Linear Ion Trap Scan Modes
This tutorial will cover the various Linear Ion Trap (LIT) scan modes described below on the Q
TRAPTM System and the 4000 Q TRAPTM System. The method described here uses direct infusion
via the syringe pump in positive ion mode using the TurboIonSpray® source on the Q TRAPTM
System or the TurboVTM Source on the 4000 Q TRAPTM System. Information on preparing
GluFibrinopeptide B standard solutions can be found in the Appendix.
Enhanced MS Scan (EMS)
The Enhanced MS scan is the standard ion trap MS scan. Ions are transmitted from the source
through the quadrupoles (quadrupoles are in RF only mode) into the ion trap. After the ion trap is
filled, the ions are scanned out axially to the detector.
Enhanced Multiply Charged Scan (EMC)
The Enhanced Multiply Charged Scan is a standard ion trap MS scan that can be used to improve
the signal/noise ratio on multiply charged ions. Ions are transmitted from the source through the
quadrupoles (quadrupoles are in RF only mode) into the ion trap. Once the ion trap is filled, the
singly charged ions are emptied from the ion trap, leaving predominantly the multiply charged
ions behind. Ions are then scanned out axially to the detector.
Enhanced Resolution (ER)
This scan mode allows for high resolution MS to be obtained of an ion of interest. The first mass
scanning quadrupole (Q1) is set to transmit a mass window of 6-8 amu around the ion of interest.
This ion is stored in the ion trap, then scanned out at a slow scan speed (250 amu/sec) over a
narrow (~30 amu) mass range. Peak widths of ~0.15 – 0.30 amu (width at half of intensity
maximum, FWHM) can be achieved in this scan mode.
Enhanced Product Ion Scan (EPI)
The Enhanced Product Ion Scan is used to obtain a high quality MS/MS spectrum on a specific
ion. The fragmentation is done in the collision cell and thus provides the information rich MS/MS
spectrum typical of collisionally activated dissociation fragmentation. In this scan mode, the
precursor ion to be fragmented is first selected in Q1 with a mass window of 1-4 amu wide,
filtering out all other ions. The precursor ion is fragmented by collisionally-activated dissociation
(CAD) in the Q2 collision cell. The fragment ions generated are captured in the ion trap; then
scanned out at one of three scan speeds, depending on the required fragment ion resolution.
MS/MS/MS Scan
The MS/MS/MS Scan can be used to obtain further information on a specific MS/MS fragment
ion. In this scan mode, the first parent ion to be fragmented is selected in Q1 with a mass window
of 1-4 amu. The ion is then fragmented in the collision cell by CAD. The fragment ions are
transferred to the ion trap where one of the resulting fragment ions is selectively isolated (isolation
width of 1-5 amu). After isolation, the ion is excited using a single wavelength excitation
frequency (~1 Da excitation width) to induce fragmentation (energy of excitation and excitation
time are controlled by user). Resulting fragment ions are captured and then scanned out of the ion
trap at one of the three scan speeds. Because the isolation and fragmentation steps are both
occurring in the linear ion trap for the second MS/MS step, the bottom 1/3rd of the spectrum will
be cut-off. This is typical of fragmentation done in an ion trap and is referred to as the 1/3rd cutoff
rule. When mass isolation is done in an ion trap (3D or linear), a specific q-value must be used for
Linear Ion Trap Scan Modes
1
that ion to be stable in the ion trap. At this q-value, only fragment ions produced with m/z values
of 30% of the parent mass and higher will also be stable in the ion trap.
Time Delayed Fragmentation Scan (TDF)
This scan mode can be used to simplify the low mass portion of an MS/MS spectrum of a peptide.
