PRM Conductor Quick Reference

PRM Conductor is an application that aids in the creation of targeted MS methods. This program filters "bad" transitions, and selects "good" peptides via a set of criteria, and helps the user to create one or more instrument methods that will be acquired with at least a required number of points per peak.

Launching PRM Conductor from the Thermo tools menu will open the application. If there was data in the Skyline document, a report will be exported from Skyline and imported to PRM Conductor. The view will look similar to below.

The user parameters are split into 3 sections; Refine Targets, Define Method, and Create Method

Refine Targets

These parameters configure a series of simple filters for the transitions, and allows to filter precursors by the number of good transitions that they have.

• TMT Modifications Present?
  • This value is No if there are no TMT modifications. If TMT modifications are present, then the exported methods can account for this, for example by choosing MS3 precursors that have TMT tags.
• Transition Mode
  • This value is Precursor/DDA if ALL the analytes have precursor transitions. The 1.0 PRM Conductor version would set this mode if ANY precursor transition was present, which was confusing. This DDA mode means that MS2 data may only be acquired once per LC peak, and so some metrics like Time Correlation are not valid. This mode is common for Small Molecule analysis when data are imported from Compound Discoverer. PRM Conductor is still useful in this case, to create instrument methods, but it may be advisable to select Keep All Precs. option, and apply filtering on a later targeted data set.
  • This value is usally Targeted/DIA, enabling the full filtering functionality of the Time Correlation filter.
• Min. Abs. Area
  • Filters transitions by requiring that they have a minimum absolute area value. This value may have to change for data acquired on different instruments. For example, the intensity scaling on Orbitrap instruments is arbitrarily scaled to a value that is ~10x the value for Ion trap and Astral data.
• Min. Signal / Backgnd.
  • Filters transitions by requiring that they have a minimum signal to background ratio. Signal is the Skyline peak area, and Background is the Skyline background value. This value should be used sparingly, because the current background metric that we use is not as good as it could be.
• Min. Rel. Area
  • Filters transitions by requiring that they have a minimum relative area to the largest transition for its precursor.
• Min. Time Corr.
  • Filters transitions by requiring that they have a minimum correlation to the median transition. This value is called Shape Correlation by Skyline,and was defined by Searle et. al., and later in some detail by Heil et. al.
• Min./Max. Width (s)
  • Filters transitions by requiring that their base LC width be within the given bounds. The bounds is computed from the Skyline FWHM and converted to a base peak width by assuming a Gaussian shape and multiplying by 2.54. See the supplement of Remes et. al. for a derivation.
• Min./Max. RT (min)
  • Filters precursors by requiring that their retention time be within the given bounds.
• Min. Good Transitions
  • Filters precursors by requiring at least this many transitions that satisfy the above filters.
• Keep All Precs.
  • This option ensures that no precursors are filtered, and the top Min. Good Transitions are kept. If there are not enough good transitions, "bad" transitions are selected according to Area x Time Corr. This option is useful when the user wants to keep all the precursors and just clean up the transitions, or is using DDA mode.

Define Method

These parameters control how many targets can be included in the assay. When parameters in this section are changed, and the 'enter' key is pressed, the graph on the right will update, showing how many precursors can be included in the assay, while respecting Cycle Time defined by the LC Peak Width and the Min. Pts. per Peak.

Analyzer

• Ion Trap
  • Scan Rate (kDa/s)
    • Sets the analysis scan rate. This value affects the acquisition rate, and therefore the assay target capacity.

• Astral

  • Currently the Astral has no analyzer-specific parameters
    

• Orbitrap

  • Resolution (k)
    • Sets the analysis resolution. This value affects the acquisition rate, and therefore the assay target capacity.
      

• Triple

  • Max #Transitions
    • Only up to this number of transitions will be selected, since this value linearly decreases the assay target capacity.
      

• Min Dwell (msec)

  • Also called injection time on many instruments, this value sets the smallest amount of precursor accumulation time per target. Typically we set this value small enough so that it does not affect the assay capacity, and rely on the dynamic AGC algorithm to boost the injection time according to the available time in the cycle. See Remes et. al. for a description of the dynamic AGC process.
  • In some cases, the experimenter may know that to achieve high quality data, a minimum dwell time is needed, and will adjust this value accordingly.

