Protein identification tools

The high resolving power of FT-ICR-MS can readily be exploited in bottom-up protein identification. A nice example is the identification of high-abundant proteins in a tryptic digest of human plasma without any prior separation. The 2745 peaks in the spectmm could be reduced to 1165 isotopic clusters and 669 unique masses, 82 of which matched tryptic fragments of albumin (93% sequence coverage) and 16 others transferrin (41%) [39]. The same group showed that a theoretically predicted retention time of a tryptic peptide can be applied as an additional protein identification tool, next to its accurate mass acquired in LC-FT-ICR-MS. [40-41]... 

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. 

TABLE 16.15 Resource sites with MS-based protein identification tools... 

The obtained peak list together with other data (biological species, possible posttransla-tional modifications of amino acids, etc.) is then submitted to a software tool (usually publicly available) and searched against a certain protein database, which leads to protein identification. The majority of available software tools also offer information on the statistical probability of protein identification. 

ExPASy Proteomics tools (http //expasy.org/tools/), tools and online programs for protein identification and characterization, similarity searches, pattern and profile searches, posttranslational modification prediction, topology prediction, primary structure analysis, or secondary and tertiary structure prediction. 

Sotriffer C, Klebe G. 2002. Identification and mapping of smaU-molecule binding sites in proteins computational tools for structure-based drug design. Farmaco 57(3) 243-251. 

Bioinformatics tools involving computer-based statistical analyses are essential for data management and analysis. When a complex biological sample containing thousands of different proteins is analyzed by multifaceted approaches, such as multidimensional protein identification technology, the identification of the proteins in the mixture is extremely complicated. Even multiple peptide identification methods, such as using both MS and... 

Developments in mass spectrometry technology, together with the availability of extensive DNA and protein sequence databases and software tools for data mining, has made possible rapid and sensitive mass spectrometry-based procedures for protein identification. Two basic types of mass spectrometers are commonly used for this purpose Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and electrospray ionization (ESI)-MS. MALDI-TOF instruments are now quite common in biochemistry laboratories and are very simple to use, requiring no special training. ESI instruments, usually coupled to capillary/nanoLC systems, are more complex and require expert operators. We will therefore focus on the use of MALDI-... 

Sechi, S. and Chait, B. T. (1998) Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal. Chem. 70,5150-5158. 

Blackburn, R. K. Moseley, M. A. Ill 1999. Quadrupole time-of-flight mass spectrometry a powerful new tool for protein identification and characterization. Am. Pharm. Rev., 2, 49-59. 

Despite its clever utility, SELDI suffers from several limitations. The SELDI MS instrumentation usually is capable of accurately detecting proteins with molecular weights less than 45,000, the detected proteins cannot be identified using this technique alone, and reproducibility in complicated experiments is suspect (86). Improvements in next-generation instruments using ProteinChip tandem MS techniques that enable direct protein identification (87), improved surface chemistries (88), and improved experimental design (89,90) should all greatly enhance SELDI s effectiveness as a powerful proteomic tool (91,92). 

The comprehensive molecular biology server, ExPASy (Expert Protein Analysis System), provides Proteomic tools (http //www.expasy.ch/tools) that can be accessed directly or from the ExPASy home page by selecting Identification and Characterization under Tools and Software Packages. The Protein identification and characterization tools of the Proteomics tools (Figure 11.4) provide facilities to ... 

Figure 11,4. ExPASy Proteomic tools. ExPASy server provides various tools for proteomic analysis which can be accessed from ExPASy Proteomic tools. These tools (locals or hyperlinks) include Protein identification and characterization, Translation from DNA sequences to protein sequences. Similarity searches, Pattern and profile searches, Post-translational modification prediction, Primary structure analysis, Secondary structure prediction, Tertiary structure inference, Transmembrane region detection, and Sequence alignment.

Identifying altered canine and bovine proteins has proved to be particularly challenging since these species are poorly represented in current genomic databases. As a result of this, new bioinformatic tools (Multildent, http //expasy.org) have had to be developed to facilitate cross-species protein identification (Wilkins et al., 1998). The most significant change observed for bovine DCM was a sevenfold increase in the enzyme ubiquitin carboxyl-terminal hydrolase (UCH) (Weekes... 

Hochstrasser, D. F. (1998). Multiple parameter cross-spedes protein identification using Multildent - a world-wide web accessible tool. Electrophoresis 19, 3199-3206. 

Tandem MS (MS/MS) has proven a particularly powerful tool, comparing fragmentation patterns of MS/MS spectra with established databases, enabling protein identification. 

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