Analyst TF 1.

6 Software
Information Dependent Acquisition Tutorial

Release Date: June 2012

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Information Dependent Acquisition Tutorial

Tutorial Overview
Information Dependent Acquisition (IDA) experiments are used to obtain both single MS and MS/ MS information about the components in your sample. IDA is useful for any workflow where you do not know the components of your sample. IDA acquisition methods can be created using the Acquisition Method Editor or by editing the template methods using the Method Wizard. For descriptions of the template methods, refer to the Template Methods in Analyst TF Software.pdf file, which is found in the Start > Programs > AB SCIEX > Analyst TF 1.6 Software > Hardware and Software Guides folder.

Topics in this tutorial:

Creating an IDA Method on page 4 Additional IDA Criteria on page 9 Scheduled MRM HR Algorithm on page 18

Objectives
In this tutorial, you will learn how to set up a basic IDA experiment using a simple workflow for high-flow analysis of small molecules in the Acquisition Method Editor.

Related Documentation
System User Guides Advanced User Guide Analyst TF 1.6 software Help system The applicable ion source guide

Required Material and Equipment

The following equipment and solutions are suggested. You can use other solutions depending on your system, peripheral devices, and experiment. Reserpine 10 ng/mL. LC system that includes an isocratic pump. Mobile phase 1:1 acetonitrile:water + 2 mM ammonium acetate + 0.1% formic acid.

Prerequisites
The instrument is tuned and calibrated. You have created an acquisition method. You have created a hardware profile containing a syringe pump. Analyst TF 1.6 Software 3 of 20

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Creating an IDA Method

This procedure describes a basic IDA workflow. To learn how to use IDA parameters for industryspecific workflows, refer to the Template Methods in Analyst TF Software.pdf file, which is found in the Start > Programs > AB SCIEX > Analyst TF 1.6 Software > Software Guides folder. 1. On the Navigation bar, under Acquire, double-click Build Acquisition Method. 2. In the Acquisition method pane, click Period. 3. On the MS tab, select TOF MS from the Scan type list. 4. In the Polarity group, make sure Positive is selected. 5. If required, click Edit Parameters and then edit the Source/Gas and Compound parameters. The following figure shows sample Source/Gas settings if you are using a flow rate of 200 L/min.

Figure 1-1

Parameter Settings dialog

Note: The source, compound, and mass range settings selected for the TOF MS survey scan will be copied to the dependent scan experiment with the exception of the CE parameter. 6. In the TOF Masses (Da) group, set the masses to Min. 100 and Max. 1000. 7. Click Create IDA Exp and then set the IDA filters as required.

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Figure 1-2

IDA Filters

Item Description 1 2 3 Number of survey scans: Set to 1 for this workflow. Maximum number of candidate ions to monitor per cycle: Set to 1 for this workflow. Dynamic background subtract: Select for this workflow.

Note: Infusion IDA is more challenging for the peak finder because of overlapping peaks. When Infusion IDA is selected, the peak finder will assign a +1 charge state and label the ion monoisotopic if the peak finder labels it an unknown (that is, the peak finder cannot determine if the candidate ion is monoisotopic or its charge state). All other user-selected criteria will apply (number of peaks to monitor and so forth). 8. Click OK to close the Create IDA Experiments dialog and create the IDA acquisition method. The resulting IDA acquisition method will have two experiments: the survey scan type (TOF MS) and the dependent scan type (Product Ion) (item 1 in Figure 1-3). The only exception is when you choose to create a Neutral Loss IDA method. In this case, there will be two survey scan type (TOF MS) experiments and one dependent scan type (Product Ion). In both cases, the dependent experiment will be repeated X times per cycle, where X is the maximum number of candidate ions to monitor per cycle, as entered in the Create IDA Experiments dialog (item 2 in Figure 1-2). The High Resolution and High Sensitivity options for the Product Ion scan are only supported for the TripleTOF 5600/5600+ instruments. For TripleTOF 4600 instruments, users can add up to 50 dependant scans in a single experiment in an IDA acquisition method. For TripleTOF 5600/5600+ instruments using the Analyst TF 1.6 software, up to 100 dependent scans can be added to an experiment in an IDA acquisition method.

