05Quad Scan Modes - Denver, Colorado · Quadrupole Scan Modes 1 Quadrupole Scan Modes This tutorial will cover the various triple quadrupole scan modes described below. The method

Quadrupole Scan Modes 1

Quadrupole Scan Modes This tutorial will cover the various triple quadrupole scan modes described below. The method described here uses direct infusion via the syringe pump using the TurboIonSpray® source on the Q TRAPTM System or the TurboVTM Source on the 4000 Q TRAPTM System.

Information on preparing a standard alpha-casein protein digest can be found in the Preparing a Standard Tryptic Digest of Alpha Casein section of this tutorial.

Neutral Loss Scan (NL)

When an ion loses a diagnostic fragment as a neutral fragment, it can be detected by using a Neutral Loss Scan. In this scan, Q1 is scanned across a specific mass range. Ions are passed into the collision cell where they are fragmented. Q3 is scanned over a similar mass range, offset by the neutral mass of the diagnostic fragment. Therefore, any molecule being passed through Q1, which loses a neutral molecule of the defined mass will then be transmitted through Q3 and detected. In positive ion mode, a phosphoserine-containing peptide can lose a neutral molecule of 98 Da (H3PO4) when experiencing mild collisions in the gas phase. Here, the original charge state of the parent is conserved. A doubly charged peptide with a phosphoserine residue can therefore produce a doubly charged product ion that is 49 m/z lower than the original mass. This can be specifically detected using a neutral loss scan.

Precursor Ion Scan (Prec)

When an ion loses a diagnostic fragment as a charged fragment, it can be detected by using a Precursor Ion Scan. In this scan type, Q1 is scanned across a mass range, and ions are fragmented in the collision cell. Q3 is set to transmit only the mass of the diagnostic fragment. Therefore, only ions that are passed through Q1 that fragment to produce the diagnostic charged fragment will be detected. In negative ion mode, a phosphoserine-containing peptide can lose a charged molecule of mass -79 (PO3) when experiencing collisions in the gas phase. This diagnostic fragment can be specifically detected using a precursor ion scan.


Multiple Reaction Monitoring (MRM)

Multiple Reaction Monitoring (also known as Single Reaction Monitoring) is the scan type with the highest duty cycle and is used for monitoring a one or multiple specific ion transition(s) at high sensitivity. Here, Q1 is set on the specific parent mass (Q1 is not scanning), the collision energy is optimized to produce a diagnostic charged fragment of that parent ion, and Q3 is set to the specific mass of that fragment. Only ions with this exact transition will be detected. Many MRM scans can be looped together into one experiment to detect the presence of many specific ions (peptides or small molecules) in a complex mixture.


5.1 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 and 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.


5.2 Infusing the Alpha-Casein Digest 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 alpha casein tryptic digest solution diluted to 500 fmol/µL into 30% acetonitrile / 0.1% formic acid 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.

In this window, the syringe diameter and flow rate can be defined. For a 1 mL syringe, the syringe diameter is 4.61 mm (see table in Table of Hamilton Syringe Diameters for other diameters). Click Start Syringe Pump once the parameters have been adjusted. Use a flow rate of 5-10 uL/min for infusion of the calibrant.

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 alpha casein tryptic digest solution diluted to 100 fmol/µL into 30% acetonitrile / 0.1% formic acid 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.

To quickly check that the digest is infusing, perform an EMS scan, choose the Enhanced MS scan type from the Scan type menu. Enter a mass range from 400 – 1400. For more information about the EMS scan, see the LIT Scan Modes tutorial.

Set all the values on the Source/Gas tab to the values shown below for the TurboIonSpray® source. If the NanoSprayTM source is being used to infuse the peptide, then set the IS to 1000 for static nanoinfusion, 2300V for nanoflow and the GS1 to 0. For the MicroIonSprayTM source head, set the voltage of 2000 – 3300 V and the GS1 to 10-15.


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 (5 cycles x 2 scans) scans performed. Turn MCA on.

