Category Archives: Method Development

Method Forge … Acquisition and Method Creation

Below is a quick video of how to use the Method Forge feature while acquiring a data file.

Method Forge can be used with either an existing data file or be used to produce a data file using an instrument method. It then extracts the data out of the file and created compounds and components from detected peaks, defined in the assigned Method Template.

The Method template will be discussed in a post next week.

If the video is blurry select the cog wheel in the lower right of the video window and adjust the resolution setting.

There Can Only Be One… Calibration Point

Even though its the most memorable line from the movie “Highlander”, this statement has come up many times for assays where semi quantitation occurs in screening analysis.

One way to accomplish a rough estimate calculated amount is to use the ability to use a single calibration level for a compound.

When you do this there are two settings in the Method setting that can be used.

Selecting Average RF allows for the average response factor for a calibration curve to be calcualted. If you use cal one point

the response of the point will be compared to the response of the compound in the sample and a rough estimate based on the

average will estimate the calcualted amount.

If using a linear curve and one point, you MUST set the Origin setting to Force. This sets the origin at Zero and draw the curve

the response of the one calibration point. Thereby giving you a curve to estimate the calcualted amount from.

There is a third way using Internal Standards, but we’ll save that for another day.

Method General Setting Page – Hmmm.. I missed that setting

One thing that TF has as part of its DNA is to be flexible. Method development is a complex process and we are always looking at ways to reduce the effort, but to give flexibility in our approach.

One page that has alot of power, but is over looked many times is the General Setting Page.

These settings are the general setting for properties in the method.

These settings overide the default settings in the Application and apply to the Method and consiquently all the data that is processed with the method. These settings can be changed in at both the Master and Local Method levels.

Two points to look at.

1) Mass precision – This applies to all the masses used in producing results. It does not apply to the reporting    properties of thethings like RT, or calcualted amounts.

2) Instrument Method Edit/Update – In the master method you have a copy of the selected instrument method, which the ultimate Master instrument method is located in the Xcaliber/Methods directory. When you select edit, you are editing the copy that is located in the Master Method folder. When you select update you are overwriting the changes you made in the Master Method Copy, to the ultimate Instrument Master method in the Xcaliber/Methods directory. When you use these button in the local method you edit the Local Method copy of the instrument method that was copied to the Local Method directory, and the update button copies those changes to the Master Method instrument method. This isolated the Ultimate Master instrument method from batch level changes that you may not want to be inherited by other Master Methods produced in the furture.

Now that I have a CDS, what do I do with it?

Well, the end game of the CDS is to supply information that can be used to create methods and acquire data.

Here we’ll look at the Acquisition List side of the equation.

 

Here in the data we retrieved from a CDS or PMD file is stored.

Note that PMD files do not contain all instrument information but this can be added to a CDS and then adjusted.

From here you can select the export SRM data in the Method View tab and an Xml file containing the instrument specific data will be produced.

 

If you open this file in a text editor such as note pad it will contain the information in the Acquisition List in a format that can be imported into the Instrument Method editor.

 

 

If on the Batch View section or the final page of the Acquisition Mode wizard you select Auto-TSRM update this information is passed to the mass spec at the time of acquisition. This allows the user to collect only the data needed to be processed and allows for manual updates in the batch to be automatically loaded to the mass spec at run time. This does not monitor the RT of peaks and automatically update the mass spec controller, but allows for user input to occur in on place versus having to open the instrument editor for a second adjustment.

Using the Compound Data Store – Variant on a Theme

The CDS once created can be used to create methods in a more simplified way.

Again the CDS is a way to use knowledge you have previously collected about a compound and it analytical conditions.

First in the Method Development section select “Create new method” from the Method view subsection.

Here you can see the select compounds from CDS is enabled.

One thing to note down: only one CDS can be open at one time in TF 2.1, which ever CDs is loaded in memory in the Configuration section is the one used. If you want to use a different CDS, load that one into memory inthe Configuration section.

Next you can select compound from the CDS to be used in a new Master Method.

You have the choice to select all, highlight a few and deselect them or pick and choose. Once you have made your selections click the “Apply”button.

This will create a new Master Method.

Once created you will need to save it witha new name. The data ported over consist of acquistion related feilds in the Acqusition List section, which we will discuss later this week, and compound processing basic data (ie. compound name, retention time, detection window, etc..) At this time you will notice the little red balls next to the row number. If you scroll over this it will tell you that the compound does not have a filter string assigned.

