Information Dependent Acquisition (IDA) 1

Information Dependent Acquisition (IDA) Information Dependent Acquisition (IDA) enables on the fly acquisition of MS/MS spectra during a chromatographic run. Analyst Software IDA is optimized to generate large amounts of useful MS/MS data through the combination of specific survey scans, high resolution scans and sensitive product ion scans. In a typical IDA experiment, an MS survey scan is used to generate a peak list of all ions present. The peak list is subjected to a set of user-defined criteria to filter out unwanted precursor ions. The remaining ions are then submitted for MS/MS. This cycle is repeated throughout the duration of the acquisition, to generate large amounts of informative data. The versatility of the scan functions available on the Q TRAP TM System and the 4000 Q TRAP TM System, makes IDA both very powerful and flexible. You can perform multiple levels of MS/MS and MS/MS/MS in a fully automated way. You can create IDA experiments manually or with the IDA Wizard. This tutorial will cover both methods. It will also describe in detail methods for two important proteomics experiments: a typical method for protein identification a suggested approach for screening peptides mixtures for phosphorylation sites Note: For more information on the individual scan types, see the LIT Scan Modes tutorial or the Quadrupole Scan Modes tutorial. For more information on creating acquisition methods with LC systems, refer to the Agilent or LC Packings tutorials. Information Dependent Acquisition (IDA) 1

7.1 Building an IDA Method for Protein Identification with the IDA Wizard Activating the Hardware Profile Launch Analyst Software and double-click Hardware Configuration Editor from the navigation bar on the left side of the screen. The IDA Wizard Choose the appropriate hardware configuration for the LC system that is connected to your MS system and activate the profile. These peripherals are automatically built into your IDA method, so they must be selected in advance. For more information on setting up hardware profiles, see the LC Packings System tutorial or the Agilent caplc system tutorial. The IDA Wizard is designed to simplify the creation of IDA experiments. The Wizard can create three types of IDA experiments: Enhanced MS >>Enhanced Product This type uses the linear ion trap s Enhanced MS scan as survey scan to trigger the Enhanced Product Ion dependent scan. Enhanced Multi-Charge >> Enhanced Product This type uses the linear ion trap s Enhanced Multiply-Charged scan as survey scan to trigger the Enhanced Product Ion dependent scan. Neutral Loss/Precursor Scan >> Enhanced Product This type uses experiments with a triple quadrupole scan (either the Precursor Ion scan or the Neutral Loss scan) as an MS survey scan to trigger the Enhanced Product Ion scan. (For more information on these scan types, see the Q TRAP TM System LIT Scan Modes tutorial or the Q TRAP TM System Quadrupole Scan Modes tutorial.) To launch the IDA Wizard, double-click the IDA Method Wizard icon in the Acquire section of the navigation bar. Information Dependent Acquisition (IDA) 2

This opens the Create IDA Experiments window shown below. Building a Method with the IDA Wizard The first IDA experiment in this tutorial will be a typical protein identification experiment, in which good quality MS/MS data will be collected on as many peptides as possible. This example will use the Enhanced MS >> Enhanced Product Ion experiment type, though either of the linear ion trap MS scans would be suitable for this experiment. In addition to choosing the type of experiment you wish to create, you can choose to perform an Enhanced Resolution (ER) scan to confirm the charge state of the precursor ion. This option is highly recommended, as better mass accuracy is obtained on the precursor ion and the charge state of the ion can be accurately determined on the fly. The advantages of this information far outweigh the small amount of time it takes to perform this scan. Another option is the use of Dynamic Fill Time (DFT), a feature specifically designed for the linear ion trap scan functions to optimize the data obtained in every spectrum. Select this option for the tutorial. (DFT is explained in more detail in Dynamic Fill Time.) Set the parameters as shown above and then click Next. Setting the parameters for the EMS scan In the next window, set the parameters for the Enhanced MS survey scan. A typical scan range for measuring peptide masses is 400 1200 amu; enter this range in the Start mass / Stop Mass fields. Because the resolution of the precursor ions will be obtained in the ER scan, use the fastest scan rate (4000 amu/sec) for the EMS scan. If there is previous knowledge about the sample amount and you do not intend to use Dynamic Fill Time in the EMS scan, you can set an LIT fill time. Decrease the fill time if it is expected that higher amounts of peptide are present. Increase the fill time if increased sensitivity is required. For the Q TRAP TM System, an LIT fill time of 20 ms is a good starting point. Click Next > to continue. Information Dependent Acquisition (IDA) 3