The parameters under the Compound tab and the Advanced MS tab should be set as shown below.

⇒ 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.


⇒ For the Q TRAPTM System, a typical EMS spectrum for Alpha-casein digest is shown below.

⇒ For the 4000 Q TRAPTM System, a typical EMS spectrum for Alpha-casein digest is shown below.

Once a good spectrum is obtained, click the Acquire button to save a spectrum to the disk. Name the data file casein EMS. Now, the triple quadrupole scan modes can be tested.


5.3 The Neutral Loss Scan (NL) When an ion loses a diagnostic fragment as a neutral fragment, it can be detected by using a Neutral Loss Scan. In this scan, Q1 is scanned across a specific mass range. Ions are passed into the collision cell where they are fragmented. Q3 is scanned over a similar mass range, offset by the neutral mass of the diagnostic fragment. Therefore, any molecule being passed through Q1, which loses a neutral molecule of the defined mass will then be transmitted through Q3 and detected.

Change the Scan type to Neutral Loss (NL) from the Scan Type menu. Enter a mass range of 500-1400 with a Time of 3 sec. Set the mass of the neutral loss fragment in the Loss of window. In this example, phosphorylated serines produce a neutral loss of 98, or a loss of 49 from the doubly charged ions. Set the number of Cycles to 5.

Settings on the Source/Gas tab can remain the same as above.

Note: Sometimes improved sensitivity can be achieved by using a CAD gas setting of Medium.

Click the Resolution tab and set the Q1 resolution and Q3 resolution values both to Low.

Click the Advanced MS tab and adjust the Step size to 0.25. The step size and the time of the scan determine the dwell time for the scan. The dwell time is the length of time spend acquiring signal at each step in the quadrupole scan. In this example, the quadrupoles are scanning a 900 amu mass range taking 0.25 amu steps, therefore there are 3600 steps across the mass range. If this takes 3 seconds to scan, the dwell time is 0.8 ms (Dwell time = 3 s / 3600 steps). It is recommended that a dwell time of over 0.5 ms be maintained.


Click the Compound tab and set the collision energy to 35 eV

Click the Acquire button and acquire an NL scan with 5 cycles. Name the data file casein NL49.

⇒ For the Q TRAPTM System, a typical Neutral loss spectrum for Alpha-casein digest is shown below.

⇒ For the 4000 Q TRAPTM System, a typical neutral loss spectrum for Alpha-casein digest will be similar to that shown above with ~5 - 10x greater sensitivity.

On the Navigation Bar, click Open Data File and select the casein EMS data file to open. Also, open the casein NL data file. If the panes are full screen, click the button to tile the panes so both spectra can be seen.

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 between the two scans, the ions that shown intensity in the neutral loss scan are producing a neutral loss of 49.

In the above neutral loss scan, the same collision energy was applied to all ions across the mass range. Often, better fragmentation is achieved if the collision energy is ramped across the mass range. To do this, right-click in the gray area inside the mass range box to open the menu, then click the Collision Energy CE.

Two additional columns will appear in the mass range box to the right of the Time (sec) column. Enter the CEstart and CEstop collision energy for the specified mass range; a recommended collision energy range would be 25 to 50 eV over the 500 - 1400 mass range.

Try acquiring a spectrum with ramped collision energy and compare to the above neutral loss scan.

This neutral loss scan was acquired using Low resolution on both Q1 and Q3, this will afford the most sensitivity to the scan. However, unit resolution can be used on either or both Q1 and Q3 to narrow the mass window passed at each mass step and increase specificity. This will cause a decrease in signal intensity as the transmission window for the quadrupole is narrowed. Try collecting a neutral loss scan with different combinations of unit and low resolution to observe the effects.


Note: The collision energies required for producing the diagnostic fragments for a particular peptide modification will vary. The range of collision energies required can be estimated by infusing standards containing the modification of interest, performing Product Ion scans or Enhanced Product Ion scans and adjusting the collision energy while monitoring the spectrum. A Product Ion Scan will give the best estimate of the collision energy range required as it most closely mimics the conditions for the neutral loss scan.