After a TDF scan or a peptide ion, predominantly y-ions are found in the lower region of the
spectrum, making this scan a good tool for determining the sequence of a peptide de novo. In
TDF, ions are activated in the lower pressure region between Q2 and Q3. Because of the lower
pressure, the relaxation rate of the activated ions is reduced. Higher energy ions (which have the
possibility of producing secondary fragment ions that complicate the low mass portion of the
spectrum) will fragment rapidly and are not stored in the trap. The slower low energy
fragmentation events are stored in the trap and will predominate the lower half of the spectrum in
a TDF scan.
After collisional activation of the parent ion, the ions are held in the ion trap and allowed to
fragment for a specified period of time (Q3 cool time). The fragment ions produced with masses
lower than the Q3 fill mass are ejected from the ion trap. After the Q3 cool time, the entire mass
range is stored in the ion trap. The trapped fragment ions are then scanned out of the ion trap.
Ion Trap MS/MS Scan
MS/MS can also be performed in the ion trap by using the MS/MS/MS scan function. In this scan,
the parent ion is first selected in Q1. No collision energy is used so the peptide mass can be
selected and isolated in the ion trap. Now, excitation energy can be applied and the peptide will be
fragmented in the ion trap. These fragment ions can then be scanned out of the trap at one of three
scan speeds. Because the isolation and fragmentation steps are both occurring in the linear ion
trap, the bottom 1/3rd of the spectrum will be cut-off. This is typical of fragmentation done in an
ion trap and is referred to as 1/3rd cutoff rule.
Q0 Trapping
Turn on Q0 trapping to increase the sensitivity of the scan in most linear ion trap scan types. When
Q0 trapping is turned on, ions produced at the source are accumulated in Q0 during the scanning
of the linear ion trap. When the ion trap is ready to be filled again, the ions from Q0 are released.
Dynamic Fill Time (DFT)
Dynamic Fill Time (DFT) is a feature specifically designed to optimize the data obtained in every
spectrum for the linear ion trap scan functions. DFT will automatically adjust the fill time used to
fill the ion trap based on the ion flux coming from the source. For more intense ions, the fill time
will be automatically reduced to ensure the trap is not overfilled with ions. For less intense ions,
the fill time will be automatically increased, ensuring that good ion statistics are obtained in the
spectrum. DFT is applicable for the following scan types: Enhanced MS (EMS), Enhanced
Resolution (ER), Enhanced Product Ion Scan (EPI) and MS/MS/MS (MS3).
AutoFrag
AutoFrag is a feature designed to improve the fragmentation quality of the Enhanced Product Ion
spectrum. The collision energy used during the filling of the ion trap is stepped across a range of
energies, ensuring that the optimal collision energy is reached. A collision energy (CE) and a
collision energy spread (CES) are specified, the collision energy is the center value and the spread
determines the energy values on either side. For example, if a CE of 30 V is specified with a CES
of 5 V, the trap fill time will be split into three and collision energies of 25, 30 and 35 V will be
used in each third of the trap fill time. AutoFrag can also be used for the first fragmentation event
in MS/MS/MS experiments.
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4.1
Creating a Hardware Profile
Open up the Hardware Configuration Editor by clicking on the Hardware Configuration entry on
the Navigation bar (left hand side of the screen).
⇒ For the Q TRAPTM System with the integrated syringe pump, click the New Profile button to
create a new hardware profile and name it ‘MS + syringe’. Select Add Device and choose the
Mass Spectrometer from the list of peripherals. Click OK to make it appear in the Edit
Hardware Profile window. Click Setup Device. Ensure the integrated syringe pump is
activated by checking the box Use integrated syringe pump at the bottom of the pane. Return
to the Hardware Configuration Editor by clicking OK, twice.
⇒ For the 4000 Q TRAPTM System, the syringe pump is external and no specific modification is
required in the hardware profile. Click the New Profile button to create a new hardware
profile and name it ‘MS only’. Select Add Device and choose the Mass Spectrometer from the
list of peripherals. Click OK to make it appear in the Edit Hardware Profile window. Return
to the Hardware Configuration Editor by clicking OK, twice.