For the 4000 Q TRAP TM System an LIT fill time of 5 ms is a good starting point. Click Next > to continue. Setting the parameters for the EPI scans In this window, set the parameters for the dependent scan. Typically, MS/MS is performed on the most intense 1-3 peptide ions at a time. Set this in the window shown below. You can set the collision energy (CE) for these Enhanced Product Ion (EPI) scans in two different ways. Up to three discrete collision energies can be defined for each precursor ion selected. For example, if the two most intense ions are selected, and two discrete collision energies are set, there will be four separate EPI scans performed per cycle. For protein identification experiments, it is better to Use Rolling Collision Energy to set the CE for each peptide dynamically. This feature will calculate the optimal collision energy for each peptide based on its m/z and charge state. Rolling Collision energy is described in the section on Setting the IDA Selection Criteria in more detail. Information Dependent Acquisition (IDA) 4

For the Q TRAP TM System, enter a scan range of 100-1700 in the Start mass / Stop mass window. Use the fastest scan rate (4000 amu/sec) for the EPI scan, unit resolution will still be obtained on the fragment ions. When Dynamic Fill Time (DFT) is being used, the LIT fill time will be automatically adjusted. When DFT is not being used, it is recommended to use an LIT fill time of 50 ms; this can be adjusted depending on the sensitivity required. Click Next > to continue. For the 4000 Q TRAP TM System, enter a scan range of 100-2000 in the Start mass / Stop mass window. Use the fastest scan rate (4000 amu/sec) for the EPI scan, unit resolution will still be obtained on the fragment ions. When Dynamic Fill Time (DFT) is being used, the LIT fill time will be automatically adjusted. When DFT is not being used, it is recommended to use an LIT fill time is 20 ms; this can be adjusted depending on the sensitivity required. Click Next > to continue. The width of the precursor isolation window (transmission window in Q1) is controlled by setting the Resolution Q1 parameter. Low resolution is the recommended setting, this will allow a 2-3 m/z window to pass through Q1 into the collision cell. The advantage of using a slightly wider window width is the sensitivity will be improved and the entire isotope envelope of the parent ion will be selected for fragmentation, preserving charge state information in the fragmentation data. Setting the IDA Criteria for the Dependent Scans The selection criteria for the IDA experiment are defined next. To set the mass range for selecting potential precursor ions, enter the lower and upper m/z boundaries in the For ions greater than and For ions smaller than boxes. The Intensity Threshold allows the threshold for the minimum intensity of the ions in the EMS survey scan to be set. Enter the desired intensity threshold in the Which exceeds _ counts (cps) box. Ions whose intensities are below that specified in the intensity threshold will be removed from the peak list and not be submitted for an ER and EPI scan. 50000-100000 cps is a good starting point for the intensity threshold. In the With charge state check box, you can specify the desired charge states of precursor ions to be submitted for MS/MS. When this selection criterion is activated, only the ions in the ER spectrum with the desired charge states will remain in the peak list to be considered for the product ion scan. If the charge state is undetermined from the ER scan, you can choose to include it in the peak list by checking the Include unknowns box. For protein digests, if the charge state is undetermined, it is most likely multiply charged and therefore of interest. Information Dependent Acquisition (IDA) 5

The Exclude Former Target Ions criteria allow you to exclude ions that have already been sent for MS/MS. These ions can be excluded for the remainder of the run by checking Always. When doing LC IDA experiments, select For _ (sec); this invokes exclusion for a limited time window, so the precursor ions eluting during that time will not be sent for MS/MS more than once. If Never is checked, dynamic exclusion is turned off. Finally, to allow an ion to be sent for MS/MS multiple times before exclusion, set the After_ occurrences. If 1 is entered here, the ion will be sent for MS/MS once, and then it is excluded. If 2 is entered, up to two MS/MS spectra will be collected on the same ion before it is added to the dynamic exclusion list. Click Finish to build the IDA method. Information Dependent Acquisition (IDA) 6