5.4 Multiple Reaction Monitoring (MRM) Multiple Reaction Montioring (also known as Selected Reaction Monitoring) is the scan type with the highest duty cycle and is used for detecting a specific molecule at high sensitivity. Here, Q1 is set on the parent mass, the collision energy is optimized to produce a diagnostic charged fragment of that parent ion, and Q3 is set to the mass of that fragment. Only ions with this exact transition will be detected. Many MRM scans can be looped together into one experiment to detect the presence of many specific ions.

In this example, use the Enhanced Product Ion scan to choose MS/MS fragment ions to use for MRM analysis. Choose the Enhanced Product Ion scan type from the Scan type menu. Enter a mass range from 100 – 1700. Ensure that the Polarity is switched back to positive ion mode.

Enter the mass of the precursor ion to be fragmented in the Products Of window, start with the doubly charged ion of mass 976.5.

The Number of scans to sum automatically defaults to 2. Set the number of Cycles to 10. This will result in a total of 20 scans.

The IonSpray Voltage (IS) on the Source/Gas tab needs to be adjusted for positive ion mode, try 5500 V.

⇒ For the 4000 Q TRAPTM System, the mass range will be automatically divided into three Start

– Stop ranges.

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, set this to 48 eV.


Under the Advanced MS tab, choose the scan speed for acquisition. Begin with the 4000 amu/sec scan speed.

⇒ For the Q TRAPTM System, click the Q0 Trapping option on.

⇒ For the 4000 Q TRAPTM System, click the Q0 Trapping option off.

Next, click the Resolution tab and set the Q1 Resolution to Low.

Click the Start button to begin acquisition.


A typical EPI spectrum for the 976.5 2+ ion is shown below.

Pick three of the more intense fragment ions to optimize the MRM settings. As an example, the ions at mass 391.3, 292.2 and 129.1 will be used. To obtain maximal sensitivity, try to optimize a few different possibilities then chose the top 1 or 2 MRM transitions that give the highest signal to be used in the LCMS experiment.

Choose the MRM (MRM) scan type from the Scan type menu. Enter the three transitions of interest in the Q1 Mass and Q3 Mass boxes. In the Time (msec) box, enter a dwell time for each transition. When choosing an appropriate dwell time, ensure that the cycle time does not become too long. Dwell times less than 10 ms are not recommended as sensitivity will decrease. For monitoring just a few transitions, 200ms is a good default

Click the Resolution tab and set the Q1 resolution and Q3 resolution values both to Low.


Now that the transitions are established, the settings for each transition must be optimized. Because different fragment ions are being investigated, the collision energy for each must be individually determined. The Ramp Parameter feature can be used to determine the collision energy for each transition. Click Edit Ramp… to open the Ramp Parameter Settings window.

Select the Collision Energy from the Parameter options. Enter the range over which the collision energy will be ramped, 20 to 100 V, and the Step size to be used.

Click OK. Notice that the Ramp Parameter box is now checked.

Now, when the scan is started, the collision energy will be ramped across the range specified for the three transitions specified. Click Start to begin the scan. Once the data has been acquired, right-click the data and select Open File. This will open the window show below.

Three curves are generated, each one representing one of the three transitions. From the shape of the curve, the optimal CE that gives the best signal can be determined for each MRM. To determine which color represents which transition, right-click the blue square in the top left hand corner of the pane to reveal the window below.


Notice that the MRM transition of 976.5 to 129.1 with a collision energy of 85V produces the highest signal.

The collision cell exit potential can also be optimized in a similar way. Click the Edit Ramp… button to open the window below. Choose the Collision Cell Exit Potential from the Parameter options. Enter the Start / Stop range and the Step size shown below for this parameter. Click OK.

Click Start to begin the scan. Once the data has been acquired, right-click the data and select Open File. This will open the window show below.