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Figure 1-3

IDA experiment (for TripleTOF 5600/5600+ instrument)

Item Description 1 2 3 Dependent scan type. Create IDA Exp. Click to open the Create IDA Experiments dialog. IDA criteria tabs. On the MS tab, the IDA experiment check box is selected. Clearing this check box or deleting one of the experiments will deactivate the IDA feature. Additional IDA criteria (item 3 in Figure 1-3) can be accessed through the additional IDA criteria tabs in the Acquisition Method Editor (Switch Criteria, Include/Exclude, and IDA advanced). 9. Set the basic switch criteria filters as follows:

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Figure 1-4 Switch Criteria TabGeneric Filters

Item Description 1 2 3 4 For ions greater than: Limits the mass range for selection of candidate ions. In this example, set to 250. For ions smaller than: Limits the mass range for selection of candidate ions. In this example, set to 750. With charge state: Limits the charge state. Not used. Which exceeds: (Intensity threshold) unit are in counts per second (cps). All spectra are normalized to cps. In this example, set to 750 cps. Exclude isotopes within: This is a symmetrical window around the candidate ion that was selected for MS/MS. Enter the halfwidth of the window (that is, an entry of 4 means that any peak that falls in the window M4 to M+4 will not be selected in the same cycle as M for MS/MS). When this feature is activated, the peak finder considers any unknown peaks as monoisotopic and +1 charge state. In this example, leave the default values. Note: This feature should only be used for small molecule applications. The peak finder algorithm has been optimized for CHNO-type molecules.

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Information Dependent Acquisition Tutorial Figure 1-4 Switch Criteria TabGeneric Filters (Continued)

Item Description 6 Mass Tolerance: This is a symmetrical window around the candidate ion that was selected for MS/MS. Enter the half-width of the window (that is, an entry of 50 mDa means that any peak that falls within the window M50 mDa to M+50 mDa will be considered to be the same peak as M, where M is the m/z of the ion). Select your preferred units by clicking either ppm or mDa. In this example, leave the default value of 50. Maximum number of candidate ions to monitor per cycle: This specifies the number of dependent scans that will be performed per cycle. In the case where there are less candidate ions than the maximum number of MS/MS per cycle, the time spent on MS/MS will be distributed among the candidate ions (that is, the total cycle time will be held relatively constant throughout the acquisition). All returned MS/MS spectra are normalized to cps. Automatically populated with a number from the Create IDA experiments dialog. Exclude former target ions: (Dynamic Exclusion) For this example, leave the default value. Never: Always consider the candidate ion for MS/MS (that is, dynamic exclusion is turned off). Always: After the candidate ion has been selected for MS/MS, never do it again. For X seconds: After the candidate ion has been selected for MS/MS, do not consider it for MS/MS for the next X seconds. It is recommended that this value be set to half the LC peak width. After Y occurrence(s): In combination with Always: Perform MS/MS on the candidate ion Y times, and then never consider it again for MS/MS. In combination with For X seconds: Allows the candidate ion to be selected for MS/MS Y times during X seconds before being put on the exclusion list. Note: Dynamic exclusion routine uses MW (not m/z). Candidate ions with the same MW (within the mass tolerance) but difference charge states will be considered to be the same candidate. The charge state that is the most intense survives on the candidate list if the number of repeats has not been met.

Note: If the real-time peak finder algorithm cannot determine the charge state of an ion, it will be considered as an unknown and will not be selected as a candidate ion unless Infusion IDA or Exclude isotopes within has been selected. 10. Save the IDA experiment.

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Information Dependent Acquisition Tutorial 11. Set up an LC method. In this example, use an isocratic pump with a flow rate of 200 L/min and with 5 L injections. 12. Create and run a batch using this acquisition method. At 609.2807, MS/MS should be triggered.

