Biomedical Engineering Reference
In-Depth Information
2. Routine solutions should be prepared in water that has a
resistivity of 18 M
cm, unless otherwise stated. The solu-
tions used for both solid-phase extraction and SIMAC pro-
tocols, along with all the solvents used in the LC-MS sys-
tem, must be LC-MS grade. LC-MS grade solvents used
here were sourced from LGC Promochem (Middlesex,
UK), but any other LC-MS grade solvents should be ade-
quate.
3. The lysis buffer used in this protocol is urea based. Urea
acts as a denaturing agent at high concentrations such as
that used in the current lysis buffer (8 M). When the
lysis buffer is mixed with the cells, the urea denatures
the proteins disrupting the cellular structures and acting
as a lysing agent. Due to its denaturing nature, urea also
protects phosphopeptides from protease degradation and
phosphatases activities present in the biological samples.
Please note that this compound is toxic; hence one must
be extremely careful when handling it.
4. For this type of experiment, it is essential that the
lysis buffer contains different protease and phosphatase
inhibitors such as Na
3
VO
4
and NaF (tyrosine phosphatase
inhibitors), sodium pyrophosphate and ß-glycerol phos-
phate (phosphatase inhibitors of broad specificity). This
will preserve the phosphosites of the cell lysate proteins.
5. Any other reagent for protein quantification may be suit-
able.
6. The murine NIH-3T3 fibroblasts used in this example
should not be maintained in culture for a long number of
passages (maximum 25-30 passages) and should be kept
between 10 and 80% confluency. This is due to the high
transformation potential of this cell line. When culturing
NIH-3T3 at 100% confluency and/or for long passages,
they stop behaving like normal fibroblast and become
transformed.
7. Cells are harvested at 75% confluency when they are
actively dividing and when proliferative signalling networks
are activated. This favours the study of kinase activity dif-
ferences between experimental conditions.
8. Phosphopeptides are very sensitive to degradation by pro-
teases and phosphatases present in the sample. For this
reason, when performing phosphoproteomic experiments,
it is very important to use prechilled solutions and to
keep the sample on ice unless otherwise stated. This will
reduce any phosphatase and protease activity that may
remain in the sample despite the treatment with urea and
inhibitors.
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