Acquisition Type

• MS2
  • The standard analysis type, where each acquisition contains data for a single precursor that is mass selected and fragmented.
• MS3
  • Currently only available when Ion Trap analyzer is selected, in this mode, each acquisition contains data for a single precursor that is mass selected, fragmented, and one or more fragments are further mass selected and fragmented.
  • The Max # Transitions parameter becomes available in this mode, limiting the number of MS3 precursors that will be simultaneous mass analyzed in the second MS stage. For purposes of the acquisition rate estimations, this mode currently assumes that resonance CID with 4 msec activation time is used for the first activation, and HCD is used for the second activation. In reality if the user selects a method template the uses resonance CID for the second activation, then the activation for each MS3 precursor is performed serially, taking at least #transitions x activation_time to be performed.

• MTM

  • Multiple target monitoring is a type of method that can be generated from 1 Th isolation window GPF DIA data. It analyzes the GPF data to determine when it is safe to expand the number of targets, or the isolation width, to encompass multiple targets. MTM can be used to expand the number of targets in an assay compared to normal PRM, or alternatively for the same number of targets, MTM can enable the use faster gradients and higher throughput, or higher injection times and better sensitivity.
  • When the Opt. check box is unchecked, then the isolation widths are multiples of the GPF isolation width, which is shown in a disabled text box (1 here). A pop-up window shows the distribution of isolation widths, and how many of them are for single or multiple targets.
  • When the Opt. check box is checked, then the isolation widths are customized for each acquisition, such that the window size is (largest_mz - smallest_mz) + MTM Min. Width (Th).
    
    

• DDIA

  • Dynamic DIA is a type of DIA method in which the precursor range shifts as a function of the experiment time. This method can be used to acquire data with a narrower isolation window than would be possible if data for the entire precursor range had to acquired on each cycle.
  • The DDIA Width(Th) sets the isolation width that will be used. Narrower widths will acquire higher quality data, for fewer precursors.
  • A pop-up plot appears that shows the density of precursors in the Skyline document, along with lines that trace the proposed precursor range as a function of experiment time.
    

Scan Range Mode

• Define m/z Range

  • Define an explicit first and last mass for the acquired spectra. The acquisition rate is inversely proportional to the size of the m/z range.
    

• Define First Mass

  • Define a constant first mass for all acquisitions. The last mass is determined based on the charge state, such that last_mass = precursor_mz * charge + 10
    

• Auto

  • The first and last mass are determined automatically. The last mass is determined as in the Define First Mass mode. The first mass is determined as the lowest mass that still retains most of the ions at the last mass, based on experimental evaluations.
    

• Optimize

  • The first and last mass are set based on the set of "good" transitions for a precursor plus a small buffer. This mode enables the fastest acquisition rates possible for the Ion trap analysis, where the mass analysis time is directly proportional to the scan range.
    

Other Define Method Parameters

• LC Peak Width (s)

  • The expected LC peak width at the base. This value is pre-populated from the input data, but can be updated as desired. The instrument maximum cycle time is this value divided by the desired points per peak.

• Points per Peak

  • The minimum desired acquisition points per LC Peak width. The cycle time is the LC peak width divided by this value.

• Cycle Time (s)

  • The desired cycle time, based on the LC Peak Width / Points per Peak

• Acquisition Width Type
  Defines the method used to set the acquisition window width around each target in the method.

  • Global
    • All targets get the same, global acquisition window size, Acq. Window (min).
      
  • Per precursor
    Each target gets an acquisition window width defined as its individual base peak width, times the Acq. Window (factor) value.
    

• Acq. Window Optimize

  • When set to Off, the acquisition windows will be exactly the ones determined based on the current Acquisition Width Type. Note how the Assay 1 density in yellow is the same as the Refined density in red in the time region from 0 to ~10 minutes.
    
  • When set to On, then the acquisition windows widths are expanded by up to some factor (5x currently), while ensuring that the acquisitions don't exceed the desired cycle time. Note how the Assay 1 density matches the cycle time for much of the region between 0 and 10 minutes now. The reason for this mode is that early eluting molecules can have variable retention times, and Adaptive RT may have little information to be able to adjust the targeted schedule at these early times. Making the widths larger in a dynamic way is a nice way to make the assay acquisition more robust. In the future we could add a UI parameter that specifies the maximum expansion factor.
    

• Max Precs. / Group

  • Sets the maximum number of peptides per protein in Skyline's Peptide mode, or molecules per molecule group in Skyline's Molecule mode. This parameter can be used to increase assay quality by removing targets from the assay that may not add additional information. The included targets are selected by their quality, defined as their Area x Time Correlation.

