Biology Reference
In-Depth Information
3. Centrifuge pooled material either in the same tube or perform
stacked centrifugations in a 1.5 mL vial in order to minimize
liquid losses. Centrifugation is performed for 2 min at
13,000 rpm to ensure that any insoluble material is not loaded
into SCX column. Repeat until the complete sample is
processed.
4. Transfer sample to a clean tube for loading onto SCX
column.
5. Fractionate labeled peptide mixture by SCX chromatography.
Gradient details are sample and LC system dependent. We use
ÄKTA Purifi er (GE Healthcare) chromatograph. 5 mL of sam-
ple are injected into a 50 mL superloop. Sample is then loaded
from the loop onto the SCX column and washed for 20 min
with 100 % SCX-A at a fl ow rate of 0.1 mL/min.
6. Next step in the program is triggering of an elution gradient
at a fl ow rate of 0.1 mL/min as follows: 5-35 % SCX-B in
30 min, 35-100 % SCX-B in 50 min, wash at 100 % SCX-B for
20 min, return to 0 % SCX-B (100 % SCX-A), and re-equilibrate
column in SCX-A until conductivity signal is fl at. 1-min frac-
tions are collected upon triggering of gradient and peptides
usually elute between 16 and 70-75 min, thus yielding 45-50
fractions. Peptide recovery is monitored using a UV detector
(two different signals at 280 nm and/or 214 nm may be used
to monitor peptide chromatographic peaks). This fraction-
ation can be adjusted dependent on the instrument and the
program conditions (
see
Note 10
).
7. The UV absorption of the different fractions and the summed
fi nal chromatographic area (mAU × min
−1
at 280 nm) are
known after SCX. Thus, it is possible to redistribute the frac-
tions in an effort to equalize the amount of peptides in each
fraction and the number of MS/MS events after each mass
spectrometric analysis. After redistribution of the peptides,
around 25 fractions are collected and analyzed by LC-MS/MS
(Fig.
2
), precluding the injection of samples too concentrated
or too diluted and, thus, improving protein identifi cation and
quantitation.
8. The combined SCX fractions are dried under vacuum and can
be stored at −20 or −80 °C until analysis by reverse-phase
LC-MS/MS. Preferably peptide cleanup can be carried out to
ensure complete desalting prior to mass spectrometry (
see
Note 11
).
9. For desalting of peptides, we use C
18
spin-cartridges (Agilent
Technologies) following the manufacturer's recommendations.
Briefl y, resuspend sample in 100-150
L of sample buffer.
Wash C
18
beads in activation solution, equilibrate the beads in
wash buffer and bind sample, wash resin at least three times in
μ
