein sequence has to be present in the database of interest. Additionally most PMF algorithms assume that 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
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
based protein identification to achieve sufficient specificity of identification (6). Therefore, the typical PMF samples are isolated proteins from
two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first ...
(2D gels) or isolated
SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. T ...
bands. Additional analyses by
MS/MS
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
can either be direct, e.g., MALDI-TOF/TOF analysis or downstream nanoLC-ESI-MS/MS analysis of gel spot eluates.
Origins
Due to the long, tedious process of analyzing proteins, peptide mass fingerprinting was developed.
Edman degradation
Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid resi ...
was used in protein analysis, and it required almost an hour to analyze one amino acid residue.
SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. T ...
was also used to separate proteins in very complex mixtures, which also employed methods of electroblotting and staining. Then, bands would be extracted from the gel and sequenced, automatically. A recurring problem in the process was that interfering proteins would also purify with the protein of interest. The sequences of these interfering proteins were compiled into what came to known as the Dayhoff database. Ultimately, having the sequences of these known protein contaminants in databases decreased instrument time and expenses involved in protein analysis.
Sample preparation
Protein samples can be derived from
SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. T ...
or
reversed phase HPLC, and are then subject to some chemical modifications. Disulfide bridges in proteins are reduced and cysteine amino acids are carbamidomethylated chemically or acrylamidated during the gel electrophoresis.
Then the proteins are cut into several fragments using proteolytic enzymes such as
trypsin
Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the dig ...
,
chymotrypsin
Chymotrypsin (, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenu ...
or
Glu-C. A typical sample:protease
ratio
In mathematics, a ratio shows how many times one number contains another. For example, if there are eight oranges and six lemons in a bowl of fruit, then the ratio of oranges to lemons is eight to six (that is, 8:6, which is equivalent to the ...
is 50:1. The proteolysis is typically carried out overnight and the resulting peptides are extracted with
acetonitrile
Acetonitrile, often abbreviated MeCN (methyl cyanide), is the chemical compound with the formula and structure . This colourless liquid is the simplest organic nitrile (hydrogen cyanide is a simpler nitrile, but the cyanide anion is not clas ...
and dried under vacuum. The peptides are then dissolved in a small amount of distilled water or further concentrated and purified 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
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of ...
. 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
Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A comm ...
analysis. LC/ESI-MS and CE/ESI-MS are also great techniques for peptide mass fingerprinting.
A small fraction of the peptide (usually 1 microliter or less) is
pipette
A pipette (sometimes spelled as pipett) is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured volume of liquid, often as a media dispenser. Pipettes come in several designs for various purposes with diffe ...
d onto a MALDI target and a chemical called a
matrix
Matrix most commonly refers to:
* ''The Matrix'' (franchise), an American media franchise
** ''The Matrix'', a 1999 science-fiction action film
** "The Matrix", a fictional setting, a virtual reality environment, within ''The Matrix'' (franchis ...
is added to the peptide mix. Common matrices are
Sinapinic acid
Sinapinic acid, or sinapic acid (Sinapine - Origin: L. Sinapi, sinapis, mustard, Gr., cf. F. Sinapine.), is a small naturally occurring hydroxycinnamic acid. It is a member of the phenylpropanoid family. It is a commonly used matrix in MALDI mass ...
,
Alpha-Cyano-4-hydroxycinnamic acid, and
2,3-Dihydroxybenzoic acid. The matrix molecules are required for the
desorption
Desorption is the physical process where a previously adsorbed substance is released from a surface. This happens when a molecule gains enough energy to overcome the activation barrier of the bounding energy that keeps it in the surface.
There ...
of the peptide molecules. Matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed. There is one predominantly MALDI-MS sample preparation technique, namely dried droplet technique. The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide fragments 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 ionisation and transition of matrix molecules and peptides from the solid phase into the gas phase. The ions are 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-to-charge ratio is proportional to their
time of flight
Time of flight (ToF) is the measurement of the time taken by an object, particle or wave (be it acoustic, electromagnetic, etc.) to travel a distance through a medium. This information can then be used to measure velocity or path length, or as a w ...
(TOF) in the drift tube and can be calculated accordingly.
Coupling ESI with capillary LC can separate peptides from protein digests, while obtaining their molecular masses at the same time.
Capillary electrophoresis
Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electr ...
coupled with ESI-MS is another technique; however, it works best when analyzing small amounts of proteins.
Computational analysis
The mass spectrometric analysis produces a list of molecular weights of the fragments which is often called a peak list. The peptide masses are compared to protein databases such as
Swissprot, which contain protein sequence information. Software performs ''in silico'' digests on proteins in the database with the same enzyme (e.g. trypsin) used in the chemical cleavage reaction. The mass of these peptide fragments is then calculated and compared to the peak list of measured peptide masses. The results are statistically analyzed and possible matches are returned in a results table.
See also
*
Protein mass spectrometry
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrume ...
*
Shotgun proteomics Shotgun proteomics refers to the use of bottom-up proteomics techniques in identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spectrometry. The name is derived from shotgun seq ...
*
Top-down proteomics
Top-down proteomics is a method of protein identification that either uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry (MS/MS) analysis or other protein purification methods ...
*
Bottom-up proteomics
Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry.
The major alternative work ...
References
External links
USA Peptides
{{Protein methods
Protein methods
Laboratory techniques
Mass spectrometry