Biology Reference
In-Depth Information
Increasingly peptide-level separations of digested protein extracts
are becoming the preferred means of sample fractionation in pro-
teomics. These approaches are of higher throughput, overcome
many of the physiochemical biases inherent in gel-based approaches,
and typically identify many more proteins including lower abun-
dance proteins that are not stained on gels. However, they lack
visual representation of the sample achieved by gel separation and
the knowledge gained of a protein's Mr and isoelectric point,
which can add an additional level of confi dence to the protein
identifi cations obtained. Gel-free techniques can also be used to
determine the mitochondrial or non-mitochondrial origin of a
protein, by harnessing the high-throughput capability of direct
RP-HPLC-ESI-MS/MS. To do this we produce triplicate samples
of low-quality, mid-quality, and high-quality mitochondrial isola-
tions. Typically these have been collected pre gradients (for low
quality), post gradients and washes (for med quality), and follow-
ing FFE (for high quality) [
9
,
17
,
51
]. These samples are then
quantitatively analyzed to determine quantitative enrichment (QE)
of mitochondrial proteins and quantitative depletion (QD) of non-
mitochondrial proteins in the increasingly purifi ed mitochondrial
samples (low
3.4.5 Gel-Free Peptide
Fractionation and
Identifi cation to Determine
Mitochondrial Purity
high). Thus any proteins seen to increase in
abundance when analyzing our increasingly pure mitochondria
sample are of mitochondrial origin and any protein seen to decrease
in abundance is a contaminant. Here we outline methods for deter-
mining the mitochondrial origin of proteins using a quantitative
enrichment/depletion (QED) approach.
→
med
→
Tryptic digestion and peptide mass spectrometry can be impaired
by the presence of salts, lipids, cellular components, and particu-
larly charged species. To remove these mitochondrial proteins are
acetone precipitated as outlined in Subheading
3.4.4
.
Sample Preparation
1. Following precipitations add resuspension buffer to the dried
pellets (1
g of mitochondrial protein) and incubate
at room temperature for 45-60 min with occasional vortexing.
2. Once the sample is in solution add IAA to 10 mM and incu-
bate in the dark for 30 min.
3. Dilute the sample to 1 M urea with dilution buffer.
4. Add digestion solution, and incubate at 37 °C for 12-16 h.
The high concentrations of urea, DTT, IAA, and
NH
4
HCO
3
in the sample are incompatible with ionization/
mass spectrometry. These are removed by a reverse-phase-
solid-phase extraction (RP-SPE) step using C18 spin columns
or C18 embedded pipette tips.
5. Add 2.5
μ
L per 10
μ
6. Charge the C18 column (or tip) by loading with 750
μ
L of FA to the sample.
L,
pipette once) of 70 % (v/v) ACN and 0.1 % (v/v) FA and spin-
ning at 150 ×
g
for 2 min, and discard the fl ow-through.
μ
L (10
μ