7.2 Fine Tuning Your MS Method Analyst Software automatically organizes the Mass Spec experiment in the left hand pane of the Acquisition Method window. Also, if peripherals of the LC system or the integrated syringe pump are defined in the hardware profile, they will automatically be built into the method. Control of these peripherals is described in the other tutorials on configuring your LC systems. Build the pump and autosampler methods into your IDA method now, as described in the other tutorials. Completing the Mass Spec portion of the method Once the pump and autosampler parameters have been defined, the Mass Spec section of the method can be completed. Click on the +EMS entry in the left hand pane of the window to view the survey scan parameters. Enter a Duration time in the right hand window on the MS tab. This time should be not be longer than the length of the pump run and should reflect the time window of the method in which the peptide MS/MS is desired. Information Dependent Acquisition (IDA) 7

Control the survey scan length by choosing Number of scans to sum. Typically, a Total Scan Time of 0.5 to 1 sec or Number of scans to sum of 1-2 should be spent collecting the survey scan. Notice when you change the Number of scans to sum, the Total Scan Time changes accordingly. Setting the ion source parameters Next, click Edit Parameters to set the settings for the source. If the Nanospray TM source and the nanoinfusion source head is being used at flow rates of ~100 500 µl/min, use an IonSpray Voltage (IS) setting of ~1500 to 2300 V (from -1300 to -1700 V for negative ion mode) and a GS1 setting of 0. If the MicroIonSpray TM source head is being used at flow rates of ~100 500 µl/min, use an IonSpray Voltage (IS) setting of ~1800 to 3500 V (from -1500 to -3000 V for negative ion mode) and a GS1 setting of 0. For more information on optimizing the source conditions for nanolc applsications, refer to the section in the tutorials Using the LC Packings Integrated System or Using the Agilent NanoLC System, or refer to your NanoSpray TM Source manual If the TurboIonSpray Source or TurboV TM Source is being used for flow rates above 3 µl/min, set IS to ~5500 V (~ -4500 V for negative ion mode) and Ion Source Gas 1(GS1) and Ion Source Gas 2(GS2) depending on the flow rate (the higher the flow rate, the higher the GS1 and GS2 flow rate should be). Changes to the Source/Gas or Compound tabs can be applied to all experiments by clicking one or both of the check boxes at the bottom of the window. Ensure the Interface Heater is turned on. Click OK to save parameters. Information Dependent Acquisition (IDA) 8

Next, click the ER scan on the left hand side of the window to check the settings for this experiment. There are two entries in the Center column so two ER scans on two separate ions from the survey will be performed. Under the Resolution tab, the Q1 Resolution is set to Open. Notice that the time required to perform the two ER scans is only 316 ms. This additional time is worthwhile because a lot of important information can be gained from these higher resolution scans. Accurate charge state determination and improved mass accuracy on m/z peaks are obtained when the Enhanced Resolution scan is used. To check the settings for the dependent scans, click on the EPI experiments in the left hand pane. Notice when IDA is used, the Product Of window is set to 30. This indicates this value will be set by the IDA software. Do not edit this value. Information Dependent Acquisition (IDA) 9

You can adjust the length of time spent on each product ion scan by changing the Number of scans to sum. A total scan time of 1-2 secs or 1-2 scans to sum are typical settings. Apart from the Number of Scans to Sum, the Total Scan Time depends on the mass range acquired and the Fill Time (which is set on the Advanced MS tab). AutoFrag For each of the ion trap scans in an IDA method, you can define a Number of Scans to sum. When scans are summed together the data quality improves. However, the overall duty cycle is less, as it takes longer to complete the scan. You must decide whether data quality or duty cycle is more important for your specific experiment. 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. You specify a collision energy (CE) and a collision energy spread (CES). (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. To turn on this feature for the EPI scans, click Edit Parameters and enter a value for CES on the Compound tab. Start with a value of 5. In an IDA method, this can be used in addition to Rolling Collision Energy. In this case, the Rolling Collision Energy feature will set the center collision energy, and the Collision Energy Spread value will determine the values on either side of this center value. Information Dependent Acquisition (IDA) 10

