Database post-translational modification

Demirev, P. A. Lin, J. S. Pineda, F. J. Fenselau, C. Bioinformatics and mass spectrometry for microorganism identification Proteome-wide post-translational modifications and database search algorithms for characterization of intact H. Pylori. Anal. Chem. 2001, 73, 4566 573. 

Database Interrogation (Profound, Sequest) Protein Identification Post-translational Modification Characterization... 

SWISS-PROT (Bairoch and Apweiler, 2000) is a protein sequence database that, from its inception in 1986, was produced collaboratively by the Department of Medical Biochemistry at the University of Geneva and the EMBL. The database is now maintained collaboratively by Swiss Institute of Bioinformatics (SIB) and EBI/EMBL. SWISS-PROT provides high-level annotations, including descriptions of the function of the protein and of the structure of its domains, its post-translational modifications, its variants, and so on. The database can be accessed from http //expasy.hcuge.ch/sprot/sprot-top.html or numerous mirror sites. In 1966, Translated EMBL (TrEMBL) was created as a computer-annotated supplement to SWISS-PROT (Bleasby et al, 1994). 

EMBL Nucleotide Sequence Database. SWISS-PROT consists of core sequence data with minimal redundancy, citation and extensive annotations including protein function, post-translational modifications, domain sites, protein structural information, diseases associated with protein deficiencies and variants. SWISS-PROT and TrEMBL are available at EBI site, http //www.ebi.ac.uk/swissprot/, and ExPASy site, http //www.expasy.ch/sprot/. From the SWISS-PROT and TrEMBL page of ExPASy site, click Full text search (under Access to SWISS-PROT and TrEMBL) to open the search page (Figure 11.3). Enter the keyword string (use Boolean expression if required), check SWISS-PROT box, and click the Submit button. Select the desired entry from the returned list to view the annotated sequence data in Swiss-Prot format. An output in the fasta format can be requested. Links to BLAST, feature table, some ExPASy proteomic tools (e.g., Compute pI/Mw, ProtParam, ProfileScan, ProtScale, PeptideMass, ScanProsite), and structure (SWISS-MODEL) are provided on the page. 

Target Information database - The Database contains information such as Protein-Protein interactions, Metabolic and Signaling Pathways, Transcription factors, post-Translational modifications, Disease information, various Drugs and Clinical compounds and the Companies of interest. 

Proteins and peptides are linear polymers made up of combinations of the 20 most common amino acids linked with each other by peptide bonds. Moreover, the protein produced by the ribosome may undergo covalent modifications, called post-translational modifications, after its incorporation of amino acids. Over 200 such modifications have been detected already [13,14], the most important being glycosylation, the formation of disulfide bridges, phosphorylation, sulfation, hydroxylation, carboxylation and acetylation of the N-terminal acid [15]. The most frequent are listed in Table 8.1 and a more comprehensive database of mass changes due to post-translational modifications of peptides and proteins is available on the Internet [16]. 

Accurate determination of the molecular mass of a protein is useful for its identification and the determination of its purity. Owing to the possibility of post-translational modifications that are not contained in the database, the correct mass often is not sufficient for identification of a protein but it does facilitate the search. 

In proteomics and biomarker discovery, complex mass spectra from single protems, protein mixtures, or protein digests are obtained. Data systems exist that aid in characterization of spectral data to identify such properties as intact protein mass, amino acid subsequences, and post-translational modifications. Fragmentation information can also be compared with peptide databases to identify structural mutations that may be present. 

Once the spots of interest in a 2-DE gel are selected, the next step is to identify the corresponding proteins in a database. This is another challenge for bioinformatics to design tools adapted to match experimental data with those in sequence databases. Even if the amino acid sequence of a protein can be predicted with a reasonable degree of confidence, post-translational protein modifications cannot always be predicted from the DNA sequence and their presence or absence can be of paramount importance for the final structure, as well as for the function or dysfunction of a protein. Powerful protein identification tools therefore have to take into account information about known post-translational modifications wherever possible. 

Increasing reproducibility of available separation techniques and sensitivity and affordability of mass spectrometers, as well as the desire and need to automate the identification process, have caused peptide mass fingerprinting and MS/MS sequencing to gain importance and to become the method of choice for many proteomics laboratories. Several tools are available to assist users in the interpretation of mass spectrometry data. Peptldent (http //www.expasy.org/tools/peptident.html) on the ExPASy server follows the concept of the other tools from the ExPASy proteomics suite, in that it takes into account annotation available in the SWISS-PROT/TrEMBL database, in particular as post-translational modifications and processing are concerned. The user can paste peptide masses (monoisotopic or average) into the Peptldent form, but peptide mass data can also be uploaded from a file on the user s local computer. Supported file formats are .pkm ... 

The science community has clearly established the essential role which protein and DNA sequences play in the understanding of biological systems. The sequences themselves are informative Indeed, many software tools are available which allow to make sense of the primary sequence information. Take those that analyse protein sequences for domains and active sites, perform similarity and homology searches, or predict the three-dimensional structure or physico-chemical parameters. However, raw sequences contain insufficient information, per se. One cannot infer any description or understanding of the level of expression of the active proteins, the content of post-translational modifications (PTMs), the tertiary structure and, what is perhaps the most relevant information, a protein s function. Like the sequences themselves, all these added value data need to be captured in various databases. These databases have to be queried by different types of users in proteomics and should therefore be easily searchable by software tools and be inter-linked in order to document the correspondences between the type of information provided by the different databases. 

Moreover, even if all proteins are identified, there is the need to characterize them further. The importance of post-translational modifications in biological activity is proven, as well as that of amino-acid substitutions. These properties are often difficult to detect automatically, as they are often missing information in databases. They are, however, of potentially huge importance for the understanding of biological activity and of the presence of a diseased state. 

Tliis method enables the rapid identification of DNA-binding proteins. Immobifized DNA probes haiboring a specific sequence motif ai e incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by MALDI-TOF. Tlie determined mo-leculai masses aie often sufficient for identification. If not, the proteins ai e subject to MS peptide mapping followed by database seaiches. Apai t from protein identification, the protocol also yields information on post-translational modifications. Tlie protocol was validated by the identification of known prokaiyotic and eukai yotic DNA-binding proteins, and is use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA. ... 

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