Choose the CXP value that is the optimal setting for each transition. The Declustering Potential DP can also be optimized for MRM transitions following the same procedure for ramping parameters.


Now, the optimal collision energy value and the optimal collision cell exit potential have been determined for each MRM transition.

These values can now be built into the method for each specific MRM scan. To do this, right-click in the gray area inside the mass range box to open the menu, then click the Collision Energy CE.

Two additional columns will appear in the mass range box to the right of the Time (sec) column. Enter the CE(volts) and CXP(volts) values for each transition determined from the parameter ramping experiment.

Repeat for the Collision Cell Exit Potential CXP.

To check the sensitivity of the optimized MRM experiments, unclick the Ramp Parameter box.


Next, click Start to observe the three MRM experiments. Once the data has been acquired, right-click the data and select Open File. This will open the window shown below.

Because this is an infusion experiment, the MRM traces are horizontal lines. If these MRM experiments were used during an LCMS experiment, these traces would reflect the chromatographic peak shape and retention time for the ions of interest.

These MRM scans were acquired using Low resolution on both Q1 and Q3, this will afford the most sensitivity to the scan. However, unit resolution can be used on either or both Q1 and Q3 to narrow the mass window passed at each mass step and increase specificity. This will cause a decrease in signal intensity as the transmission window for either quadrupole is narrowed. Try collecting the MRM scans with different combinations of unit and low resolution to observe the effects.


5.5 The Precursor Ion Scan (Prec) When an ion loses a diagnostic fragment as a charged fragment, it can be screened for using a Precursor Ion Scan. In this scan type, Q1 is scanned across a mass range, and ions are fragmented in the collision cell. Q3 is set to transmit only the mass of the diagnostic fragment ion. Therefore, only ions that are passed through Q1 that fragment to produce the diagnostic charged fragment will be detected.

Change the solution in the syringe for negative ion mode infusion.

⇒ For the Q TRAPTM System, fill the 1 mL syringe with the alpha casein tryptic digest solution diluted to 500 fmol/µL into 30% acetonitrile / 2.5% ammonium hydroxide solution and place it in the syringe pump on the Q TRAPTM System.

⇒ For the 4000 Q TRAPTM System, fill the 1 mL syringe with the alpha casein tryptic digest solution diluted to 100 fmol/µL into 30% acetonitrile / 2.5% ammonium hydroxide solution id and place it in the external syringe pump.

Note: Negative ion mode infusion can be performed in 0.1% formic acid but often better sensitivity is achieved in negative ion mode when using a more basic solution.

Choose the Precursor Ion (Prec) scan type from the Scan type menu.

Change the Scan type to Precursor Ion (Prec) from the Scan Type menu. Enter a mass range of 500-1400 with a Time of 3 sec. Change the Polarity to negative by clicking on the appropriate radial button. Set the mass of the precursor ion in the Loss of window. In this example, phosphoserines produce a diagnostic fragment of mass –79. Set the number of Cycles to 5.

The IonSpray Voltage (IS) on the Source/Gas tab needs to be adjusted for negative ion mode. For the TurboIonSpray® source, set the value to -4500. If the NanoSprayTM source is being used to infuse the peptide, then set the IS to -800 for static nanoinfusion, from -1500 to - 2100V for nanoflow, and the GS1 to 0. For the MicroIonSprayTM source head, use a voltage from –1500 to -3300V and aGS1 of 10-15. Negative ion mode requires a lower IonSpray voltage due to corona discharge.

Note: Sometimes improved sensitivity can be achieved by using a CAD gas setting of Medium.


Click the Advanced MS tab and adjust the Step size to 0.25. The step size and the time of the scan control the dwell time for the scan. The dwell time is the length of time spend acquiring signal at each step in the quadrupole scan. In this example, the quadrupoles are scanning a 900 amu mass range taking 0.25 amu steps, therefore there are 3600 steps across the mass range. If this takes 3 seconds to scan, the dwell time is 0.8 ms. It is recommended that a dwell time of over 0.5 ms is maintained.