Additional IDA Criteria

Topics in this section:
Create IDA Experiments on page 9 Include List Criteria on page 10 Exclude List Criteria on page 11 Charge State on page 13 IDA Advanced Tab on page 13 IDA Advanced TabMatch Isotopes on page 13 IDA Advanced TabMass Difference on page 15 IDA Advanced TabNeutral Loss on page 16

Mass Defect Filters on page 17

Create IDA Experiments

Use this dialog to set the parameters for the IDA experiment. To access this dialog, click Create IDA Exp on the MS tab of either the TOF MS or Product Ion scan types.

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Figure 1-5

Create IDA Experiments Dialog

Item Description 1 2 Number of survey scans: Set to 2 for Neutral Loss IDA experiments. Maximum number of candidate ions to monitor per cycle: Determines the number of MS/MS candidates to trigger. In the acquisition method, only one Product Ion scan is displayed. Dynamic background subtraction: Select to prioritize the fastest rising ions in the spectra for MS/MS acquisition. This filter ensures MS/MS is acquired near the LC peak top. The Dynamic Background Subtract function can be used with standard filters and mass defect. Rolling collision energy: Calculated based on m/z and charge state of the ion (applicable to peptide experiments). Adjust CE when using iTRAQ reagent: Automatically increases the magnitude of the CE by 9 V. Rolling CE is automatically activated. Infusion IDA: The software will assign a +1 charge state and call the ion monoisotopic if the peak finder presumes it is an unknown (peak finder cannot determine if the candidate ion is monoisotopic or its charge state).

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Include List Criteria

In the Include List on the Include/Exclude tab, enter the masses for which you want to trigger dependent scans. To access this tab, click the IDA Experiment check box on either the TOF MS or Product Ion scan types.

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Inclusion List Criteria

Item Description 1 Include List: Select the Include List check box to activate. Candidate ions that are on the inclusion list and meet the inclusion list criteria will be preferentially selected for MS/MS. Mass (Da): Enter the m/z for the ions that you want to preferentially perform MS/MS on. RT (mins): Enter the LC retention time for the ion of interest. If the ion is detected at the specified retention time, it will be preferentially selected for MS/MS. Entering a value of 0 means that the ion will be preferentially considered throughout the acquisition. For X (sec): This is the window centered around the specified retention time. This helps to account for any drifts in LC retention time. If the ion on the inclusion list is detected in the window around the specified retention time, it will be preferentially selected for MS/MS. Intensity (cps): This is the intensity threshold for the ion on the inclusion list. If the candidate ion surpasses the threshold (in cps), it will be preferentially selected for MS/MS. Import: Click to select a txt file to import ions for the inclusion list. Charge State: Click to open the Charge State dialog. The values entered will automatically populate the inclusion list with the specified charge states for a given molecular weight. Refer to Charge State on page 13.

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Exclude List Criteria

In the Exclude List, enter the masses that you want excluded from being selected for dependent scans. To access this tab, click the IDA Experiment check box on either the TOF MS or Product Ion scan types.

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Figure 1-7

Exclusion List Criteria

Item Description 1 Exclude List: Select the Exclude List check box to activate. Candidate ions that are on the exclusion list will not be selected for MS/MS at the retention time specified. Mass (Da): Enter the m/z for the ions that you want to exclude from MS/MS. RT (mins): Enter the LC retention time for the ion of interest. If the ion is detected at the specified retention time, it will not be selected for MS/MS. Entering a value of 0 means that the ion will be excluded throughout the acquisition. For X (sec): This is the window centered around the specified retention time. This helps to account for any drifts in LC retention time. If the ion on the exclusion list is detected in the window around the specified retention time, it will not be selected for MS/ MS. Import: Allows the user to select a .txt file to import ions for the exclusion list. Charge State: Click to open the Charge State dialog. The values entered will automatically populate the exclusion list with the specified charge states for a given molecular weight. Refer to Charge State on page 13.

