as well as prior to MS/MS experiments. This is done by directly
injecting a solution containing a set of standards of known m/z
ratio which act as reference points. For the Impact HD QTOF
(Bruker™) used in our analysis, the calibration should result in
a standard deviation of 0.20 ppm or less compared to reference
standards, with this parameter dependent on the instrument
used. The instrument should be switched from standby to
operate for at least 30 min prior to calibration. For our instrument, this process is controlled using the mass spec operating
software called “Otofcontrol.”
3.2.2 Starting Up
the MS-LC System Prior
to Running Samples
1. Initiating the LC system. Using Chromeleon, manually set the
flow rate to 40 μL/min. The system should initially be in 100%
Mobile Phase B, as the C18 analytical column is ideally stored
in acetonitrile. From there, ramp the system into 3% Mobile
Phase B over the course of 10 min. If the system has not been
used in several days, purging should be performed prior to
initialization (see Subheading 3.2.1, step 2).
2. Blank Methods. At the start of each day, two blank MS methods
(one ~20-min run, followed by a ~8-min run, see Fig. 2a) are
run to minimize carryover from the previous day, as peptides
may accumulate within the immobilized pepsin and analytical
columns (see Notes 6 and 7). The pre-optimized MS-LC
methods are loaded in the integrated MS control software,
which is Bruker instruments program called “HyStar.” The
methods loaded in Hystar control the various parameters
(MS parameters, data collection) via Otofcontrol and the LC
system (gradient specifications) via Chromeleon. A separate
method must be loaded in “Chronos,” which controls the
RTC PAL. The latter controls the valves within the PAL, thus
determining the flowpath of the sample through the pepsin
column, trap vanguard pre-column, and analytical column (see
Fig. 1). Prior to initiating the runs, the sample loop is washed
with 100 μL 0.1% formic acid. Calibration is performed following the startup methods.
3.2.3 Sample Running
When running samples, the 100 μL Hampton glass syringe used for
sample loading and the 0.1% FA used for washing the sample loop
are kept on ice so as to minimize back exchange of deuterated
samples.
1. Similar to the startup process, the LC (shown in Fig. 2a) and
MS methods (see Note 8) are loaded in both Hystar and
Chronos. The MS run will elute the majority of peptides from
the sample; the short blank run further elutes residual peptides
from the system to reduce carryover between samples (see
Notes 6 and 7). The sample files should be named correctly
and saved in the proper folders.
474
Jordan T. B. Stariha et al.
injecting a solution containing a set of standards of known m/z
ratio which act as reference points. For the Impact HD QTOF
(Bruker™) used in our analysis, the calibration should result in
a standard deviation of 0.20 ppm or less compared to reference
standards, with this parameter dependent on the instrument
used. The instrument should be switched from standby to
operate for at least 30 min prior to calibration. For our instrument, this process is controlled using the mass spec operating
software called “Otofcontrol.”
3.2.2 Starting Up
the MS-LC System Prior
to Running Samples
1. Initiating the LC system. Using Chromeleon, manually set the
flow rate to 40 μL/min. The system should initially be in 100%
Mobile Phase B, as the C18 analytical column is ideally stored
in acetonitrile. From there, ramp the system into 3% Mobile
Phase B over the course of 10 min. If the system has not been
used in several days, purging should be performed prior to
initialization (see Subheading 3.2.1, step 2).
2. Blank Methods. At the start of each day, two blank MS methods
(one ~20-min run, followed by a ~8-min run, see Fig. 2a) are
run to minimize carryover from the previous day, as peptides
may accumulate within the immobilized pepsin and analytical
columns (see Notes 6 and 7). The pre-optimized MS-LC
methods are loaded in the integrated MS control software,
which is Bruker instruments program called “HyStar.” The
methods loaded in Hystar control the various parameters
(MS parameters, data collection) via Otofcontrol and the LC
system (gradient specifications) via Chromeleon. A separate
method must be loaded in “Chronos,” which controls the
RTC PAL. The latter controls the valves within the PAL, thus
determining the flowpath of the sample through the pepsin
column, trap vanguard pre-column, and analytical column (see
Fig. 1). Prior to initiating the runs, the sample loop is washed
with 100 μL 0.1% formic acid. Calibration is performed following the startup methods.
3.2.3 Sample Running
When running samples, the 100 μL Hampton glass syringe used for
sample loading and the 0.1% FA used for washing the sample loop
are kept on ice so as to minimize back exchange of deuterated
samples.
1. Similar to the startup process, the LC (shown in Fig. 2a) and
MS methods (see Note 8) are loaded in both Hystar and
Chronos. The MS run will elute the majority of peptides from
the sample; the short blank run further elutes residual peptides
from the system to reduce carryover between samples (see
Notes 6 and 7). The sample files should be named correctly
and saved in the proper folders.
474
Jordan T. B. Stariha et al.