Basic Design of MRM Assays for Peptide Quantification - LC-MS/MS in Proteomics: Methods and Applications - page 178

dimensions. To determine the instrument and source parameter
settings, a peptide standard, such as Glu-fibrinopeptide, can be
infused. Once the mass spectrometer is detecting the peptide, the
parameters can be adjusted to optimize signal strength. This infu-
sion standard can also be used to track instrument performance
over time.
Scan-based parameters focus on settings that are specific to
each of the transitions included in the method. These settings
affect the sensitivity, specificity, and duty cycle of the MRM
method. Besides the precursor and fragment (Q1/Q3) masses
that specify an MRM, the transmission windows for the Q1
and Q3, the collision energy (CE) for each transition, and the
dwell time of each measurement can be optimized within the
experiment. The transmission window sets the resolution of the
quadrupole, that is, the mass range that is transmitted through
Q1 and Q3 for a transition. Transmission window settings can be
low, high, or unit ( Fig. 10.4 ) . When a transmission window is
set to “unit resolution,” the quadrupole transmits 0.7-1 atomic
mass unit (amu) of mass range; low resolution is typically set to

High Resolution Unit Resolution Low Resolution

400
380
360
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0

461.7515(2)

462.2518(2)

462.7539(2)

460.0 460.5 461.0 461.5 462.0 462.5 463.0 463.5 464.0 464.5 465.0 465.5 466.0 466.5
m/z, Da

Fig. 10.4 The transmission window sets the resolution of the quadrupole. Q1 settings at high, unit, and low resolution
affect the selection of the isotopic envelope from the precursor ion.