Click the Resolution tab and set the Q1 resolution and Q3 resolution values both to Low. Ensure that the MCA option is clicked on.

Finally, click the Compound tab and set the collision energy to –60 eV. This should provide enough energy to fragment the peptides in the collision cell and create the diagnostic fragment of–79 m/z.

Click the Acquire button and acquire a precursor ion scan with 5 cycles. Name the data file casein prec79.


⇒ For the Q TRAPTM System, a typical precursor ion spectrum for Alpha-casein digest is shown below.

⇒ For the 4000 Q TRAPTM System, a typical precursor ion spectrum for Alpha-casein digest is shown below.

The ions that show intensity in the precursor ion scan are producing a diagnostic ion of mass –79.

On the Navigation Bar, click Open Data File and select the casein prec79 data file to open. Also, open the casein EMS file. If the panes are full screen, click the button to tile the panes, now both spectra can be seen.

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 between the two scans, the ions that show intensity in the precursor ion scan are producing a diagnostic fragment of–79 m/z and are likely phosphorylated.

In the above precursor ion scan, the same collision energy was applied to all ions across the mass range. Often, better fragmentation is achieved if the collision energy is ramped across the mass range. To do this, right-click in the gray area inside the mass range box to open the menu, then click the Collision Energy CE.

Two additional columns will appear in the mass range box to the right of the Time (sec) column. Enter the CEstart and CEstop collision energy for the specified mass range; a recommended collision energy range would be -40 to -90V over the 500 - 1400 mass range.

Try acquiring a spectrum with ramped collision energy and compare to the above precursor ion scan.

This precursor scan was acquired using Low resolution on both Q1 and Q3, this will afford the most sensitivity to the scan. However, unit resolution can be used on either or both Q1 and Q3 to narrow the mass window passed at each mass step and increase specificity. This will cause a decrease in signal intensity as the transmission window on either quadrupole is narrowed. Try


collecting precursor ion scans with different combinations of unit and low resolution to observe the effects.

Note: The collision energies required for producing the diagnostic fragments for a particular peptide modification will vary. The range of collision energies required can be estimated by infusing standards containing the modification of interest, performing Product Ion Scans or Enhanced Product Ion scans and adjusting the collision energy while monitoring the spectrum. Product Ion Scan will give the best estimate of the collision energy range required as it most closely mimics the conditions for the neutral loss scan.


5.6 Appendix Preparing a Standard Tryptic Digest of Alpha Casein

Alpha Casein protein can be purchased from Sigma (C-6780).

Trypsin can be purchased from Promega (Cat# V5111).

1. Prepare a 1 nmol/uL solution of Alpha Casein protein in 25 mM Ammonium Bicarbonate (pH should be ~8.0 without adjusting the pH). The molecular weight of alpha casein is 22960 g/mol.

2. Dissolve 20ug of Promega trypsin (1 vial) in 20 uL of 25 mM Ammonium Bicarbonate. (This enzyme solution can be pre-warmed at 37C for 15 minutes before use to enhance initial activity)

3. Combine 30 µL of protein stock solution with 20 µL of enzyme and 2.5 µL of acetonitrile. Place this mixture at 37 °C for four hours to digest. The digest should be complete after four hours.

4. Add 450 µL of a 2% acetonitrile / 0.1% trifluoracetic acid solution to quench the reaction and dilute to a working stock concentration of ~60 pmol/µL.

5. Store at –20C.

Table of Hamilton Syringe Diameters

Volume (µµµµL)

Diameter (mm)

1000 4.61

500 3.26

250 2.3

100 1.46

50 1.031

25 0.729

10 0.46

Documents

05Quad Scan Modes - Denver, Colorado · Quadrupole Scan Modes 1 Quadrupole Scan Modes This tutorial will cover the various triple quadrupole scan modes described below. The method