Once back in the Hardware Configuration Editor, click Activate Profile. The computer will
establish communication with the devices in the profile and checkmark will turn green. Close the
editor.
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4.2
Infusing the GluFibrinopeptide B
Create a new project named Training by selecting Projects>Create Project from the Tools menu.
Once the hardware profile has been activated, click Tune in the navigation bar (left-hand panel),
then click the T icon to put the instrument into ‘Ready’ mode. You will hear the gases come on as
the instrument becomes active.
Double click Manual Tuning in the navigation bar to open a blank template.
⇒ For the Q TRAPTM System, fill the 1 mL syringe with the 500 fmol/µL GluFibrinopeptide B
solution and place it in the syringe pump on the Q TRAPTM System. To start the syringe
pump, select Syringe Pump Method from the menu by MS Method.
Define the syringe diameter and flow rate in this window. For a 1 mL syringe, the syringe
diameter is 4.61 mm (see the Table of Hamilton Syringe Diameters in the appendix for other
diameters). Use a flow rate of 5-10 µL/min for infusion of the calibrant. Click Start Syringe Pump
once the parameters have been adjusted.
Note: If the flow rate or syringe diameter must be changed once the syringe has been started, enter
the new value and click Set Flow Rate. Changes will not take effect until the Set Flow Rate button
is clicked.
⇒ For the 4000 Q TRAPTM System, fill the 1 mL syringe with the 50 fmol/µL GluFibrinopeptide
B solution and place it in the external syringe pump. Set the diameter on the syringe pump to
4.61 and the flow rate to 5-10 µL/min.
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4.3
The Enhanced MS Scan (EMS)
The Enhanced MS scan is the standard ion trap MS scan. Ions are transmitted from the source
through the quadrupoles (quadrupoles are in RF only mode) into the ion trap. After the ion trap is
filled, the ions are scanned out axially to the detector.
Choose the Enhanced MS scan type from the Scan type menu. Enter a mass range from 400 – 1300
amu.
Set all the values on the Source/Gas tab to the values shown below for the TurboIonSpray® source
or TurboVTM Source.
The Number of scans to sum automatically defaults to 2. Set the number of Cycles to 5. This will
result in a total of 10 scans. Click MCA on to average together all of the scans.
The parameters under the Compound tab and the Advanced MS tab should be set as shown below.
The Declustering Potential can be adjusted to improve the declustering in the sample interface.
Values of 40-50 V work well when ionizing peptides.
Under the Advanced MS tab, the scan speed is chosen. Begin with the 4000 amu/sec scan speed.
Note: Whenever the scan speed is changed (Advanced MS tab), the number of cycles will need to
be readjusted. In this window, the duration time is held constant and the number of cycles is
adjusted.
Do not use Q0 trapping for this scan type. Do not adjust the Step Size.
Note: Typically, the Q3 Entry Barrier does not need to be adjusted. However, if you are
working with easily fragmented ions, decreasing the Q3 Entry Barrier can minimize fragmentation
of molecular ions.
Adjust the Fixed LIT fill time based on the strength of your ion signals. For concentrated samples,
lowering the trap fill time will reduce space charge and provide better resolution. Increase the fill
time to sample more ions for low concentration samples.
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⇒ For the Q TRAPTM System, the default fill time of 20 ms is a good starting point.
⇒ For the 4000 Q TRAPTM System, reduce the fill time to 5 ms as a starting point.
Now that the acquisition method has been created and the syringe pump is infusing, data
acquisition can begin. Click the Start button, acquisition will begin and two new panes will appear
in the Manual Tune window. The Total Ion Chromatogram (TIC) for the acquisition (left pane)
represents the total number of ions reaching the detector in each scan. The right hand pane (shown
below) contains the current spectrum that is being acquired.
⇒ For the Q TRAPTM System, a typical EMS spectrum for GluFibrinopeptide B is shown below.