7.3 Dynamic Fill Time Dynamic Fill Time (DFT) is a feature specifically designed to optimize the data obtained in every spectrum for the linear ion trap scan types. 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. You can adjust the DFT settings by selecting Tools- > Settings -> Method Options in Analyst Software to display the dialog box below. The TIC Target defines the optimal ion current for each scan type. The trap fill time will be adjusted such that the TIC obtained in each scan is close to this value. The Min and Max Fill Time define the time boundaries of the trap fill time settings. Under Fixed Fill Time, the default fill times for each scan type can be defined, these are only used when DFT is turned off in an acquisition method. Dynamic Fill Time can be applied to all three of these experiment types within an IDA experiment, or only to selected experiments. Information Dependent Acquisition (IDA) 11

Under the Advanced MS tab for each scan type, the option to use DFT or Q0 trapping is available. Q0 trapping To use DFT, click the Dynamic Fill Time radio button. When this is activated, a quick DFT scan will occur before each scan type to measure the ion flux from the source. The trap fill time will be adjusted accordingly for each scan based on this ion flux. Within one IDA cycle, the signal obtained from the previous trap scan can be applied to calculate trap fill times for subsequent scans without having to repeat the DFT scan. This feature is called Use precursor area and is activated by clicking the radio button. It is recommended that this feature be used mainly for the Enhanced Product Ion scan. When DFT is not selected, a fixed trap fill time will be used. You can adjust this fixed time by changing the value in the Fixed LIT fill time field. You can use Q0 trapping instead of DFT. The decision to use DFT or Q0 trapping should be made based on the sensitivity required for each experiment. If there are reasonable amounts of sample available and maximum sensitivity is not required, then dynamic fill time is the best option for ion trap filling. If maximum sensitivity is important, then use Q0 trapping. 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. A typical experimental design for LCMS/MS experiments where a medium to low amount of sample is present would be to apply DFT to the EMS, DFT to the ER scan (to ensure best resolution for assigning charge state), and to use Q0 trapping for the EPI scans (to ensure maximal sensitivity on MS/MS). Note: if Q0 trapping is used for an EPI scan during IDA, use 2 scans to sum. This ensures that the advantages of Q0 trapping are applied to all ion trap mass ranges. Information Dependent Acquisition (IDA) 12

7.4 Setting the IDA Selection Criteria Setting IDA First Level Criteria Click on the IDA Criteria section of the method to observe the settings that were passed from the IDA Wizard. To set the mass range limitations for potential precursor ions, enter the mass range to be considered in the For ions greater than and For ions smaller than boxes. In the With charge state check box, you can specify the desired charge states of precursor ions to be sent for MS/MS. When this selection criterion is activated, only the ions in the ER spectrum with the desired charge states will be remain in the peak list to be considered for the product ion scan. If the charge state is undetermined from the ER scan, you can choose to include it in the peak list by checking the Include unknowns box. For protein digests, if the charge state is undetermined, it is most likely multiply charged and therefore of interest. Note: Make sure the Use Enhanced Resolution Scan to confirm charge state box is checked, otherwise the EMS survey scan is used for charge state determination. The Intensity Threshold allows the threshold for the minimum intensity of the ions in the EMS survey scan to be set. Enter the desired intensity threshold in the Which exceeds _ (cps) box. Ions whose intensities are below that specified in the intensity threshold will be removed from the peak list and not be sent for an ER scan. 50000-100000 cps is a good starting point for the intensity threshold. The Exclude former target ions criteria allows the option to exclude ions that have already been sent for MS/MS. These ions can be excluded for the remainder of the run by checking Always. When doing LC IDA experiments, select For _ (sec); this invokes exclusion for a limited time window, so precursor ions of the same m/z value eluting during that time won t be sent for MS/MS more than once. If Never is checked, dynamic exclusion is turned off. Finally, to allow an ion to be sent for MS/MS multiple times before exclusion, set the After _ repeat occurrences. When 1 is entered here, the ion can be sent for MS/MS one more time before it is excluded, meaning there can be up to two occurrences of MS/MS on that specific peptide ion. Information Dependent Acquisition (IDA) 13