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Charge State

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Figure 1-8

Charge State Dialog

Item Description 1 Depending on the polarity of the acquisition method, the assumption of proton gain (positive polarity) or proton loss (negative polarity) is made. The software automatically adds or subtracts the correct number of protons based on the charge state. Molecular Weight: Enter the molecular weight of the neutral. Charge State From X to Y: Select the minimum and maximum charge state to calculate.

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Selecting OK will automatically populate the Include or Exclude List with the m/z of the ions are the specified charge states.

IDA Advanced Tab

The Use Pattern criteria will trigger MS/MS on a candidate ion if the ion is part of a user-specified pattern. The specified pattern can be based on a distinctive isotope pattern (Match Isotopes), a known mass difference outside of the normal isotope pattern (Mass Difference), or a Neutral Loss (specific to NL-IDA). Depending on the criteria selected, the available user-editable fields will change.

IDA Advanced TabMatch Isotopes

On the IDA Advanced tab, select Match Isotopes to use isotope matching to identify the peak of interest. When this option is selected, the rest of the settings in the Use Pattern field are enabled. You can manually enter, or use the isotopic calculator to enter, the isotope pattern that you want to use to trigger MS/MS. The first entry must have a mass difference of 0 Da and 100% abundance. Subsequent entries in the Abundance column can be greater that 100% if the monoisotopic peak is not the most abundant peak in the isotope pattern.

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Figure 1-9

IDA AdvancedMatch Isotopes

Item Description 1 2 Use Pattern: Select the Use Pattern check box to activate the Use Pattern criteria. When Match Isotopes is selected, you can either manually enter the isotope pattern or use the isotopic calculator (3) to calculate and populate the isotope pattern that you want to use to trigger MS/MS. When entering the pattern manually, the first entry must have a mass difference of 0 Da, and 100% abundance. Subsequent entries in the Abundance column can be greater that 100% if the monoisotopic peak is not the most abundant peak in the isotope pattern. Select Isotopic calculator to automatically calculate the isotope pattern based on user-specified formula. Enter a formula in the Isotopic Distribution Calculator and click calculate. Then click OK to close the Isotopic Distribution Calculator dialog. The Mass Diff and Abundance columns are automatically populated. Note: It is recommended to delete any row with an abundance less than 1%.

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Information Dependent Acquisition Tutorial Figure 1-9 IDA AdvancedMatch Isotopes (Continued)

Item Description 4 Tolerance: (Mass Difference) This is the mass tolerance in Da. The mass difference must be within plus or minus the mass tolerance to be a match for the pattern. Tolerance (% Abundance): This is the abundance tolerance in %. The abundance for the specified peak in the pattern must be within the abundance tolerance in order to be a match for the pattern. Isotope to use for: (MS/MS) Select which peak in the isotope pattern on which you want to perform MS/MS (for example, monoisotopic, 1st isotope peak and so forth).

IDA Advanced TabMass Difference

This feature is useful for triggering MS/MS on candidate ions that are outside a normal isotope pattern, for example, iCAT reagent labeling. The pair of ions that satisfy the mass difference criteria will both be considered for MS/MS. On the IDA Advanced tab, select to use mass difference to identify the peak of interest. In the Mass Diff. (Da) column, type the difference in mass between peaks to be used as a threshold to activate the IDA experiment. The algorithm looks for two candidate ions that differ by the specified mass difference. If the mass difference is satisfied, both candidate ions will be considered for MS/MS.

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Figure 1-10 IDA AdvancedMass Difference Item Description 1 2 Use pattern: Select the Use pattern check box to activate the Use Pattern criteria. Mass Diff. (Da): Enter the mass difference that you want to use to consider the candidate ion for MS/MS. For single survey scans the algorithm looks for two candidate ions that differ by the specified mass difference. If the mass difference is satisfied, both candidate ions are considered for MS/MS. Note: The charge state of the candidate ions will be taken into consideration. 3 Tolerance (Mass Difference): This is the mass tolerance in Da. The mass difference must be within plus or minus the mass tolerance for the ion pair to be considered for MS/MS.

