Chromatography qaporter  2019-03-15

Hi, everyone. Does skyline allow you to enter chromatography and can skyline predict unknown retention time based on line with no standard for small molecules, because I read the tutorial on IRT Retention Time but, that was based on peptide, so I want to know if you can do that with small molecules?

Nick Shulman responded:  2019-03-15
Skyline-Daily can do iRT with small molecules. Skyline 4.2 only knows how to do iRT for peptides.

iRT support for small molecules is a very new feature in Skyline-Daily, and we are still trying to figure out all of the details about how it should work. It might be a little confusing right now, because the user interface still uses the word "peptide" in a lot of places where "molecule" would be more correct.

Let us know if you get stuck anywhere trying to use iRT with small molecules in Skyline-Daily.
-- Nick
qaporter responded:  2019-03-15
What about chromatography, is there feature, where you can enter information about chromatography dealing with your small molecule?
Nick Shulman responded:  2019-03-15
What do you mean by "information about chromatography"?

If you know what the retention time of your molecule is, but you do not want to bother creating an iRT database, you can use the "Explicit Retention Time" column.

If you are inserting a small molecule transitions list:
Edit > Insert > Transition List
after you choose "small molecules", you can push the Columns... button and add "Explicit Retention Time" to the columns that you are specifying.

Also, you can add the Explicit Retention Time to a report in the Document Grid, and change the value for precursors that you have already added to your Skyline Document.
-- Nick
qaporter responded:  2019-03-18
The final goal of what we are trying to do is to use Skyline for retention time prediction of unknown modified RNA molecules due to the lack of standards available for purchase.

1. Is Skyline a useful tool for this?
2. If so, do you need to input any parameters about the chromatography specifically used to acquire the data (i.e. stationary, mobile phase, or gradient)?
Nick Shulman responded:  2019-03-18
Skyline does not know anything about the physics of chromatography. When Skyline talks about "retention time prediction", Skyline is talking about using a library which has been populated with earlier experiments in which the exact same analyte was observed.

Skyline is not the correct tool for predicting the retention times of analytes that you have not measured before.
qaporter responded:  2019-03-20
So, in the tutorial about "adding IRT values for new target peptides", was that not discussing a way to add peptides without IRT values? I attached the picture with the information.
Nick Shulman responded:  2019-03-20
After you have extracted chromatograms for your analytes, and chosen all of your peaks correctly, you can add those results to your iRT database.
That's what's happening in the iRT tutorial down at the bottom of page 15 in the "Calculating iRT Values" section.

In Skyline-Daily you should be able to do that same thing with small molecule analytes. You need to have a Skyline document which contains extracted chromatograms for your iRT standards and your other analytes of interest, and you need to make sure that all of the correct peaks have been chosen.
-- Nick
qaporter responded:  2019-04-18
Title: Retention Time of the Unknown
Hi, long time no see. I have a question about this comment, when you say extracted chromatogram for all your analytes and choose the peaks, do you mean this, I attached a picture below. Also, what does the article mean to predicted retention time, if the time is already unknown? Another question, I have is what material is needed to predicted retention time, if the retention time is unknown?
Nick Shulman responded:  2019-04-18
I was saying that you can tell Skyline to extract chromatograms using the menu item:
File > Import > Results

After Skyline extracts chromatograms, Skyline does its best job to choose which peak along the chromatogram corresponds to each analyte. You might be able to look at the chromatogram and do a better job choosing peaks than Skyline did. You might have some prior knowledge about what the molecule's retention time will be, or you might know what the peak shape is supposed to be, or anything like that.

Then, I was saying that after you have chosen the best peaks that you can, you can add those results to your iRT database.

I do not understand your question about "to predicted retention time, if the time is already unknown". Is that a sentence that you found in a document somewhere?
-- Nick