Biology Reference
In-Depth Information
3. Remove destain solution and discard, and add another 50
L
of detaining solution. Cover the 96-well plate and shake on
orbital rocker at 700 rpm for 30-45 min.
4. Remove destain solution and discard. Dry gel pieces at 50 °C
for 20 min with the lid open (
see
Note 33
).
5. Add 15
μ
L of digestion solution to each dried gel spot (visu-
ally check to ensure that each gel spot is immersed in solu-
tion). Cover and incubate at 37 °C for 12-16 h for
digestion.
6. Add 10-15
μ
L 100 % (v/v) acetonitrile to each well and cover
and shake on orbital rocker at 750 rpm for 15 min.
7. Take supernatant from each sample and place into a new
96-well plate.
8. Add 10-15
μ
L of extraction solution to gel pieces. Cover and
shake on orbital rocker at 750 rpm for 15 min.
9. Take supernatant from each sample and place into the same
well of the 96-well plate from
step 7
above.
10. Repeat
steps 4
and
5
.
11. Dry down samples in the new plate
μ
using
a vacuum centrifuge.
The identifi cation of proteins that differ in abundance following
the isolation of mitochondria by two differing isolation techniques
allows us to determine the degree of contamination from a number
of likely sources including other organelles or cellular compart-
ments. Recently we showed that the top six sources of contaminat-
ing protein of mitochondria following two-density gradient
isolation were the chloroplast (45 % of contaminating proteins),
plasma membrane (35 % of contaminating proteins), cytoplasm
(12 % of contaminating proteins), vacuole (5 % of contaminating
proteins), extracellular proteins (2 % of contaminating proteins),
and peroxisome (1 % of contaminating proteins) [
24
]. Although
protein tandem mass spectrometry is a highly variable process
depending on both the type of ionization and confi guration of
mass spectrometry used, we have outlined a general procedure for
both MALDI-MS/MS and ESI-MS/MS to the point of sample
ionization following which machine-specifi c parameters/knowl-
edge are required for each hardware setup.
Identifi cation of Proteins
That Differ in Abundance
Following DIGE
A. Method using MALDI-MS/MS.
Peptide samples derived from trypsin digestion of excised
gel spots to be analyzed by MALDI-MS/MS are “spotted”
onto a MALDI plate with an appropriate matrix using a dried
droplet method.
1. Appropriate machine-specifi c calibration standards should
always be spotted close to the samples being analyzed.
