Biology Reference
In-Depth Information
5. Analyze peptide ions using the data-dependent “triple-play”
method: (1) full mass spectrum scan, (2) zoom scan of the
major ions with higher resolution to determine their charge,
and (3) fragmentation of these ions. Acquire mass spectra from
mass/charge 200-2,000.
6. Perform protein identifi cation with adequate software and reli-
able data evaluation (
see
Note 21
). Download
A
.
thaliana
protein sequences from the National Center for Biotechno-
logy Information FTP site (
http://www.ncbi.nlm.nih.gov/
)
.
Set cysteine carbamidomethylation to static modifi cation and
methionine oxidation and N-terminal acetylation to possible
modifi cations. For precise analysis, do not specify the protease
used (
see
Note 22
).
4
Notes
1. We store the grains in a refrigerator, in Eppendorf tubes (1 g
of grains per tube) thoroughly closed and sealed with parafi lm.
2. At fi rst, concentrated NaOH (10 M) can be used, but when
getting closer to the fi nal pH, it is better to use diluted NaOH
solutions. Proceed similarly when acidifying solutions with HCl.
3. Dilution of Tween 20 is complicated because of its high vis-
cosity. Use a 10 mL graduated test tube. Fill the tube with
9 mL of ultrapure water and make up to 10 mL with Tween
20. For the manipulation with Tween 20 use a Pasteur pipette.
Store the solution in a dark place.
4. Be careful not to exceed the fi nal volume (20 mL) of the solu-
tion. Add water in small doses and slightly heat the mixture to
support urea dissolution. Before further use, the solution must
be slowly returned to room temperature.
5. Work cautiously with acrylamides and their solutions due to
their high toxicity.
6. Acrylamide hydrolyzes to ammonia and acrylic acid, if stored
for longer time.
7. Be careful when weighing large amounts of SDS. Avoid moving
rapidly the open container because SDS irritates mucosa when
inhaled.
8. Avoid storing ACN in plastic vessels; ACN, as an organic sol-
vent, may extract polymers hindering LC-MS/MS analysis.
9. We use Sequencing Grade Modifi ed Trypsin purchased from
Promega and
-Chymotrypsin from bovine pancreas pur-
chased from Sigma-Aldrich.
10. Usually, the OB layer is solid enough to be easily collected with
spatula.
α
