Sequence information

Tandem mass spectrometry (MS/MS) produces precise structural or sequence information by selective and specific induced fragmentation on samples up to several thousand Daltons. For samples of greater molecular mass than this, an enzyme digest will usually produce several peptides of molecular mass suitable for sequencing by mass spectrometry. The smaller sequences can be used to deduce the sequence of the whole protein. 

Determination of DNA Sequence Information. Almost all DNA sequence is determined by enzymatic methods (12) which exploit the properties of the enzyme DNA polymerase. Whereas a chemical method for DNA sequencing exists, its use has been supplanted for the most part in the initial deterrnination of a sequence. Chemical or Maxam-Gilbett sequencing (13) is mote often used for mapping functional sites on DNA fragments of known sequence. 

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

DNA sequence information is the starting poiat for other appHcations, including the expression of a gene product, the search for related sequences ia biological samples, in vitro mutageaesis of the sequeace, and stmcture—function studies of gene expression. 

TR Defay, EE Cohen. Multiple sequence information for threading algorithms. J Mol Biol 262 314-323, 1996. 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

So efficient is the automated dideoxy method that sequences up to 1100 nucleotides in length, with a throughput of up to 19,000 bases per hour, can be sequenced with 98% accuracy. After a decade of work, preliminary sequence information for the entire human genome of 2.9 billion base pairs was announced early in 2001- Remarkably, our genome appears to contain only about 30,000 genes, less than one-third the previously predicted number and only twice the number found in the common roundworm. 

The multiprotein unit that synthesize RNA by copying the sequence information from the leading strand of the DNA. Its activity is tightly controlled by phosphorylation of the C-termal domain (CTD), access to DNA and interaction by general and sequence specific transcription factors and coactivators and corepressors. 

However, these observations are not proof of the role of a donor-acceptor complex in the copolymcrization mechanism. Even with the availability of sequence information it is often not possible to discriminate between the complex model, the penultimate model (Section 7.3.1.2) and other, higher order, models.28 A further problem in analyzing the kinetics of these copolyincrizations is that many donor-acceptor systems also give spontaneous initiation (Section 3.3.6.3). 

The TIC trace from this analysis, shown in Figure 5.5, exhibits a maximum at ca. 19 min, and a representative electrospray spectrum is illustrated in Figure 5.6. Transformation of the latter produces the spectrum presented in Figure 5.7 which indicates the presence of two species with relative molecular masses (RMMs) of 56 548.5 and 58 161.4 Da. These masses are lower than the value of 59.1 kDa calculated from previously obtained sequence information. 

Enzymes are now being used in conjunction with LC-MS to provide sequence information as an alternative to the Edman degradation procedure. 

This is a task for which electrospray ionization is well suited although, as discussed earlier in Section 4.7, this is a soft ionization technique that yields almost exclusively molecular ions with little fragmentation and consequently, in the case of biopolymers, little sequence information directly. 

In order to obtain complete sequence information for a particular protein, it may be necessary to carry out digestion with more than one enzyme. 

When extracting sequence information from mass spectra, not only is the m/z value at which the ions occur of importance since these provide an indication of the amino acid composition of the peptide giving rise to the ion, but so is the mass difference between adjacent ions. This indicates the particular amino acid residue that has been lost and thus provides the sequence information required. The mass differences arising from each of the amino acids are shown in Table 5.6. 

Sequence information for the remaining fragments was obtained by Edman degradation (see Section 5.3.1 above) after isolation of the individual peptides using preparative HPLC - the chromatographic resolution being sufficient to allow this, and thus enabled the complete sequence to be determined. 

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. 

The use of MS-MS to provide sequence information has been described [13] for the study of proteins extracted from yeast (Saccharomyces cerevisiae). The procedure was somewhat complex and consisted of the following steps ... 

Trypsin digests of 17 proteins were carried out and the peptides produced then studied by using LC-MS-MS. In summary, these produced between one and six peptides which gave some sequence information by MS-MS. Of these peptides, the number of amino acid residues sequenced ranged from three to thirteen. 

Figure 5.20 LC-MS-MS spectrum of a tryptic peptide derived from spot 13 (see Figure 5.18) of the silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae, showing the sequence information that may be extracted. From Poutanen, M., Salusjarui, L., Ruohonen, L., Penttila, M. and KaUddnen, N., Rapid Cotn-mun. Mass Spectrom., 15, 1678-1692, Copyright 2001. John Wiley Sons Limited. Reproduced with permission.

Peptide mapping The process of considering the amino acid sequence information from peptides obtained by enzyme digestion in an attempt to derive the (amino acid) sequence of the parent protein. 

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