This is important because at Thermo we need a filter string assignment to tease the data about the peak out of the RAWFILE. From the Master Method file menu item select “Associate new raw data file”

By selecting a file, that has data for the compounds you previously selected fromthe CDS, and enabling the “Update scan filters for all peaks” button; TF will use the data in the Acqusition list to automatically update the filter selection box for each peak.

Any conflicts and the red ball will remain, to let you know that you must manually select a filter for that peak. This is where the SRM, XIC and SIM experiment type come into play. It specifies which type of filter to select from as in each method you can have blended experiment types.

Tune in next time for “AUTO-SRM UPDATE”

Compound Data Store or Compound Database – Kinda the same thing….

TF has the concept of bring in a data you know about compounds and storing it for later use.

This we call the Compound Data Store (CDS), but what’s in a name… Its a an XML file that contains information about the compound.

The picture above shows this data in excel. It contains information like the compound name, experiment type, collicion energy, retention time, fragments relationships.

Attached here is a copy of the csv template TEMPLATE.

This information can be edited in excel and uploaded into TF to add to a database or to alter the data base. The changes will be reflected in the TF CDS module.

Once the data is in the CDS, a method developer can utilize it to create processing methods which in turn can be used in syncing acquistion methods in the Master Method.

A blog post to come later this week will highlight the use of the information in the Auto-SRM feature.

A quick note is that the CDS is not automatically turned on at installation.

The user will have to configure the option and restart TF to take advantage of this feature.

 

 

Can LCQuan and TraceFinder Co-exist

In a word, Yes.

Well, to be a little more detailed. TF 2.0 and above can be installed on the same PC, but they have to be versions that are supported by the same Thermo Foundation base application. So LCQuan 2.7 is the needed version, today.

If you have the QuickCalc application, you can export the method out to a .pmd file then import into TF.

That export though does reset the retention time to 1.0 minutes so they will have to be reset in TF or you can reset the RT in Xcalibur.

You can also export the Sequence to a .sld file that can be imported into TF, if you want to look your already acquired data in TF for comparison.

Then you can simply  right click in the Batch View Grid, browse to the data and and map raw files to samples. This will copy the files into the Tf directory and set the file references.

Then just submit the batch for Processing.

If you want to use a report much like the Long and Short Summary, use the TF Custom Report: Xcalibur Style Summary Report, that is located not he Custom Reports Template page.

Enable/Disable Ion Ratio

In TF Methods Ion Ratios can be set but updated not to calculate if needed.

In this case the user may find that they have set up a sort of generic method with two confirming ions.

The body they are doing the work for requires only one as part of their data set.

The user can simply go to the method and select an ion and disable the calcualtion by unchecking the “Enable” box on the ratios page.

The ratio wont be calcualted and the chromatogram will not be displayed in data review or on reports.

The Sum of All Peaks

One of the key features of TF is the use of a compound being composed of multiple components.

So the gist, of it is that a specified compound can consist of up to 6 quantitative peaks, each of those can have up to 10 confirming ion ratio peaks.

So A compound can consist of up to 66 chromatographic peaks when using mass spectral data.

This means that you can visualize under the compound name quantitative peaks and their associated ion ratio confirming ions.

 

The compound is composed of these multiple peaks, which have summed areas. But they can be visualized separately. The total response of adding all the, lets say peak areas, is the total response of the compound. And by using the multiple quan peak feature, these chromatographic peaks don’t have to be at the same retention time. So if you were doing a chiral separation but wanted the total response, you would just make them individual quan peaks under the compound.

 

Now if you have coeluting peaks and want one big peak to represent your compound, then you can use the signal pane’s mass grid to accomplich this type of mass intergration.

All the responses are summed, but no individual visualizations.

What the Heck is a Ion Range????????

So one thing that has been resurfacing is confirmations that I know what think is my data really is my data.

TF uses one such tool in Ion Ratio calcualtions.

This is a ratio of the selected response of one defined peak that is connected to another defined peak.

In TF, we define that as, a Confirming Ion has a Ratio to the Quan ION/Peak, and we can set some acceptance threshold around that ratio.

Now, we have a few ways of defining the Ratio.

Thats the Ion Range.

Now we can use:

Manual – Where you type in the Target Ratio.

Average – Where the Ratio is deteremined by Averaging the ratios found in the calibration curve for these peaks and then producing an average. This automatically sets the Target Ratio, so you don’t have to.

Weighted Average – The same as above but its weighted and usually used for really large orders of magnitude methods.

Level – Again its like the above but it only finds the Ratio for the one specified calibration level.

The last three allows the data to define your criteria, and keeps you from having to adjust the ratios manually. This can be done for processing on the fly or in post acquisition processing.