Because MCA is turned on for this acquisition, the intensity of the fragment ion peaks will
increase as the data is summed for 5 cycles.
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⇒ For the 4000 Q TRAPTM System, a typical EMS spectrum for GluFibrinopeptide B is shown
below. Because MCA is turned on for this acquisition, the intensity of the fragment ion peaks
will increase as the data is summed for 5 cycles.
To zoom in on a region of the spectrum, drag the mouse below the X-axis around the desired
region. To zoom back out, double-click below the X-axis. Zoom in on the ion at 786 m/z to view
the isotopic cluster of this peptide. If the view has been expanded too far, hold down the control
key and right click the spectrum. Selecting Undo Zoom will zoom out one level.
Click the Acquire button and save a spectrum to the disk, using 5 cycles, MCA turned on and a
Trap Fill time of 20 ms. Name the data file GluFib EMS 4000amu
Now, try adjusting the ion trap fill time and observe the changes in the signal intensity and peak
shape. Notice that if the ion trap fill time is decreased, the peak resolution may improve. When
more concentrated samples are measured, peak broadening may be observed and can be due to the
effects of space charge within the ion trap. Reducing the fill time lets fewer ions into the ion trap
and reduces the space charge effects.
Try the 1000 amu/sec scan speed and observe the changes in the peak width. Reducing the scan
speed can improve the resolution of the ions, but the scan will take longer.
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4.4
The Enhanced Multiply Charged Scan (EMC)
The Enhanced Multiply Charged Scan is a standard ion trap MS scan that can be used to improve
the signal/noise ratio on multiply charged ions. Ions are transmitted from the source through the
quadrupoles (quadrupoles are in RF only mode) into the ion trap. Once the ion trap is filled, the
singly charged ions are emptied from the ion trap, leaving predominantly the multiply charged
ions behind. Ions are then scanned out axially to the detector.
Change the Scan type to Enhanced Multi-Charge from the Scan Type menu. All of the MS tab
settings can remain the same.
Click the Advanced MS tab and observe the Q3 Empty Time defaults to 100 ms. This setting
controls the length of time the ions are held in the ion trap and the singly charged ions are
emptied. The longer this time is, the more the enhancement in signal/noise will be.
Note: the multiply charged ions will also decrease in intensity, but at a slower rate than the singly
charged ions.
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⇒ For the Q TRAPTM System, start with a LIT fill time of 20 ms and a Q3 Empty time of 100 ms
with Q0 Trapping clicked on. A typical spectrum is shown below.
⇒ For the 4000 Q TRAPTM System, start with a LIT fill time of 10 ms and a Q3 Empty time of 50
ms with Q0 Trapping off. There is an additional parameter called MCS barrier (multiple
charge separation) on the 4000 QTRAP system for this scan type. This value is normally close
to 2, but can be optimized to improve S/N for multiply charged ions. A typical spectrum is
shown below.
Click the Acquire button and acquire an EMC scan with 5 cycles and name it GluFib EMC
Q0trapping.
Notice the background ions have decreased relative to the peptide ion. Also, the total number of
counts of the GluFibrinopeptide B parent ion is also higher in this scan relative to the EMS scan in
the previous section. This is because the Q0 trapping has been turned on. Try clicking off the Q0
trapping and observe the signal intensity of the parent ion. The Enhanced Multiply Charged scan
generally will produce the largest improvement in signal to noise when Q0 trapping is turned on.
On the Navigation Bar, click Open Data File and select the GluFib EMS 4000 amu data file to
open. Also, open the GluFib EMC Q0 trapping file. If the panes are full screen, click the
button to tile the panes, now both spectra can be seen.
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Click the Truck icon
(it will turn gray in color) to drag one spectrum into the other spectrum
pane. Click the spectrum to be moved, then move the mouse to the edge of the pane (a cross with
arrows will appear). Click and hold the mouse, then move into the other pane and release. The two
spectra will be in the same pane. They can be arranged side by side or up and down by clicking on
the cross at the edge of a pane and moving the gray box into the desired position. Multiple panes
can be trucked into the same pane. Compare the signal to noise seen between the two scans.