The Mass Tolerance sets the error tolerance for the various include/exclude features and is defaulted to 250 mmu. A reasonable mass tolerance range is 250-1000 mmu, depending on the expected mass accuracy of the MS data. Choosing Exclude isotopes within will specifically exclude 13 C isotope masses above a certain window from the selected precursor ion. A value of three to four amu is usually useful for this feature. This will apply to the specific ion as long as it appears on the variable exclusion list. In determining the related isotopic peaks, the specified mass tolerance is used. Only related isotope peaks will be specifically excluded, not the entire mass window. When Rolling Collision Energy is invoked during IDA, the collision energy will be optimized for each peptide sent for MS/MS based on its m/z and charge state. The equations used to define these m/z vs. CE relationships can be observed by clicking Settings. The software comes with these predefined relationships (linear line defined by a slope and intercept) that are optimized for the fragmentation of normal tryptic peptides. You can adjust these values within each method. Generally, if more fragmentation is desired, the value of the intercept can be increased. +1 CE +2 +3 m/z Setting Inclusion/Exclusion Lists If you know additional information about a sample, Inclusion or Exclusion lists can be created to ensure that the desired information is obtained under the Include/Exclude list tabs. Both the Include and Exclude list are time-filtered and you must enter the ion must as an m/z value (Note: if multiple charge states are to be included/excluded, each m/z value must be entered). Click the Always box to exclude an ion for the entire duration of the run. An ion can be specifically included / excluded for a small window of time, at any point in the run, by entering a retention time and a width for the time window. Specify the intensity threshold (in counts) for the Inclusion list in the Intensity text box. This value is the intensity that the ion must exceed in the survey scan to be considered acceptable for MS/MS. This setting takes precedence over the intensity threshold specified in the First Level Criteria. Information Dependent Acquisition (IDA) 14

The Import button on the Inclusion/Exclusion allows you to import simple text files (space delimited text, with the.txt file extension) to populate either list. Setting Isotope Pattern Criteria In the Isotope Pattern tab, check Match isotopes to use very specific isotopic patterns in the survey scan as switch criteria. Only when this pattern is present that will an ion be sent for MS/MS. Enter the tolerances for the mass difference and the percentage abundance in the left and right Tolerance fields, respectively. These values are the tolerances within which the isotopic pattern can be found for a product ion scan to be carried out on the first peak. Information Dependent Acquisition (IDA) 15

If the exact isotope ratios are not known for a class of compounds, use the Isotope Calculator to calculate them. Click Isotope calculator to open the window shown below. (Refer to the Calculators Tutorial for more information on how to use this calculator.) Enter the formula of interest (Use C 34 H 68 Cl 2 as an example for this data file) and choose Elemental from the menu. Enter the Num of charges in the required polarity to define the charge state of the compound. The isotopic distribution can be viewed as a spectrum or as a text file by clicking on the Graph or Text tab. Click OK to automatically enter the mass differences and abundances into the Match isotopes window. Click to save the method. See the section on Running an IDA Method in this tutorial to use this method to collect data by infusion. To perform an IDA experiment using an LC system, refer to the Agilent LC tutorials or the LC Packings tutorial. Information Dependent Acquisition (IDA) 16

7.5 Manually Building an IDA Method for Phosphopeptide Identification The next IDA experiment will be a typical phosphopeptide identification experiment, where a negative ion precursor ion scan will be used as a survey scan. This type of method can also be built using the IDA Wizard but is shown here to demonstrate how to manually build and IDA method. In this experiment, a precursor ion scan of 79 will be performed. Any ions above an intensity threshold will be considered for MS/MS. The instrument will change to a positive polarity and perform a quick Enhanced Resolution scan to confirm the charge state. If the ion is multiply charged, an Enhanced Product Ion scan will be done. After the cycle is finished, the Q TRAP TM System or the 4000 Q TRAP TM System switches back to negative mode for the next precursor ion scan. All three triple quadrupole MS/MS scan types (precursor ion scan, neutral loss scan and multiple reaction monitoring scan, MRM) can be used as survey scans for IDA. IDA methods using these scans as survey scans are built in a similar way as described in this section or can be built through the IDA Wizard. Please see the Quadrupole Scan Modes tutorial for more information on setting up the individual triple quadrupole scans. In this example, the IDA method will be constructed manually. This type of method can also be constructed using the IDA Wizard. As before, you must first activate the Hardware Profile before you can build the method. Double-click Build Acquisition Method in the Acquire section of the navigation bar. Notice that the Mass Spec experiment is shown in the left hand pane of the Acquisition Method window. Also, if peripherals of the LC system or the integrated syringe pump are defined in the hardware profile, they will be built into the method. Again, control of these peripherals is described in the other tutorials on configuring your LC systems. Information Dependent Acquisition (IDA) 17

