Protein identification, sequence database

Sequence Comparisons Proteins called molecular chaperones (described in Chapter 4) assist in the process of protein folding. One class of chaperone found in organisms from bacteria to mammals is heat shock protein 90 (Hsp90). All Hsp90 chaperones contain a 10 amino acid signature sequence, which allows for ready identification of these proteins in sequence databases. Two representations of this signature sequence are shown below. 

Liebler DC (2002) Introduction to Proteomics. Tools for the New Biology. Humana Press, Totowa, New Jersey Mann M, Hojrup P, Roepstorff P (1993) Use of mass spectro-metric molecular weight information to identify proteins in sequence databases. Biol Mass Spectrom 22 338-345 Rabilloud T (2000) Proteome Research two dimensional gel electrophoresis and identification methods. Springer Ver-lag, Berlin Heidelberg... 

The first resources for computer modeling of protein structure are the nucleic acid and protein sequence databases (see Table 6.1), curated by the European Molecular Biology Laboratory (EMBL) in Europe, the National Center for Biotechnology Information (GenBank at the NCBl) in the United States, and the DNA Database of Japan (DDBJ) in Japan. These databases are accessible via the Internet, and most likely one s own scientific institution maintains a local version, which is updated through CD-ROMs released quarterly. Perhaps the predominant protein sequence database is SWISS-PROT. - Others include the nonredundant protein sequence database (OWL) and the protein identification resource database (PIR). ... 

Various forms of tandem mass spectroscopy (MS/MS) have also been used in the analysis of biomolecules. Such instruments consist of an ionisation source (ESI or MALDI or other) attached to a first mass analyser followed by a gas-phase collision cell. This collison cell further fragments the selected ions and feeds these ions to a second mass detector. The final mass spectrum represents a ladder of fragment ions. In the case of peptides the collision cell usually cleaves the peptides at the amide bond. The ladder of resulting peptides reveals the sequence directly [496]. Thus, tandem MS instruments, such as the triple quadrupole and ion-trap instruments have been routinely applied in LC-MS/MS or ESI-MS/MS for peptide sequencing and protein identification via database searching. New configurations, which have been moving into this area include the hybrid Q-TOF [498], the MALDI-TOF-TOF [499] and the Fourier transform ion cyclotron resonance instruments [500]. 

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. 

The PepSeq program of Micromass s ProteinLynx software package was used for de novo analysis of the sequence (MS/MS) data. MS-Pattern of ProteinProspectror55 used the sequence tag determined from PepSeq to search the nonredundant database of the National Center for Biotechnology Information (NCBI) for protein identification. 

Perkins, D. N., Pappin, D. J. C., Creasy, D. M., Cottrell, J. S. (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18), 3551-3567. 

D. N. Perkins, et al., Probability-Based Protein Identification by Searching Sequence Databases Using Mass Spectrometry Data. Electrophoresis, 20, no. 18 (1999) 3551-3567. 

Fig. 7. Protein identification with electrospray tandem mass spectrometry and a triple quadrupole mass spectrometer. Fragment spectra of several peptides are generated during one investigation. From the fragment spectra short sequence stretches can be read. Together with their mass location in the peptide of the measured mass, they can be used to specifically identify a protein in the database. Because the protein identification depends only on one peptide, several proteins can be identified from one sample.

Selected entries from Methods in Enzymology [vol, page(s)] General Protein kinase classification, 200, 3 protein kinase catalytic domain sequence database identification of conserved features of primary structure and classification of family members,... 

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... 

The basic identification process is analysis of the sequence or mass of six amino acids unique in the proteome of an organism, then to match it in a database. Put another way, protein identification is achieved converting proteins to peptides, determining the sequence of peptides, and then, matching with corresponding proteins from matching sequences in a database (Figure 5.3). 

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