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4.5
The Enhanced Resolution Scan (ER)
This scan mode allows you to obtain high resolution MS for an ion of interest. The first mass
scanning quadrupole (Q1) is set to transmit a mass window of 6-8 amu around the ion of interest.
This ion is stored in the ion trap, then scanned out at a slow scan speed (250 amu/sec) over a
narrow (~20 amu) mass range. Peak widths of ~0.15 – 0.3 amu (width at half of the intensity
maximum) can be achieved in this scan mode.
Choose the Enhanced Resolution (ER) scan type from the Scan type menu. Enter the mass of the
ion of interest, 785.9 in the Center window as shown below. Multiple ER scans can be performed
in one experiment by entering multiple masses in different rows of this window. Set the number of
cycles to 5. Ensure that MCA is clicked on.
Click the Resolution tab and notice that the default Resolution on Q1 is Open.
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Next, click the Advanced MS tab and note that the default scan speed is set to 250 amu/sec.
Ensure Q0 Trapping is clicked off.
⇒ For the Q TRAPTM System, start with a Fixed LIT fill time of 20 ms.
⇒ For the 4000 Q TRAPTM System, start with a Fixed LIT fill time of 10 ms.
Click the Start button, collect one scan then click Stop. (Sometimes when Q0 trapping is on in a
previous scan, the first cycle of the next scan will contain the stored ions which are released from
Q0.)
Click the Acquire button and name the data file GluFib ER. Click OK to begin data acquisition.
⇒ For the Q TRAPTM System, a typical spectrum is shown below.
Turn Q0 Trapping back on. Click the Start button, collect one scan then click Stop. (When Q0
trapping is turned, the first cycle of the next scan will not contain any stored ions as it takes one
cycle to turn the Q0 trapping on) Click the Acquire button and name data file called GluFib ER
Q0T on.
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⇒ For the 4000 Q TRAPTM System, a typical spectrum is shown below.
⇒ Turn Q0 Trapping back on. Click the Start button, collect one scan then click Stop. (When Q0
trapping is turned, the first cycle of the next scan will not contain any stored ions as it takes
one cycle to turn the Q0 trapping on) Click the Acquire button and name data file called
GluFib ER Q0T on.
Notice the increase in intensity in the Enhanced Resolution spectrum when Q0 trapping is turned
on. Also, notice the peaks are broader in the more intense spectrum. There is space charge
occurring in this spectrum.
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Measuring Peak Width and the Graph Info Window
To measure the peak width on the peaks, open the Graph Info Window under the View menu.
When the mouse is dragged across a peak on the spectrum, the resolution and m/z of the
centroided peak will appear in this window.
You can dock this Graph Selection Info window and make it an integral part of the view by rightclicking on the window and checking Allow Docking. Now the window can be moved to the left
hand side of the screen under the Navigation Tool Bar.
To keep the Graph Selection Info window open and save this screen set-up, hold down the control
key and press the ‘0’ (zero) key while.
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4.6
The Enhanced Product Ion Scan (EPI)
In this scan mode, the precursor ion to be fragmented is first selected in Q1, filtering out all other
ions. The precursor ion is fragmented by collisionally-activated dissociation (CAD) in the Q2
LINACTM collision cell. The fragment ions generated are captured in the ion trap; then scanned out
at a fast scan speed.
Choose the Enhanced Product Ion scan type from the Scan type menu. Enter a mass range from
100 – 1700. Notice as you enter the mass range, the range is split into two separate ranges (three
mass ranges for the 4000 Q TRAPTM system. This feature automatically optimizes the quadrupole
transmission steps for the mass range chosen. The scan range can be adjusted by changing either
the low or high end of the mass range, then clicking the Optimize Masses button. Alternatively, the
entire mass range can be deleted and re-entered by deleting the rows in the table. To delete a row
in this table, click the gray box containing the row number and hit the Delete key.