Build the pump and autosampler methods into your IDA method now, as described in the LC system tutorials. Completing the Mass Spec portion of the method Once the pump and autosampler parameters have been defined, complete the Mass Spec section of the method. Click on the first MS scan row underneath Period in the left hand pane. Choose the Precursor Ion scan from the Scan type menu. Now set up the parameters for the precursor ion survey scan. Typical settings for a precursor ion scan for phosphopeptides are shown. Click Negative, under Polarity, to set the instrument polarity. Enter the mass of your fragment ion (79) in the Precursor Of window. A good mass range for peptides is 500 1200 amu in 3 seconds. Note: Another alternative is to enter the same mass range three times in the same window with time settings of 1 sec each, as shown below. These three scans will then be averaged together before the IDA criteria are applied. This may improve the signal to noise quality of the data. It is also recommended that the collision energy be ramped across the mass range rather than using one fixed collision energy for all phosphopeptides. To do this, right-click in the gray area inside the mass range box to open the menu, then click on the Collision Energy CE. Two additional columns 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 is 45 to 95 V over the 500-1200 mass range. Information Dependent Acquisition (IDA) 18

Set the Duration time according to your LC Pump method, making sure the MS method ends before the pump method finishes. Click Edit Parameters to set the Source/Gas parameters as described in the section on Fine Tuning Your MS Method. Remember that this is a negative ion mode survey scan so use an appropriate ionspray voltage for negative ion mode. Setting the Advanced MS parameters Click the Advanced MS tab to set more precursor ion parameters. Set a Step size between 0.1 and 0.45. The mass range, scan time and step size all determine the dwell time for each step in the scan. Typically, use a larger step size so the precursor ion survey scan is faster. The Resolution settings you choose for Q1 and Q3 depend on the sample you will be measuring. Tighter resolution will result in a more specific scan, but the signal intensity may be reduced. Note: This is a triple quadrupole scan and therefore does not require DFT. To add another experiment to the IDA method, right-click on the Prec row in the left hand pane and choose Add experiment from the menu. A new row is added to the method. Click on this new row and change the Scan type to Enhanced Resolution and change the polarity back to positive mode. In the box for masses on the right, enter the value 1 in the first row. This will indicate to the software that it is an IDA method and to use the mass of the most intense peak from IDA peak list. If more than one precursor ion is to be selected in each cycle, add an extra row for each precursor ion to be selected and number the rows 1, 2, 3 etc (up to 8 precursor ions can be selected in an IDA cycle). Information Dependent Acquisition (IDA) 19

Click Edit Parameters to set the Source/Gas parameters as described in the section on Dynamic Fill Time. Remember that this is now a positive ion mode scan, so use an appropriate ionspray voltage for positive ion mode. No other parameters need to be changed, but open the Advanced MS tab and notice the settling time. When switching between polarities, there is a 700 ms minimum settling time between scans to allow the power supplies to settle. This settling time is only required in the scan where the polarity has switched, in this case, the ER scan. Finally, you can choose to apply DFT to the ER scan in this IDA experiment by clicking on the radio button. (See section on Dynamic Fill Time for more information.) Information Dependent Acquisition (IDA) 20

