Basic Local Alignment Sequence Tool

A sequence tag from an unknown protein allows a number of further options for characterisation. Even a short stretch of amino acid sequence provides a powerful means of interrogating a protein database, and may provide a useful alternative to PMF in poor quality samples that may have peptides derived from more than one protein. More advanced database searching systems will find proteins with homologous sequences to that of the peptide tag. A good example of this type of database searching system is BLAST (Basic Local Alignment Sequence Tool), (Altschul et al., 1997). A modified form of this (MS BLAST) was used in conjunction with PMF to characterise the proteome of the yeast Pichia pastoris, whose genome has not been fully sequenced... 

The aim of the fust dimension breadth is to reveal sequence-function relationships by comparing protein sequences by sequence similarity. Simple bioinformatic algorithms can be used to compare a pair of related proteins or for sequence similarity searches e.g., BLAST (Basic Local Alignment Search Tool). Improved algorithms allow multiple alignments of larger number of proteins and extraction of consensus sequence pattern and sequence profiles or structural templates, which can be related to some functions, see e.g., under http //www. expasy.ch/tools/ similarity. 

Sequence similarity searching Basic Local Alignment Search Tool (BLAST) http //www.ncbi.nlm.nih.gov/BLAST/ Comparison of novel sequences with known genes. 

A sequence alignment is a way of determining the similarity between two strings. This is a classical question in computer science, and has an exact solution referred to as the Smith/Waterman alignment. Unfortunately, this exact solution can be slow when analyzing large sequences, and therefore, approximate methods, such as Basic Local Alignment Search Tool (BLAST), have been developed to identify very similar sequences. 

BLAST Basic Local Alignment Search Tool. One of the more widely used tools in genome informatics, for similarity searches of protein or DNA sequences. 

Therefore, and also to compare the results with existing information, one may still want to search the database with the results obtained by de novo sequencing. The approach to follow is a BLAST search (Basic Local Alignment Search Tool, [133-134]). Sequence alignment is a powerful way to compare novel sequences with previously characterized sequences and search for similarities. The BLAST search is much slower than the database searches outlined in Ch. 17.6.2. A nice example is the de novo identification of the bacterium Shewanella putrefaciens proteome by 2D-GE, in-gel digestion, direct infusion nano-ESl, and nano-LC-MS [135]. The results were BLAST searched against the incomplete genome of the bacterium. 

Fig. 2. Alignment of human proliferating cell nuclear antigen (PCNA) and Escherichia coli DNA polymerase III (3-subunit. The sequence alignment obtained in the fifth iteration of Position Specific Iterative Basic Local Alignment Search Tool using query human PCNA protein NP 002583.1 to the sbjct E. coli DNA polymerase III (3-subunit NP 418156.1 is displayed.

Fig. 3. Sequence alignment of an uncharacterized protein to phosphinothricin N-acetyltransferase. The sequence alignment obtained in the third iteration of Position Specific Iterative Basic Local Alignment Search Tool of the query uncharacterized protein BAE56987.1 to the sbjct phosphinothricin N-acetyltransferase 1VHS is displayed. The Q/RxxGxG/A motif found in a large number of coenzyme A-binding proteins is highlighted by a box.

BLAST Basic local alignment search tool for discovery of matching patterns of DNA or protein sequences. 

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