• Priority File

  • Double click to select a file with a .prot extension that you've made. Just create a text file, and save it with the .prot extension. On each line, you can enter a protein name, or molecule list name. Skyline puts either the protein name or molecule list name in the Report column called "Molecule List". Any precursors that belong to the protein or molecule list will be accepted into the assay, even if they don't pass all the filters.
  • Using a Priority file is a good way to incorporate iRT compounds into an assay, when doing an initial search for heavy-labeled standards.
  • If the Balance Load check box is not checked, then each of the multiple assays that may be generated will contain the prioritized targets.
  • New for v1.1, you can also enter peptide sequences or molecule names. These are the values found in the Skyline report columns called, "Modified Sequence Monoisotopic Masses", or "Molecule", in the Skyline Protein or Molecule modes, respectively.
  • For example, the file could have 2 peptide sequences and a protein name.
    LPALFC[+57.021464]FPQILQHR
    ESYGYNGDYFLVYPIK
    SPEA_ECOLI

Create Method

These parameters control aspects of how the method will be created.

• Balance Load

  • When checked, a single assay will be created that respects the currently defined Cycle Time. The number of Refined precursors added will depend on the particular choice of Define Method parameters. Precursors are added to the assay iteratively from each protein or molecule group in turn, where the precursors from each group are added in order from highest to lowest quality. Quality is currently defined as area x time correlation. A balanced load refers to how the precursor time density becomes uniform at the limit of many potential precursors, instead of peaked in the middle of the run.
  • When not checked, multiple assays will be created that analyze all Refined precursors. Each assay will be of the traditional, non-balanced type. If a Priority File is specified, prioritized precursors will be added to all of the assays.

• 1 Z/Pep.

  • When checked, only a single precursor from each peptide (or molecule, in Skyline's Molecule mode) will be accepted into the assay. The selected precursor will have the highest quality, defined as area x time correlation.
  • When not checked, multiple precursors having different charge states could be in the assay, if they all passed the various filters.

• Abs. Quan.

  • When checked, a light-version of each heavy-labeled peptide will be added to the assay. This is a nice way to create a light+heavy assay from an initial heavy-only assay. The instrument time plot will update to have 2x the number of targets, allowing to visualize whether enough points per peak will be acquired.

• Export Skyline File

  • When checked, a new Skyline file having the precursors with their corresponding filtered transitions will be created when the Export Files button is pressed. Because creating the new Skyline file takes some 10's of seconds, it can be annoying, and it was made into an option. The good part about using this option is that the new Skyline file will have its Transition Settings set to PRM with Stellar-specific parameters.
  • When not checked, no new Skyline file will be created when the Export Files button is pressed. Often we use this mode, and rely on using the Sync to Skyline button to update the current Skyline document, and later save a new Skyline document manually.

• Combine DIA Windows for Reference

  • This is a special option that only appears if the input data are DIA with isolation width less than 15 Th, and no large window Adaptive RT information is available.
  • When checked, and if the .meth has specified Adaptive RT for Dynamic Time Scheduling, then the exported method will have an rtbin file that is made by combining acquisitions in silico. The DIA alignment acquisitions will have an isolation width of near 50 Th, and will adjust the target schedule accordingly.
  • This is a nice option when creating Stellar MS targeted assays from Astral DIA data, which are likely acquired with small isolation widths, such as 2, 3, or 4 Th.
    

• Base Name

  • Output files will have this value in them. For example, the .meth, .sky, and .csv files associated with a method will all have use this name.

• Method Template

  • Double click this text box to open a file chooser for a .meth file that will serve as a template for exported assay files. The template file should have a tMSn method in it, and the appropriate LC parameters, and Method and Experiment durations. Other parameters like activation type should also be specified. If Adaptive RT is specified in the Dynamic Time Scheduling, then an rtbin file will be created during export and saved in the .meth file. This is the preferred way to add Adaptive RT functionality to a method.
  • You can make methods on your non-instrument computer if you have downloaded the Workstation version of the instrument software. This software is available at the Thermo Flexnet site.
    • In this case, you can use the standalone method editor found at C:\Program Files\Thermo Scientific\Instruments\TNG\Stellar\1.1\System\Programs\TNGMethodEditor.exe.
    • The standalone method editor only has access to MS information, not the LC driver information, but you can copy a .meth file from an instrument that has the LC information in it, and then resave and modify it on your laptop.

• Sync to Skyline

  • Pressing this button updates the current Skyline file by filtering the transitions, precursors, and proteins that are not in the final assay.

• Export Files

  • Pressing this button will export precursor lists, transitions lists, .meth files if specified in the Method Template field, and a .sky file if Export to Skyline is checked.