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, 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 us ...

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 (2D gels) or isolated

SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...

bands. Additional analyses by

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 residu ...

was used in protein analysis, and it required almost an hour to analyze one amino acid residue.

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

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 d ...

, chymotrypsin 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 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 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

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 pipetted onto a MALDI target and a chemical called a matrix 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 2,3-Dihydroxybenzoic acid is a natural phenol found in ''Phyllanthus acidus'' and in the aquatic fern ''Salvinia molesta''. It is also abundant in the fruits of '' Flacourtia inermis''. It is a dihydroxybenzoic acid, a type of organic compound. T ...

. 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 elect ...

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.

References

External links