Biology Reference
In-Depth Information
and then lyophilized. They could also be desalted by gel fi ltration or
through dedicated membranes under centrifugation.
There are also various possibilities to elute proteins captured by
CPLL beads and thus render the solution directly compatible with
the analysis. This is the case with protein desorption using SDS-
Laemmli buffer under boiling conditions. Here the sample can be
used directly for SDS-PAGE analysis and then after band slicing and
trypsination the peptides are analyzed by LC-MS/MS [
34
].
If the proteomic analysis is performed by 2D electrophoresis, the
most compatible elution is to use TUC solution. However this does
not elute 100 % of proteins from the beads and should be completed
by another orthogonal elution. Alternatively the TUC solution could
comprise some amounts of cysteic acid that produces an almost
exhaustive desorption of proteins. In this case due to the very low pI
value of cysteic acid, which collects at the anode, 2D electrophoresis
can also be performed [
40
]. This elution is also compatible with the
application of isoelectric focusing separation of components.
When 2D-DIGE is used as 2D electrophoresis analysis, the only
elution that is compatible with this technique is the use of 20 mM
Tris buffer containing 7 M urea, 2 M thiourea, and 4 % CHAPS,
pH 8.5 (sodium carbonate could also be used instead of Tris).
For direct ELISA-based assays of eluted proteins, the denatur-
ing desorption agents that can be selected are 0.2 M glycine-HCl,
2 % NP-40, pH 2.4; 0.1 M acetic acid, 2 % NP-40; 1 M NaCl, 2 %
NP-40; or 0.1 M acetic acid containing 40 % ethylene glycol. In
case a single eluent does not desorb all proteins from the beads
these solutions could be used as a sequence and the eluates blended.
It is here recalled that protein elution from beads may not be
necessary. This is the case when proteins are just analyzed after
trypsin digestion. This treatment can be operated directly on the
beads and the obtained peptides directly analyzed by LC-MS/MS
[
36
]. This approach is recommended especially when dealing with
small samples involving small volumes of beads with time saving
and largely reduced protein losses.
4
Notes
1. It is recommended not to reuse hexapeptide beads because (a)
some level of carryover may appear, with consequent misinter-
pretation of data, and (b) some peptides may have been modi-
fi ed as a consequence of stringent elution conditions from
previous operations.
2. The sample should be clear and not contain lipids in suspension
such as in milk or in bile fl uid. Large amounts of nucleic acids or
viscous polysaccharides, when present, should also be removed
using current methods. Samples should not contain a large amount
of detergents or denaturing agents. For example, nonionic deter-