IDA Advanced TabNeutral Loss

On the IDA Advanced tab, select to use a neutral loss to trigger dependent MS/MS scans. This check box is automatically selected when you select two survey scans in the Create IDA Experiment dialog. In the Mass Diff. (Da) column, type the neutral loss(es) to be used to activate the IDA experiment. The neutral loss should be specified to at least the fourth decimal place. A 10 ppm tolerance is used to determine whether the neutral loss is satisfied.

Figure 1-11 IDA Advanced TabNeutral Loss

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Mass Defect Filters

Select to use mass defects to trigger dependent MS/MS scans. This filter is useful in metabolism studies where multiple mass defects can be used to trigger MS/MS acquisitions on phase I and phase II metabolites.

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Figure 1-12 Mass Defect Filters Item Description 1 Exclusively: The mass defect filter is applied exclusively if this check box is selected. MS/MS is performed only on candidate ions that have a mass defect that falls within the given criteria. If not selected, candidate ions that meet the mass defect criteria have higher priority in the candidate list over other ions that meet the rest of the switch criteria specified (with the exception of the inclusion list: Candidate ions satisfying the inclusion list that have greater intensity than the ions meeting the mass defect filter have higher priority) Peptides: MS/MS will be triggered on any peptide that meets the theoretical mass defect window. The IDA algorithm converts the m/z of the candidate ion to molecular weight and compares its mass defect to the theoretical mass defect window. The theoretical mass defect and mass defect window are based on an equation that was derived from theoretical peptides contained in a database. Metabolism: MS/MS will be triggered on any ion that meets the specified mass defect criteria. The IDA algorithm converts the ion to molecular weight and compares the mass defect criteria. Multiple mass defects can be used.

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Information Dependent Acquisition Tutorial Figure 1-12 Mass Defect Filters (Continued) Item Description 4 Tolerance: (mDa) This is the tolerance around the mass defect. Ions must have a mass defect that falls within the Mass Defect the user entered Tolerance to be considered for MS/MS Formula: Enter the molecular formula of the representative compound of interest (for example, your drug, a predicted cleavage metabolite of the drug, drug + glucuronide). Multiple formulae can be entered to allow for real-time multiple mass defect filtering. Width (Da): When a formula is entered, the software automatically calculates the associated molecular weight (MW). The Width (Da) is the window centered around the calculated MW. If a candidate ion has a mass defect that meets the mass defect the mass defect tolerance, and falls within the window around the MW, it will be considered for MS/ MS.

Scheduled MRM HR Algorithm

The Scheduled MRM HR algorithm helps acquire high quality MS/MS data from compounds with known masses and retention times. This algorithm can also be used to extract fragment mass with very narrow widths (0.02 Da) from TOF MS/MS spectra. The narrow extraction gives much better selectivity. To create a Scheduled MRM HR algorithm acquisition method, do the following: 1. Create an IDA method in the Acquisition method editor. 2. On the Switch Criteria tab, set the Which exceeds threshold to two million or higher.

3. On the Include/Exclude tab, select the Include List check box.

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4. In the For field, specify the window size in seconds. 5. Set the Intensity to 0. 6. Enter m/z and retention time values, or import large lists using the Import button.

7. Using the m/z and retention time values, calculate the maximum number of concurrent product ions that are scheduled for acquisition. Note: The user can also use the Computing sMRM Concurrency.xls spreadsheet to easily calculate the maximum number of concurrent product ions. Contact support@absciex.com to obtain a copy of this spreadsheet. 8. On the Switch Criteria tab, set the Maximum number of candidate ions to monitor per cycle field to a value higher than the number of concurrent scheduled product ions calculated in step 7.

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9. Save the method.

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