small molecules: Error with calibration curve: all of the external standards are missing one or more peaks

small molecules: Error with calibration curve: all of the external standards are missing one or more peaks ayson  2020-03-27

Hello! What are some of the criteria of having calibration curve? Currently, I only have standards and also blanks but no quality control samples. I have already labeled my standards as standards and blanks as blanks and I have also put in the concentration. But whenever I am opening calibration curve this is what I get: Error: All of the external standards are missing one or more peaks

Is it because some of my standard samples especially the lower region do not have any peaks detected? Thank you!

Nick Shulman responded:  2020-03-27
Can you send me your Skyline document? ("File > Share" etc)

The problem is likely that some of your transitions do not have a matching chromatogram in your .raw file, and none of your replicates have a complete set of chromatograms.
Another thing that might be going on is that you're normalizing to the heavy form, but Skyline is not able to figure out which light transition is supposed to correspond to which heavy transition.

-- Nick
ayson responded:  2020-03-27
THanks, Nick! Very helpful! And so I actually deselected light from molecule setting/label and I am seeing my calibration curve now. =) Is there a way I can get %CV's area for replicates for each transitions?
Nick Shulman responded:  2020-03-27
If you want to see CV values across groups of replicates, you can use the Pivot Editor in the Document Grid. Here is a page showing how to do that:

Another way to do that is with the CV Histogram graph:
View > Peak Areas > 2D Histogram

The histogram graph in Skyline 20.1 would allow you to group by a replicate annotation value that you have defined, but Skyline-Daily allows you to group by other values such as "Analyte Concentration".
-- Nick
Brendan MacLean responded:  2020-03-27
You can in the Peak Areas plot, through the right-click menu, and also in the Customize Report form under TransitionResultsSummary.Area.CvArea. If you click on the binoculars (find) button in the Customize Report form, then you can search for Cv to find all the different CV options. And, finally, you should be able to achieve interesting transition level CV reporting using the Pivot button to the right of the Reports menu in the Document Grid, which would also allow you to group your CVs by some other value such as Analyte Concentration (which you can also do in the Peak Areas plot).

Hope this helps. If it is too vague, maybe choose one and we can explain in more detail.

ayson responded:  2020-03-27
Thanks Nick and Brendan! This is very helpful!

@Nick should I use skyline-daily instead so I can preview histogram by other values such as "analyte concentration"? I like the how to pivoteditor and I was able to create my own %CV data with the analyte concentration included. Is there away I can include pivot table with the original data showing values for replicate? If not, I'll probably just join tables through Spotfire or Excel sheet.

@Brendan, Thank you for all of the tips and tricks as well. But it seems like the %CV in the transitionresults summary do not include analyte concentration for grouping. Is there a way I can change the grouping for that calculation? As I said above, I am really interested in getting output with the individual replicate values and as well as %CV's per analyte, precursor, product and analyte concentration. I would really like to know how to view the replicate CV's by analyte concentration in the histogram. Let me know if anyone of you can help me out with this.

You guys are so awesome! Keep safe. Thank you again!

Nick Shulman responded:  2020-03-27

Actually, I have discovered that the CV Histogram window in Skyline-Daily has a few bugs when it comes to grouping on things other than Annotations. I will fix these bugs, but I would recommend that you wait until the next update of Skyline-Daily if you want to try out the new features of the Area CV Histogram graph.

I don't understand what you mean by "include pivot table with the original data showing values for replicate".
-- Nick
ayson responded:  2020-03-27
THanks, Nick! I will hold off downloading skyline-daily until the bugs have been fixed. Will we get emails when new version is read for update?

For your followup question, in the document report CV area, CV height, CV retention time do not include grouping by concentration and so the %CV for the height and the area is high. And so, the only way I can get %CV with analyte concentrate is when I do pivot table. I would like to combine pivoted table with the %CV to the transition results summary. Another way to get around is if we can have CVarea, CVheight, CV retention time in the transition summary list that also includes analyte concentration. Please let me know if this is still not clear. I can visualize it for you. Thanks!