Right-click on the ER scan in the left hand pane and choose Add IDA Criteria Level to the method. See section on Setting the IDA Selection Criteria for a detailed explanation of the criteria. Remember, if multiple ER scans and EPI scans are to be done in one experiment, you must adjust the Select 1 to X most intense peaks to represent the number of scans. This should equal the number of rows entered in the ER scan window. You should set the intensity threshold in this type of experiment much lower than the previous experiment because the survey scan is a triple quadrupole scan mode. Typical settings are 200-500 cps for the Q TRAP TM System and 2000 5000 for the 4000 Q TRAP TM System, but you should observe the intensities you are getting in your experiment and adjust the threshold accordingly. Note: It is recommended to set the Mass Tolerance higher in this type of method to take into account the positive to negative polarity switch. A value of 2000 2500 is typical. If multiple precursor ions are to be selected in an IDA cycle, be sure to enter same number into the Select X to X most intense peaks (i.e.. if three rows numbered 1,2,3 have been entered for the ER scans, then Select 1 to 3 must be entered in the IDA criteria page). Finally, right-click on the IDA Criteria and add another experiment. Change this experiment to an Enhanced Product Ion scan type. Do not change the Product Of window in this scan type, it will use the masses determined in the ER scan above. Ensure the positive ion polarity is selected. Information Dependent Acquisition (IDA) 21

Enter the desired mass range; 100-1700 is typical. Notice as you enter the mass range, the range is split into two separate ranges. This feature automatically optimizes the quadrupole transmission steps for the chosen mass range. Change the number of scans to sum to adjust the length of time spent performing MS/MS on each ion. Choose the 4000 amu/sec scan speed on the Advanced MS tab and select DFT or Q0 trapping, depending on the level of sensitivity required. Note: The settling time in the EPI experiment should be 0 as there has been no polarity switch between the ER and the EPI scans. Notice that the settling time has been automatically adjusted in the precursor ion scan to account for the switch back from positive mode to negative mode between the EPI scan and the next precursor ion scan. Click Edit Parameters to set the Source/Gas parameters as described in the section on Fine Tuning Your MS Method. Remember that this is now a positive ion mode scan, so use an appropriate ionspray voltage for positive ion mode. If multiple precursor ions are to be selected and multiple ER scans are being performed, make sure to enter the same number of EPI scans into the cycle. Once one EPI has been created, you can easily duplicate the EPI by right-clicking on the EPI row in the left hand column and selecting Copy experiment. Click on the first precursor ion scan experiment and view the Advanced MS tab. Notice that a 700 ms settling time is applied now, because there is a polarity switch in going from the positive ion mode EPI scan to the next negative ion precursor ion scan in the next cycle. Click to save the method. Information Dependent Acquisition (IDA) 22

7.6 Running an IDA Method You can run an IDA method by either infusion or LC/MS/MS; the procedure for starting the IDA method is similar in both cases. For LC/MS/MS IDA experiments, please refer to the LC Packings or Agilent LC tutorials for setting up the LC portion of the acquisition method. For infusion experiments using an integrated syringe pump on the Q TRAP TM system, start the syringe pump from the Manual Tune window. 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 (see table in Appendix for other diameters). 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 do not take effect until you click Set Flow Rate. For infusion experiments using an external syringe pump the 4000 Q TRAP TM system, enter the correct syringe diameter (see table in Appendix for other diameters) and flow rate on the key pad and press Run. Open Manual Tune from the Tune section of the navigation bar and enter the appropriate ionspray voltage depending on the ion source being used. Set the EMS Mass Range to 400-1500. Click Start to start monitoring the spectrum. Ensure MCA is unchecked so the TIC can be monitored for fluctuation. Adjust the IS in 100V increments until a stable spray is achieved. The position of the spray tip relative to the orifice can also be adjusted until a stable spray is observed. Be careful not to spray straight down the orifice. In addition, the Curtain Gas (CUR), GS1 and GS2 can be adjusted to optimize the signal, use the highest curtain gas setting that doesn t diminish the intensity of the sample ions. Once a stable spray has been achieved, be sure to enter the source parameters determined into the IDA method to be run. Save the method file with the modified parameters. Refer to the Batch Editor tutorial to automatically run the LC method. Information Dependent Acquisition (IDA) 23

Appendix Table of Hamilton Syringe Diameters Volume (µl) Diameter (mm) 1000 4.61 500 3.26 250 2.3 100 1.46 50 1.031 25 0.729 10 0.460 Information Dependent Acquisition (IDA) 24