Proteins, introduction posttranslational modification

Although the total synthesis of a protein allows complete control over the structure, including posttranslational modifications and introduction of labels at desired sites in the sequence, it is still a major undertaking for which most laboratories whose main interest is in the biology of their target proteins are not equipped. In certain cases, for example when the site of introduction of a specific chemical modification is near the C-terminus, a combination of molecular biological and chemical methods has proved to be very powerful. 

The last example shows that chemical synthesis of proteins can be a useful tool to study pertinent questions in biochemistry. Its main advantage so far relates to its capability to introduce unnatural amino acids, specific isotope labels, biophysical monitors, or very important posttranslational modifications. The latter modification the introduction of a GPI anchor or a GPI anchor mimic onto PrP, has been the subject of further research. These experiments were not solely directed towards specific questions of PrP research but also towards development of a general method to modify proteins with a GPI anchor. 

Collagen, a family of fibrous proteins, is produced by a variety of cell types but principally by fibroblasts (cells found in interstitial connective tissue), muscle cells, and epithelial cells. Type I collagen [collagen(l)], the most abundant protein in mammals, is a fibrous protein that is the major component of connective tissue. It is found in the extracellular matrix (ECM) of loose connective tissue, bone, tendons, skin, blood vessels, and the cornea of the eye. Collagen(l) contains approximately 33% glycine and 21% proline and hydroxyproline. Hydroxyproline is an amino acid produced by posttranslational modification of peptidyl proline residues (see Chapter 7, section V.C., for an earlier introduction to collagen). 

The introduction of soft ionization techniques such as ESI and M ALDI brought tremendous progress in on- and offline characterization of electrophoretically separated peptides and proteins by MS [50]. Combination of CE with MS techniques allows not only high-accuracy molecular mass determination of peptides and proteins separated by CE, but also it provides important structural data on amino acid sequence, the sites of posttranslational modifications, peptide mapping, and the noncovalent interactions of peptides and proteins. 

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