Biology Reference
In-Depth Information
7. Equilibrate the C18 column by loading with 750
L,
pipette twice) of 2 % ACN (v/v) and 0.1 % (v/v) FA and spin-
ning at 150 ×
g
for 3 min, and discard the fl ow-through.
8. Load the C18 column (or tip, pipette ten times from well) with
sample, spin at 150 ×
g
for 3 min, and discard the fl ow-through.
9. Wash the sample by loading the C18 column with 400
μ
L (10
μ
μ
L
(10
L, pipette once) of 2 % (v/v) ACN and 0.1 % (v/v) FA and
spinning at 150 ×
g
for 3 min, and discard the fl ow-through.
μ
10. Repeat the previous step.
11. Elute your sample by loading the C18 column with 750
μ
L
L, pipette ten times into well) of 70 % (v/v) ACN and
0.1 % (v/v) FA and spinning at 150 ×
g
for 2 min, and retain
the fl ow-through.
12. Repeat the previous step, and pool the two fl ow-throughs
together.
13. Dry down sample in vacuum centrifuge.
(2-10
μ
Complex peptide mixtures such as those derived from trypsin
digestion of whole mitochondria can simply be fractionated and
analyzed by RP-HPLC-ESI-MS/MS; however to increase the
number of proteins identifi ed from the sample other fractionation
techniques can be implemented prior to the RP. Two common
approaches are strong cation exchange (SCX) where peptides are
fractionated by their net surface charge and off-gel electrophoresis
(OGE) where peptides are fractionated by their total net charge.
Protocols for these approaches will not be covered in this review,
but both techniques generate many fractions and are directly ame-
nable to subsequent RP-HPLC-ESI-MS/MS.
Sample Fractionation and
Mass Spectrometry
1. The extracted peptides are resuspended in 2
μ
L 5 % (v/v) ace-
tonitrile and 0.1 % (v/v) formic acid.
2. The resuspended sample is loaded into the HPLC fl ow (5 %
(v/v) acetonitrile, 0.1 % (v/v) formic acid) prior to the in-line
C18 column by direct injection by use of an HPLC sampler
(
see
Notes 15
and
36
).
3. Once the sample has bound the C18 column a gradient of 5 %
(v/v) acetonitrile and 0.1 % (v/v) formic acid to 60 % (v/v)
acetonitrile and 0.1 % (v/v) formic acid is run to sequentially
elute bound peptides directly into the mass spectrometer and
MS and MS/MS spectra collected.
4. The resulting MS and MS/MS spectra can then be interpreted
by standard proteomics approaches using software packages
such as Mascot (Matrix Sciences), Seaquest (Thermo
Scientifi c), or X!tandem (
www.thegpm.org/tandem/
).
5. The column is then washed briefl y at 80 % (v/v) acetonitrile and
0.1 % (v/v) formic acid and re-equilibrated with 5 % (v/v) aceto-
nitrile and 0.1 % (v/v) formic acid prior to the next sample.
