Tag sequences

Although comprehensive sequence information on protein domains is not available on most instruments, shorter segments are often revealed by PSD, CAD, or other techniques. The concept of a sequence tag is based on using the partial sequence of a peptide digestion product, usually composed of a few residues, in combination with the masses of the adjoining N- and C-terminal fragments to efficiently search protein databases for the identity of unknown proteins [18,19]. 

For example, let us assume that we find three b series fragment ions, miz 908.4, 1021.5, and 1108.5 in the CAD spectrum from the tryptic digest of the human hemoglobin a subunit that belong to the peptide parent ion with m/z 1833.9 (see Fig. 5). This is the peptide between residues 41 and 56 in Table 1. 

The mass differences in the b series reveal the presence of L/I followed by S in the sequence. This information is sufficient to attempt a sequence tag search. Searching the SwissProt database for H. sapiens proteins by entering m/z 1833.9 for the parent ion and 1108.5, 1021.5, and 908.4 for the b series fragments in the MS-Seq searching tool of Protein Prospector [11] turns up a single protein, human hemoglobin a subunit with primary accession number P69905. 

The initial 252,616 entries in the database are reduced to 1328 by the parent mass filter. Using the three fragment masses the number of matching proteins for all species is 80. At this point, all the hits are related to the hemoglobin a subunit. Introducing the information on the species produces a single hit. Note, however, that the sequence coverage of the protein is only 11.3%. This limitation curtails the value of sequence tag identifications in the presence of multiple posttransla-tional modifications. 

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EST Expressed sequence tag. An EST is a partial sequence (typically less than 40C bases) selected from cDNA and used to identify genes expressed in a particuh tissue... 

ITS Sequence tagged site. A short DNA sequence that occurs just once in the human genome and whose location and base sequence are known... 

G. J. Opiteck, J. W. Jorgenson, M. A. Moseley III and R. J. Anderegg, Two-dimensional mia ocolumn HPLC coupled to a single-quadrupole mass spectrometer for the elucidation of sequence tags and peptide mapping , 7. Microcolumn Sep. 10 365-375 (1998). 

Expressed Sequence Tag. A short DNA sequence usually representing the most terminal regions of a cDNA clone. 

This process, sometimes known as sequence tagging, may be a more convenient method of obtaining sequence information but it has to be said that it is unlikely to allow as many residues to be determined as when using the classical Edman approach. 

Sequence tagging The use of MS-MS to investigate the amino acid sequence of a peptide. 

Abbreviations STS, sequence tagged site RFLP, restriction fragment iinked poiymorphism SNP, singie nucieotide poiymorphism YAC, yeast artificiai chromosome BAC, bacteriai artificiai chromosome PCR, poiymerase chain reaction. 

When the Arabidopsis Expressed Sequence Tag (EST) Database was searched with the tomato fmit Psubunit protein sequence two related cDNAs were identified (Figure 11). cDNA 2 is near full length and has been completely sequenced, cDNA 1 has also been sequenced but currently lacks approximately 100 amino acids of coding region. The two Arabidopsis cDNAs are 81% identical at the protein level and have lower identity to the protein encoded by tomato gene 1, 64 and 63% for cDNA 1 and cDNA 2, respectively. However, both cDNAs encode... 

The major advantage of the tandem mass spectrometry approach compared to MALDI peptide fingerprinting, is that the sequence information obtained from the peptides is more specific for the identification of a protein than simply determining the mass of the peptides. This permits a search of expressed sequence tag nucleotide databases to discover new human genes based upon identification of the protein. This is a useful approach because, by definition, the genes identified actually express a protein. 

Mann, M. (1996). A shortcut to interesting human genes peptide sequence tags, expressed-sequence tags and computers. Trends Biochem. Sci. 21, 494-495. 

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. 

Mann, M. Wilm, M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 1994, 66,4390-4399. 

Mortz, E. O Connor, P. B. Roepstorff, P. Kelleher,N. L. Wood,T. D. McLafferty, F. W. Mann, M. Sequence tag identification of intact proteins by matching tandem mass spectral data against sequence data bases. Proc Nat. Acad. Sci. USA 1996, 93, 8264-8267. 

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

Tetteh, K.K.A., Loukas, A., Tripp, C. and Maizels, R.M. (1999) Identification of abundantly-expressed novel and conserved genes from infective stage larvae of Toxocara canis by an expressed sequence tag strategy. Infection and Immunity 67, 4771-4779. 

Welage, L. Barnett, C. P. Landowski, D. Foster, D. Fleisher, K.-D. Lee et al. Comparison of Human Duodenum and Caco-2 Gene Expression Profiles for 12,000 Gene Sequences Tags and Correlation with Permeability of 26 Drugs., Pharm. Res. 2002, 29, 1400-1416... 

Sun, D., et al. Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm. Res. 2002, 19, 1400-1416. 

Expressed Sequence Tags (ESTs) and Computational Biology ... 

Xiao H, Merril, CR, Wu A, Olde B, Moreno R, Kerlavage AR, McCombie WR, Venter JC. Complementary DNA sequencing Expressed Sequence Tags and the Human Genome Project. 

Adams MD et al. 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet 1993 4 256-267. 

Ewing B et al. Analysis of expressed sequence tags indicates 35,000 human genes. Nature Genet 2000 25 232-234. 

C, Charmley P, Kaiser R, Hood L. Identification of clusters of biallelic polymorphic sequence-tagged sites (pSTSs) that generate highly informative and... 

Ulrich CM, Bigler J, Velicer CM et al. Searching expressed sequence tag databases discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev 2000 9 1381-1385. 

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