Enter the mass of the precursor ion to be fragmented in the Products Of window. It is
recommended that this number be entered to the first decimal place, especially if the BioAnalyst
Software manual sequencing tools are to be used on the resulting spectrum.
Set all the values on the Source/Gas tab to the values shown below for the TurboIonSpray® source
or TurboVTM Source.
The Number of scans to sum automatically defaults to 2. Set the number of Cycles to 50. This will
result in a total of 100 scans. Click the MCA option off.
⇒ For the 4000 Q TRAPTM System, the mass range will be automatically divided into three Start
– Stop ranges.
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The parameters under the Compound tab and the Advanced MS tab should be set as shown below.
The collision energy will affect the amount of fragmentation obtained in the collision cell, this can
be adjusted during acquisition. Set this to 20 eV to start. After acquisition is started, the collision
energy can be increased until a nice fragmentation pattern is observed
Under the Advanced MS tab, the scan speed for acquisition is chosen. Begin with the 4000
amu/sec scan speed.
⇒
For the Q TRAPTM System, start with a Fixed LIT fill time of 20 ms. To further improve the
sensitivity of this scan, click the Q0 Trapping option. When Q0 trapping is activated, ions
from the source will be stored in the Q0 region while the linear ion trap is scanning out the
ions for detection.
⇒ For the 4000 Q TRAPTM System, start with a Fixed LIT fill time of 20 ms. Do not turn on Q0
trapping as there should be plenty of sensitivity.
Next, click the Resolution tab and set the Q1 Resolution to Low.
Note: The width of the Low Resolution transmission window in Q1 can be adjusted by the user.
Under the Tools menu, select Settings and Tuning Options and adjust the resolution offset drop for
low resolution. A resolution offset drop of 0.1 will pass a window width of approximately 2-3 Da
wide, a good setting for peptide MS/MS.
Click the Start button to begin acquisition. Increase the collision energy until a fragmentation as
shown below is obtained. Notice as the collision energy is increase, the intensity of the parent ion
decreases and fragment ions are observed. For GluFibrinopeptide B, the optimal collision energy
will be between 33-37 eV. Once the collision energy has been determined, turn MCA on and set
Linear Ion Trap Scan Modes
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the number of cycles to 5. Click Acquire, name the data file GluFib EPI 4000amu and click OK
to start scanning.
⇒ For the Q TRAPTM System, a typical Enhanced Product Ion spectrum is shown below.
⇒ For the 4000 Q TRAPTM System, a typical Enhanced Product Ion spectrum is shown below.
Try collecting the spectrum with and without Q0 trapping to compare the difference in sensitivity.
Also, try the 1000 amu/sec scan speed and compare the peak resolution.
Linear Ion Trap Scan Modes
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4.7
Time Delayed Fragmentation Scan (TDF)
This scan mode can be used to simplify the low mass portion of an MS/MS spectrum of a peptide.
In TDF, ions are activated in the lower pressure region between Q2 and Q3. After collisional
activation of the parent ion, the ions are held in the ion trap and allowed to fragment for a
specified period of time (Q3 cool time). The fragment ions produced with masses lower than the
Q3 fill mass are ejected from the ion trap. After the Q3 cool time, the entire mass range is stored in
the ion trap. The trapped fragment ions are then scanned out of the ion trap.
Change the Scan type to Time Delayed Fragmentation Scan from the Scan Type menu. Set the
Product Of window to the mass of the parent ion to be fragmented, 785.9. It is recommended to
enter the first decimal place of the parent ion. Set the Q3 Fill Mass to 700 (this mass should be at
least 50 amu lower than the parent ion, spectrum below this mass will be affected by the time
delay).
Note: The default value for the Q3 fill mass is always half the precursor mass.
