Biology Reference
In-Depth Information
2. The extracted peptides are resuspended in 1-2
μ
L 5 %
(v/v) acetonitrile and 0.1 % (v/v) TFA.
3. Pipette 1-2
L of resuspended peptides on the MALDI
plate and let dry until the volume has reduced by ~50 %.
4. Add 1-2
μ
μ
L of matrix solution on the sample and mix (
see
Note 34
).
5. Let the spot dry.
6. The sample plate is then placed in the mass spectrometer
and MS and MS/MS spectra obtained.
7. 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/
)
.
B. Method using ESI-MS/MS.
Peptide samples derived from trypsin digestion of excised
gel spots to be analyzed by ESI-MS/MS require fractionation
to allow separation of the various peptides in time to allow
identifi cation. This is crucial to allow the assessment of peptide
abundance (MS mode), selection of a precursor peptide (MS
mode), and its fragmentation and detection of fragment ions
(MS/MS mode). If peptides are not fractionated then the
duty cycle of the mass spec would not allow the identifi cation
of suffi cient peptides to confi rm identifi cation. This fraction-
ation is typically achieved by reverse-phase (RP)-HPLC using
a C18 column (
see
Note 14
).
1. The extracted peptides are resuspended in 10
μ
L 5 % (v/v)
acetonitrile 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
Note 35
).
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) acetoni-
trile and 0.1 % (v/v) formic acid and re-equilibrated with
5 % (v/v) acetonitrile and 0.1 % (v/v) formic acid prior to
the next sample.
