Chemical fragmentation methods

There are also a number of chemical methods to specifically cleave peptide bonds. For example, cyanogen bromide (CNBr) specifically cleaves Met residues at the C-side, forming a peptidyl homoserine lactone (Fig. 7.13). To ensure that all Met residues are cleaved, the reaction takes place under denaturing mildly acidic conditions. 

If the resulting peptide fragments are still too long for sequencing, a further fragmentation step may be required using different cleavage processes. 

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In another method for sequencing, the DNA is labeled at internal base positions by enzymes which repHcate DNA strands in the presence of specific chain terrninators, thus producing an array of labeled fragments which reflect the sequence of the DNA. The rest of the procedure is the same as described for the chemical cleavage method (22). 

Despite the good results obtained in some accuracy tests, one have to keep in mind that fragmental methods suffers from the absence of peculiar fragments that could limit their applicability domain. The prediction performance of fragmental methods also largely depends on the chemical diversity of the molecular structures used to develop the model. 

Methods based on molecular properties attempt to avoid the drawbacks of the fragmental methods, such as their failure to calculate log P for structures with missing fragments. However, in some cases, models were derived from sets of compounds with moderate chemical diversity limiting the accuracy of predicted log P. 

Some researchers who use parameters derived from quantum chemical calculations on the whole solute molecule (Bodor, 1989) place their methods in a "theoretically-based" class. These are seen as preferable to methods which add the values assigned to structural parts, which then requires the assignment of interaction factors or corrections/ depending on how the parts are attached to each other. Fragment methods then are said to be in a purely empirical class and therefore obsolete. ... 

This is the main easily accessible method for direct prediction of a property from chemical structure alone. Fragment methods view a molecule as composed of specified parts, which contribute individually to the compound property. 

The thin-layer gel system used for resolving the different fragments produced by the chemical degradation method is essentially that of Sanger, Nicklen and Coulson (1977) as described in Chapter 4. An 8% polyacrylamide gel is usually used though for special purposes a 10 or 12% gel can give an improved resolution of the smallest fragments. For the standard 8% gel the polymerization mixture contains 7.6% (wt/vol) acrylamide, 0.4% (wt/vol) bisacrylamide, 50% (wt/vol) urea, 100 mM Tris-borate, pH 8.3, 2mM EDTA, 0.07% (wt/vol) ammonium persulphate and TEMED catalyst. This solution is poured or injected into a 0.4 x 200 x 400 mm mold to form a gel slab. 

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