Remember to adjust the Collision Energy (CE) back to 10 on the Compound tab as the
fragmentation is not done in the collision cell.
Check the Resolution tab and ensure the Q1 transmission is set to Low.
Note: The Collision Gas (CAD) setting on the Source/Gas tab can be adjusted to Medium to
decrease the pressure in the collision cell and therefore in the Q2 –Q3 region. This may increase
the effects of the TDF scan as well.
Click the Advanced MS to set the fragmentation energy, TDF CE, to 27 V. This setting cannot be
changed during acquisition. The TDF CE is generally 2/3 to 1/2 the value for collision energy
required in an Enhanced Product Ion scan of the same peptide.
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The Q3 Cool time is the length of time only the higher mass fragment ions are captured in the ion
trap. This can be adjusted to change the appearance of the spectrum, between 2-10 ms, but 5 ms is
a good general default for this setting.
Click the Acquire button and name the data file GluFib TDF 4000amu and click OK to start
acquisition. Notice the lower region of the spectrum is much simplified over the EPI spectrum of
the same peptide.
⇒ For the Q TRAPTM System, a typical Time Delayed Fragmentation spectrum is shown below.
⇒ For the 4000 Q TRAPTM System, the Time Delayed Fragmentation spectrum will look similar
to that shown above. A shorter Q3 Cool Time may be required (2ms).
On the Navigation Bar, click Open Data File and select the GluFib EPI 4000amu data file to
open. Also, open the GluFib TDF 4000amu file. Truck the two spectra into the same pane for
comparison, as described in the The Enhanced Multiply Charged Scan (EMC) section.
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Compare the fragmentation seen between the two scans. In the low mass region of the TDF
spectra, the higher energy fragments like b-ions and internal fragments are removed from the
spectrum, leaving mostly y-ions in the spectrum. This is useful information when trying to
manually determine a de novo sequence on a peptide.
Try altering the activation energy TDF CE and Q3 Cool time, to see how these parameters change
the spectra. As the Q3 Cool time increases the number of ions in the low mass region will
decrease. For example, if you set the Q3 Cool time to 50 ms, you shouldn’t see any ions below
the Q3 Fill mass. The precursors that have survived in the 50 ms will not have enough energy to
fragment
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4.8
MS/MS/MS Scan
In this scan mode, the primary parent ion to be fragmented is first selected in Q1. This ion is
fragmented in the collision cell by CAD. The fragment ions generated are then transferred to the
ion trap where one of the resulting fragment ions is selectively isolated (isolation width of 1-5
amu). After isolation, the ion is excited using a single wavelength excitation frequency (~1 Da
excitation width) to induce fragmentation. The energy of excitation can be controlled by user.
Resulting fragment ions are captured and then scanned out of the ion trap.
Change the Scan type to MS/MS/MS (MS3) from the Scan Type menu. Set the 1st Precursor to
785.9. Next, enter the mass of the secondary fragment to be selected in the Q3 linear ion trap. In
this example, enter the mass of the y4-ion, 480.3 as the 2nd Precursor.
Note: It is recommended to enter the mass to the first decimal place for both ions, but especially
for the 2nd precursor as the excitation width is only 1 amu wide.
The Collision Energy (CE) must be entered on the Compound tab to create the secondary ion, use
the value determined for the Enhanced Product Ion scan (~37 V).
The Excitation Energy (AF2) is the amount of energy used to fragment the second precursor ion.
The mass range to be scanned can be reduced if desired.
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Click the Advanced MS tab and observe the new setting for MS/MS/MS fragmentation. The
Excitation Time defaults to 100 ms. This setting is appropriate for most cases.
Under the Resolution tab, the Q1 resolution should be changed to Low resolution to open up
transmission in the Q1 quadrupole. This will allow the C12/C13 isotopes of the peptide to pass
through Q1, giving higher ion counts and additional charge state information. The Q3 Resolution
can also be set to LIT, to isolate the C12/C13 isotopes of the secondary fragment in linear ion trap.
