Peptide mass databases

Routinely use peptide mass database algorithms for protein identification 39 26%... 

Because of the advances in the gas-phase ionization of biomacromolecules, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) has become a powerful tool for detection, identification, and structural analysis of proteins, peptides, and polynucleotides. The molecules ionized in a gas phase by these methods are subsequently analyzed by sector, quadrupole, ion-trap, or time-of-flight mass spectrometers. In particular, the MS systems consisting of ESI and triple-stage quadrupole (ESI/TSQ) or ion-trap (IT) mass spectrometry and MALDI time-of-flight (MALDl/TOF) mass spectrometry have been most widely applied to the field of protein chemistry for the accurate determination of molecular mass of proteins and peptides, determination of amino acid sequence, identification of proteins by peptide mass databases, and analysis of posttranslational modifications such as phosphorylation and glycosylation. In general, current techniques allow detenni-... 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

Computer algorithms facilitate identification of the open reading frames that encode a given protein by using partial sequences and peptide mass profiling to search sequence databases. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

This procedure is called Peptide Mass Mapping (PMM), which is defined as a means of protein identification by comparing observed masses (m/z values) with predicted masses of digested proteins contained in a database. 

Finally, knowledge of the peptide masses that resulted from the PIR conjugation provides information to identify the parent proteins from which they originated. Peptide mass and sequence databases now are sufficiently developed to provide rapid confirmation of protein-protein interaction partners. 

ProteinProspector (http //prospector.ucsf.edu), database search tools, searching by peptide mass, with MS/MS or Edman data, etc. 

Proteins fall into three classes when characterized with mass spectrometry proteins whose complete sequence is given in a database, proteins whose sequence is partially represented in expressed sequence tag databases, and proteins whose sequence is unknown. Proteins of the first class can be identified by peptide mass mapping in a very high through-... 

Even though the MALDI peptide mass mapping technique is very powerful, it has limitations. It requires well-separated proteins, is less sensitive than identifications based on electrospray tandem mass spectrometry, can only identify proteins whose complete sequences are available in databases, and does not produce redundant information. 

If a protein mixture was digested, the database query may give an ambiguous result because there is no single protein that produces, on digestion, the measured set of peptides. Sometimes this can be compensated for by sequentially subtracting the peptide masses of the first identified protein from the mass set and rerunning the database search (Jensen et al., 1997) but if one protein was less abundant, it is likely to be missed completely in the database search. 

The second approach is the tandem mass spectrometric method (Wilm et ah, 1996 Link et ah, 1999 Yates, 2000). This method relies on fragmentation of individual peptides in the tryptic peptide mixture to gain sequence information. Its main advantage is that sequence information derived from several peptides is much more specific for the identification of a protein than a list of peptide masses. The sequence data can be used to search not only protein sequence databases but also nucleotide databases such as expressed sequence tag (EST) databases and, more recently, even... 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Keywords Proteomics MALDI-TOF mass spectrometry SDS-PAGE 2D-gel in-gel digestion peptide mass fingerprint protein identification database searching. 

The peptide tolerance is a window around each mass value in the peak list within which a theoretical database peptide mass must fall, in order to be matched. 

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