Biomedical Engineering Reference
In-Depth Information
transmit 1.5-2.5 amu and high resolution is typically set at 0.3-
0.5 amu. As the resolution of the transmission window increases,
the transmitted mass window decreases. This results in an over-
all decreased sensitivity of the scan, while the specificity increases.
A high complexity sample such as digested cell lysate, plasma, or
serum will require higher selectivity of the MRM assay since the
potential for isobaric peptides interference will be high. Settings
of 0.7 amu (unit resolution) for Q1 and 0.7 amu (unit) for Q3
are frequently used during initial optimization of the MRM assay.
Peptides with different amino acid sequences, different charge
states, and different mass-to-charge ratios (
m
/
z
) will fragment
optimally at different collision energies (CE). If a software tool is
used to select the transitions (such as MRMPilot or PinPoint), the
CE for the peptide will automatically be calculated based on the
above variables. If the transition of a precursor is selected man-
ually, the following equation can be used to determine a reason-
able collision energy (note that these equations are charge-state
dependent):
z
2
+
:CE
z
3
+
:CE
[
m
/
=
(
m
/
z
)0.044
+
5;
m
/
=
(
m
/
z
)0.05
+
4;
z
4
+
:CE
3].
The dwell time is the amount of time in milliseconds that
the instrument spends on each transmission. When first design-
ing an MRM method, starting with 25 ms/transition is reason-
able. This number can then be increased or decreased depend-
ing on the method. The number of transitions in an MRM
method multiplied by the dwell time will give the approximate
duty cycle of the MRM scan (not including instrument over-
head). For example, if a method has 100 MRMs with a dwell
time of 25 ms each, the theoretical duty cycle for the MRM scan
is 2.5 s (2500 ms) (note that instruments from different ven-
dors have different overhead time which is added time due to
optimization and chromatographic performance. One of the key
factors in designing a reproducible and reliable MRM experiment
is the number of measurements the mass spectrometer records
for a given transition as the target peptide elutes from the LC
column. It is generally advisable to measure more than 10 points
MS experiment where a 45 min gradient is run from 5 to 30%
organic buffer using a 75
and
m
/
=
(
m
/
z
)0.05
+
12 cm LC column, the peak
width of most peptides will be approximately 30-40 s at baseline.
For the MRM method discussed above, the duty cycle is 2.5 s,
so the number of points that will be measured across a chro-
matographic peak will equal 12-15. This is an acceptable num-
ber of measured points and should allow for good peak definition
and integration during sample processing. Peak integration is the
μ
m
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