The application of Peptide Mass Fingerprinting in Protein Identification Process

Posted by sherrycryer on September 4th, 2018

Peptide Mass Fingerprinting (PMF) is an essential technique for rapid identification of proteins in which allows the mass of an unknown protein to be identified. More companies are committed to developing comprehensive protein identification pipeline which is typically accomplished by Peptide Mass Fingerprinting service.

A pure protein is digested using a proteolytic enzyme (often trypsin) to cleave the protein (a single protein or a complex mixture of different proteins) into constituent peptides. The peptide masses are then measured very accurately by mass spectrometry and searched against a theoretical digest of all protein maintained in a database. In most cases, one or more stages of chromatography are applied to regulate the flow of peptides into the mass spectrometer. Statistical scoring algorithms match the measured set of unique peptides against the theoretical set of unique peptides, and an identification is achieved.

Peptide Mass Fingerprinting (PMF) grew from a need for a faster, more efficient method to identify proteins. At present, PME is employed by the majority of proteomic studies. According to the articles published in Proteomics (August 2005 to July 2007) and Proteome Science (January 2003 to September 2007). Out of the 581 articles surveyed, approximately 35% of the studies only used PMF-based protein identification, 32% of the studies only utilized MS/MS, and 33% of the studies used both PMF and MS/MS for protein identification. Thus, 68% of the studies utilized PMF for protein identification. Therefore, what deserves more investigation is the advantages of the PMF method. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large amount protein and when high-throughput MS/MS instrumentation is not readily available. Besides, in comparison to MS/MS analysis on a single peptide, identification of proteins using PMF has two significant advantages that are often ignored. Firstly, in PMF, unsuspected post-translational modifications lead to only a marginal loss in the quality of data and do not affect the outcome. In contrast, in MS/MS, unspecified post-translational modifications can adversely affect the matching and scoring process, thus precluding unambiguous protein identification. Secondly, although MS/MS analysis can be used to determine the amino acid sequence of a peptide, that peptide may be unique to a specific protein or common to several different proteins (for example, enzymes from the same family). Conversely, the use of multiple peptides for protein identification in PMF allows more extensive coverage of the protein, thereby increasing the confidence in a positive identification. Thus, in some cases, the MS/MS analysis on a single peptide may be less specific than PMF.

In addition, the results from other experiments also demonstrated that peptide mass fingerprinting was a rapid method that was useful in identifying known proteins as there is no requirement for a free amino terminus. Because a protein frequently can be identified from a subset of the peptide masses, the correct protein may be identified even if some of the peptides contain post-translational modifications, or if the sequence database entry contains errors. Mass differences between the measured and predicted peptides may provide evidence for the identity of peptide modifications. The individual proteins in a mixture of up to three or four components can be identified. The method is easily automated for high throughput operation, and robotic workstations are now available for all the steps as with modern instrumentation able to measure peptide mixtures at the sub-femtomole level.

With the advancement in Peptide Mass Fingerprinting (PMF) technology, protein identification is no more complex and time-consuming. Recently, it is widely used in various scientific areas, for example, PMF can be used in cultural objects and identify whale species. Besides, scientists demonstrate that Protein identification help with Cancer Diagnostics.

Researchers are still working on providing a comprehensive protein identification by applying Peptide mass fingerprinting in which help shorten the time of protein identification and enhance the accuracy of protein sequencing.