Biology Reference
In-Depth Information
1. Calibrate and prepare/tune the LC-MS system according to
the manufacturer's instructions. We highly recommend the
use of UPLC chromatography systems and HR/AM mass
spectrometers for optimal results. We use a LTQ-OrbiTrap XL
mass spectrometer both from Thermo Scientifi c.
2. Dissolve the peptides in 8
3.5 LC-MS ( See
Note 1 )
L 5 % (v/v) ACN, 0.1 % (v/v) FA
in dH
2
O. Pellet insoluble debris by centrifugation at 14,000 ×
g
for 10 min. Carefully transfer 7
μ
L of clear phosphopeptide
solution to the sample container appropriate for the LC auto
sampler.
3. Program the LC-MS method to inject 5
μ
L of phosphopep-
tide solution. For chromatography we use a Chromolith
CapRod monolithic column with a C18 stationary phase with
a length of 150 mm and an inner diameter of 0.1 mm. We use
0.1 % (v/v) FA in dH
2
O and 90 % (v/v) ACN, 0.1 % (v/v) FA
in dH
2
O for gradient elution and reverse phase separation of
phospopeptides.
4. Program the LC-MS method to deliver a suitable fl ow rate
and elution gradient. When using packed columns, small par-
ticle diameters will allow higher fl ow rates without adversely
affecting resolution allowing shorter analysis time. Longer
columns will achieve higher resolution and have a higher peak
capacity however the inlet pressure will be limiting. We used a
linear gradient from 5 to 35 % organic mobile phase content
in 120 min and a fl ow rate of 500 nl/min.
5. Equilibrate the column delivering an isocratic fl ow with the
same mobile phase composition as the phosphopeptide solu-
tion, in our case 5 % (v/v) ACN, 0.1 % (v/v) FA in dH
2
O for
2 or more column volumes.
6. Set up the LC-MS junction via an appropriate emitter and ESI
source and establish a stable electrospray. This step can be dif-
fi cult. Adjust the spray voltage and emitter position, replace
the emitter, check the ion optics settings and ensure suitable
potential gradients and RF amplitudes for the ions to be able
to overcome possible potential barriers. If problems persist
check the calibration, run instrument diagnostics and if neces-
sary clean and tune the system according to the manufactur-
er's instructions.
7. Program the LC-MS method to acquire MS data throughout
the duration of phosphopeptide elution from the LC system. As
mentioned above many mass spectrometry methods can be
applied to the analysis of phosphopeptides dependent on the
capabilities of the LC-MS system and the desired results. We
used data-dependent analysis in which the fi ve most intense ions
recorded in a HR/AM full scan of the total ion population in the
μ
