Biology Reference
In-Depth Information
(e.g. glucose-6-phosphate) in 25:75 methanol:water to the ESI source using a
syringe pump. Set slow-changing parameters (spray voltage and all gas flows and
temperatures) to medium values. Then, optimize these parameters one after the
other to improve signal intensity and the signal-to-noise ratio.
For each metabolite of interest, optimize fast-changing parameters (tube lens
voltage, collision energy, fragment ion mass). Provide a continuous flow of
20
L min
1
of a 100
M solution in 50:50 methanol water to the ESI source
using a syringe pump. Most manufacturers provide automated software routines
for the optimization of the compound-specific parameters. If possible, record the
optimized tube lens voltage and collision energy for multiple fragments.
Generally, the most intense fragment will provide the most sensitive
quantification. However, in some cases, the use of alternative fragments can
prevent interference with metabolites of similar retention time. For instance,
galactose-1-phosphate and glucose-1-phosphate both yield a 241-
m
/
z
fragment
ion, while fructose-6-phosphate and mannose-1-phosphate, which elute close by,
do not, even though all four compounds are structural isomers.
Ion suppression is a well-known source of error in ESI-MS. However,
U-
13
C-labelled internal standards can compensate this potential problem.
With thismethod, metabolites can elute in a timewindowof 10-60 s. Reliable peak
integration requires at least 20 data points per scan. Depending on the number of
target metabolites, it might be necessary to not scan for all metabolites during the
entire duration of the gradient but rather to divide the gradient into time segments.
Thus, in one segment, only the metabolites that are eluted are quantified.
m
m
In the long run, regular cleaning and maintenance is essential for metabolite
quantification. In our experience, the ion entry (orifice or ion transfer capillary)
should be cleaned weekly. Monthly cleaning of the Q00, skimmer, tube lens and,
depending on the device, Q0 can be performed by an experienced user.
In positive mode, tributylamine causes a strong and persistent signal at 186
m
/
z
that can interfere with the tyrosine signal regularly used for mass axis calibration.
If available, using a different ESI source for mass axis calibration significantly
alleviates this problem. Otherwise, intensive cleaning is required before the mass
spectrometer can be used in positive mode.
5.3
Peak integration of LC-MS/MS data
When working with manufacturer-provided software, manual peak integration might
be necessary for analytes with two or more phosphate groups and for groups of
isomers.
Organic acids (e.g. fumaric acid) can suffer from interference with background
signal that can hamper automatic base line detection.
If unexpected or non-Gaussian peak shapes occur, check the signal of the
corresponding U-
13
C-labelled analyte. If the retention time and peak shape
match, the peak can usually be used.