The efficiency of isolation in the ion trap can be monitored by checking on the No Fragmentation
button on the MS tab. Click the No Fragmentation button, then click Start to begin acquisition.
Observe that both the C12/C13 isotopes are isolated in the ion trap.
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⇒ For the Q TRAPTM System, the isolation of the second precursor ion is shown below.
⇒ For the 4000 Q TRAPTM System, the isolation of the second precursor ion is shown below.
To begin MS/MS/MS, stop acquisition and click off the No Fragmentation button. Set the
Excitation Energy (AF2) to 130 and start acquisition. Observe the fragments generated in the ion
trap. The excitation energy can be adjusted during acquisition. To observe the effects, turn off
MCA, increase the number of cycles and try adjusting the AF2 excitation energy setting.
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⇒ For the Q TRAPTM System, a typical MS/MS/MS spectrum is shown below.
⇒
For the 4000 Q TRAPTM System, a typical MS/MS/MS spectrum is shown below.
Linear Ion Trap Scan Modes
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4.9
Ion Trap MS/MS Scan
MS/MS can also be performed in the ion trap by using the MS/MS/MS scan type. In this scan, the
parent ion is first selected in Q1. No collision energy is used so the peptide mass can be selected
and isolated in the ion trap. Now, excitation energy can be applied and the peptide will be
fragmented in the ion trap. This is interesting to try as it illustrates the difference between
fragmentation obtained by CAD in a collision cell and fragmentation obtained from excitation in
an ion trap. In some cases, the ion trap MS/MS might also yield information complimentary to the
Enhanced Product Ion Scan MS/MS.
Set the 1st Precursor to 785.9 and set the 2nd Precursor to isolate the same mass again in the ion
trap. Set the mass range from 100 – 1500. Make sure the Collision Energy (CE) is set to 10 V as
you don’t want to fragment the parent ion in the collision cell before it is isolated in the linear ion
trap.
⇒ Adjust the Excitation Energy (AF2) to get a nice fragmentation pattern of the peptide in the
ion trap, 120 should work well for this peptide. Once a good energy is set, click Acquire and
name the data file GluFib in trap. For the Q TRAPTM System, a typical MS/MS/MS
spectrum is shown below.
Linear Ion Trap Scan Modes
25
⇒ For the 4000 Q TRAPTM System, a typical MS/MS/MS spectrum is shown below.
On the Navigation Bar, click Open Data File and select the GluFib EPI 4000amu data file to
open. Also, open the GluFib in trap file. Truck the two spectra into the same pane for
comparison, as described in The Enhanced Multiply Charged Scan (EMC) section. Compare
the fragmentation observed in the two scans.
Notice that the spectrum follows the 1/3rd cut-off rule at low mass. This means that fragment ions
at m/z values below 30% of the 2nd Precursor m/z value will not be observed. Because the
isolation of the 2nd Precursor is done in the ion trap, a specific q-value must be used for that ion to
be stable in the ion trap. At this q-value, only fragment ions with m/z values of >30% of the parent
m/z and higher will also be stable in the ion trap.
Linear Ion Trap Scan Modes
26
4.10
Appendix
[Glu1]-Fibrinopeptide B
This peptide is a good standard peptide and is available from Sigma (cat. # F-3261). The
recommended dilution solution is 30% acetonitrile in water with 0.1% formic acid.
⇒ For the Q TRAPTM System, prepare a 500 fmol/uL solution of this peptide.
⇒ For the 4000 Q TRAPTM System, use a 50 fmol/uL solution.
These dilutions will give a good strong signal for performing the various scan functions.
Table of Hamilton Syringe Diameters
Volume
(µ
µL)
Diameter
(mm)
1000
4.61
500
3.26
250
2.3
100
1.46
50
1.031
25
0.729
10
0.46
Linear Ion Trap Scan Modes
27