Post-translational modifications are the chemical modifications of proteins subsequent to their biosynthesis. The modification of certain amino acid residues may result in changes of the protein conformation and/or its capacity to interact with other proteins or ligands, to be active or inactive in the case of an enzyme, to allow or interfere with gene expression and many other biological functions. These modifications have been found by using radioactively labeled compounds, to yield a protein(s) carrying a radioactive tag located in the modifying group and that can be detected by methods such as autoradiography, liquid scintillation, and other methods after resolution of the different compounds by electrophoresis, chromatography and other separation procedures.
Confirmation of the modification is carried out by chemical synthesis of the modified amino acid and its structural confirmation by mass spectra. Once the nature of the modification has been establish, the most straightforward method of detecting it is by the use of specific antibodies against the chemical modification. Use of tagged antibodies would allow to detect the distribution of the modification in tissues by using histochemical methods and its evaluation by immune assays such as ELISA.
Although there are many post-translational modifications, some have shown to be crucial for several biological functions, such as:
·Phosphorylation or the addition of a phosphate group to tyrosine, serine, threonine or histidine. This modification affects the activity of certain oncoproteins, eg. Her and EGF.
·Acylation or the addition of an acetyl group to the lysine residues of a protein and/or its terminal amino group. This modification happens usually in histones and regulates the expression of certain genes in a mechanism know as epigenetic effects.
·Prenylation is the addition of a prenyl groups that are hydrophobic to the C-terminal cysteine residues of a protein. It occurs in several oncoprotein and the most common prenyl groups are farnesyl and geranyl-geranyl groups.
·Glycation is the addition of reducing sugar to a lysine amino group via a Schiff base. This modification plays an important role in diabetes.
·ADP-ribosylation is a modification in which ADP-ribose moieties are incorporated the arginine, glutamic or aspartic acid residues of a protein. This modification is involved in signaling and control processes such as DNA repair and apoptosis.
·Post-translational modifications also involve conjugation of a protein to other proteins or peptides, such as ubiquitylation or linkage to the protein ubiquitin. This modification tags an obsolete protein for degradation by the proteosomes.
Other modifications include the formation and cleavage of disulfide bonds, deamination of glutamine, asparagine and arginine. Modifications that contribute to changes in the structural properties of a protein.
An important post-translational modification is the proteolytic cleavage of the lipophilic amino acid sequence in some protein precursors, which is needed to translocate the protein across the lipid bilayer of cellular membranes.
Glycosylation of proteins to yield glycoproteins is a modification that plays an important role in the movement and recognition of cells from the immune system. Glycosylation also have a role in differentiation as well as cancer metastasis. Addition of the sugar residues, take place at the asparagine, serine ot threonine amino acids.
In addition, there are some post-translational modifications that are introduce into purified proteins, such as pegylation, to limit the immunogenicity of a protein. Free amino groups from proteins can be converted to carboxylic grops by succinylation, a modification that changes a cationic protein to an anionic one.
As mentioned before, the use of specific antibodies to these modifications is the easiest way to detect and follow them. BioSynthesis monospecific antibodies are prepared using our own modified antigens and using proprietary immunostimulants to obtain high affinity antibodies. These antibodies can be supply as antisera or as purified antibodies and their conjugates with fluorescent tags and enzymes.