Precursor Report: Duplicate lines?

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Precursor Report: Duplicate lines? alejandro.cohen  2020-11-19
 

Skyline people... again requesting your help. Here a curve ball:

I'll try to summarize:

I'm developing a targeted SIM (tSIM) method for 18 steroid hormones using a LC-QExactive and Skyline's Small Molecule interface. For each molecule, I have a light and deuterated standard. ALSO, for each molecule, I'm monitoring both the [M+H] and [M+Na] precursors. Reason being, these samples are ocean water derived, so I'm concerned Na might not be easy to completely removed during sample prep, and signals will likely be split (I'm clearly seeing this in the data I've analyzed so far, even with the standards).

I've followed the following tutorials which were very helpful:

https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_small_quant
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_hi_res_metabolomics

So, I have one question and one concern.

Q: For the calibration curves for any of the molecules, I see that they remain constant regardless whether I select the [M+H] or the [M+Na] (Curves DO change selecting different molecules, of course) . Does that mean the areas of both [M+H] and [M+Na] are summed up to make the final areas? Does this also apply to their Internal standards and the ratios? I'm finding it hard to navigate the tables to see how the data is compounded together.

Concern: in my precursor-Quant table, for each Replicate, I see TWO lines having the same column content (Rep, Rt, Precursor, Molecule, Ion Formula, Precursor Adduct, etc etc) EXCEPT the Area, Area Ratio and Background. Attached a screenshot.

Could you help me out with this? Thanks again for your fabulous work with Skyline.

 
 
Nick Shulman responded:  2020-11-19
The reason that you are seeing multiple lines per precursor in your report is that the report includes some columns that come from Transitions. Those columns are:
Retention Time
Area
Area Ratio
Background

If you remove those columns from your report then you should get one row per precursor and replicate.

Calibration Curves are always calculated at the Molecule level.
You should tell Skyline how you want to normalize your molecule. You can tell Skyline the default normalization method to use at:
Settings > Molecule Settings > Quantification

You can also override that default normalization method by using the Document Grid to set the Normalization Method for the molecules that need a different normalization method.

If the Normalization Method is "None" and your molecule has heavy and light, then I think the numbers that get plotted on the calibration curve usually end up being the light peak areas. Skyline sums across all of the precursors whose Isotope Label Type is not an Internal Standard. The Internal Standards can be specified at Settings > Molecule Settings > Labels

-- Nick
 
alejandro.cohen responded:  2020-11-20
Nick

Thanks, this clarifies. I am now able to monitor and trace how Individual Transition Areas (M and M+1) add-up, and confirm how the areas for [M+H] and [M+Na], for both Std and Int Std are compounded together. However, in the Molecule Report, I cant verify/confirm how each of these ([M+H] and [M+Na]) are added-up, and how the Internal standards are treated (I'd like to confirm the [M+H] and [M+Na] areas of the Int Stds are added before used to normalize the data.)

Is there any column on the Molecule Report that shows up the Summed Areas of all precursors (both light and heavy). If I add the Total Area column, I only get the values of the independent Precursors. (See attached image).

I hope I managed to clarify... it's hard putting this into words.

Thanks again, and again
 
Nick Shulman responded:  2020-11-20
The value that gets plotted in the Calibration Curve is always the "Normalized Area" column.
The column in available in the Document Grid at "Molecule Results > Quantification > Normalized Area"

There is no column in the Document Grid which shows the Total Area summed across precursors. You can use the Pivot Editor in the Document Grid to calculate such a value by grouping by making "Molecule" and "Isotope Label Type" the Row Headers and have the value be the Sum the "Total Area" column.
This page has information about how to use the Pivot Editor:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=PivotEditor
-- Nick
 
alejandro.cohen responded:  2020-11-20
Thanks Nick,

Will do!

Maybe you could pass along this suggestion to developers: Would it be possible to have the Molecule tables or Results Grid show additional columns for the summed Areas precursors (say [M+H]+[M+Na]) for the Light and Heavy Precursors? It would be great to keep track of them for QA/QC purposes. Pivot table should work, but it appears quite time consuming when Skyline must be calculating these values already

Best,

Alex