Biology Reference
In-Depth Information
are separated by HPLC in a reversed phase C18 nanocolumn. A gradient consisting
of 5-35% acetonitrile in 80 min was performed to elute peptides from the column to
a nanospray emitter for ionization and fragmentation in the mass spectrometer.
Depending on the target protein sequence, a variety of endopeptidases (such as chy-
motrypsin and endopeptidase V8) can be used to yield peptide species amenable for
MS analysis. It is also possible to use multiple proteolytic enzyme combinations to
produce different fragment profiles. For specific protocols, see
Buey et al. (2007)
and
Field et al. (2012)
.
It is important to note that all ion-filtering experiments are performed in low res-
olution, and it is therefore difficult to characterize the exact modification site(s), es-
pecially when the ions of interest are larger than 2000 Da. In these cases, after the
first ion filtering experiments, a targeted or semitargeted high-resolution MS
experiment can be performed. High-resolution fragmentation spectra are much more
easily interpreted, as the mass and the charge of all fragments are properly deter-
mined. For these experiments, an LC-MS analysis is performed using hybrid mass
spectrometers based on orbital analyzers (Orbitrap) (
Field et al., 2012
). The combi-
nation of low-resolution ion filtering and high-resolution targeted experiments rep-
resents a powerful tool to study and determine the MSA modification of MTs.
19.2.6.1
Protein preparation and digestion for MS analysis
Depending on the conditions, a ligand may modify tubulin at different residues.
Tubulin can exist in a number of different aggregation states which can result in
differential binding of MSAs. ZMP is able to covalently modify MTs, tubulin olig-
omers, and dimeric tubulin at the same two residues, indicating binding to the taxoid
site in all tubulin aggregation states (
Field et al., 2012
). In contrast, cyclostreptin
labels two different residues in MTs, but only one of these amino acids is labeled
in dimeric tubulin (
Buey et al., 2007
). It is therefore important to determine if the
test compound labels the same or different residues in different preparations of
tubulin.
19.2.6.1.1
Assembled microtubules
MSAs have high affinity for MTs and much lower affinity for unassembled tubulin.
Use the following method to assemble MTs if the MSA is a strong inducer of assem-
bly (determined in
Section 2.1
).
1.
Prepare 20 mg tubulin in GAB buffer as described in
Section 1.1
. Add 6 mM
MgCl
2
to the buffer and increase the concentration of GTP to 1 mM.
2.
To 20
M ligand or an equivalent volume of DMSO and
incubate for 1 h, 37
C. Different concentrations can be used but ensure the
ligand is in 10% excess over the number of binding sites to ensure site saturation.
3.
Pellet the MTs by ultracentrifugation (20 min, 25
C), discard the supernatant,
and resuspend the pellet in 200
m
Lof50mMNH
4
HCO
3
. Add 20
m
L of the pellet
solution to 1
m
g/
m
L trypsin (sequencing grade) and add another 20
m
Lof
NH
4
HCO
3
.
m
M tubulin, add 25
m
