Post-translational
Modification analyses
Characterization of post-translational modifications (PTMs)
plays is a very important role in proteomic analyses. Most
proteins undergo some form of modification after translation
which results in mass changes that can be detected during
MS analysis. PTMs can influence protein structure and function
and are partially responsible for the large variety of protein
isoforms found in nature. The Q-TRAP (linear trap) instrument
and the Matrix-assisted laser desorption/ionization time of
flight (MALDI ToF) can be used to identify modifications by
measuring loses of PTM-specific residues after the PTM containing
peptide is introduced into the mass spectrometer. Classic
modifications that are routinely analyzed include the detection
of metastable losses from carboxamidomethylated cysteines,
oxidized methionines, phosphorylated and glycosylated amino
acid residues.
Probably the best-studied post-translational modification
is reversible phosphorylation. This modification is found
on threonine, serine and tyrosine residues, and plays central
roles in the regulation of many cellular processes including:
cell cycle, growth, apoptosis and differentiation. Mass spectrometry
(MS) obtained from tryptic protein digests has become a powerful
tool for characterization. Due to the general low abundance
and often poor ionization of phosphopeptides is a general
need to significantly enrich samples for phosphopeptide content.
Immobilized metal affinity chromatography (IMAC) is an established
technique that is used for purification of phosphorylated
compounds and can be coupled to HPLC and mass spectrometry.
One of the most common classes of post-translational modifications
is glycosylation. This involves the covalent attachment of
oligosaccharides to proteins at specific amino acid residues.
Asparagine-linked (N-linked) or serine/threonine-linked (O-linked)
oligosaccharides are very common and are often seen on proteins
that make up major structural components including cell surface
molecules and secreted proteins. Glycosylations are important
to many cellular processes including protein sorting, immune
recognition, receptor-ligand binding, and pathogenicity.
The Centre will set up custom experiments based in individual
client needs. Please contact the senior lab manager to initiate
a PTM analysis on your sample.
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