You only need to specify an isolation scheme if Skyline does not know how to read the isolation windows from your .raw files.
There are some mass spectrometers for which all Skyline is able to see is the center of the isolation window. For those types of instruments, you need to tell Skyline what the windows are, and Skyline will figure out which window that m/z must have come from.
You would also need to specify the isolation windows if you are using Skyline to generate your instrument method.

If you are importing results from a Thermo .raw file, you can choose the Isolation Scheme called "Results Only".

By the way, the numbers in the "Prespecified Isolation Windows" grid are editable, so, if Skyline gave you the wrong numbers there, you are free to edit them.
I do not know why Skyline might have given you the numbers that it did when you imported the windows from your .raw file. It probably has something to do with margins, but I do not know.

If you would like us to take a look at your .raw file you can upload it here:
https://skyline.ms/files.url

But, everything will probably work for you if you just choose the Isolation Scheme called "Results Only", or if you choose "Use results data isolation targets" on the "Edit Isolation Scheme" window.

Hi Nick,

Thanks for your reply. I tried uploading the raw file for you to take a look, but it said that I don't have the privilege to access the directory. Do I need to zip the file before uploading it?

Hong

You did successfully upload your .raw file. The web site tells you "Permission denied" when it really means "File already exists". We are working on fixing that error message.

Now that I have your .raw file, I am not sure what I am supposed to be looking at.

Is there anything that does not seem to be working right for you?

Hi Nick,

You mentioned that it probably has something to do the margins. Can you look at what margin I can set to get the same isolation scheme in Skyline with the one generated by the Thermo method editor?

Thanks,
Hong

Have you tried the "Import" button above the isolation scheme grid? This button will allow you to point at a raw data file and Skyline will attempt to read it an determine the isolation scheme in it. Once it has done that, you can then click on the "Graph" button to review what Skyline felt the isolation scheme was and decide whether you agree.

This is probably the most reliable way to use an isolation scheme created with another tool and then used to acquire raw data.

--Brendan

Hi Brendan,

Thanks for your suggestion. I will try that.

Hong

Hi,

Sorry to bother you again. I have one more question. I would like to export TIC to normalize the peak areas of peptides.
I go to File > Export > Chromatograms > TICs. The exported .tsv contains a column Total Areas and another column Intensities.
Is Total Areas the summation of Base Peak Areas? If I want to calculate the TICs for normalization, I need to sum up all intensities. Do I understand correctly? Is there another way to export the TICs? Thanks!

Hong

When Skyline extracts chromatograms from MS1 data, Skyline remembers the total ion current.
You can see this value in the Document Grid.
It's under:
Replicates > Files > Total Ion Current Area

This column will contain the integral of the Total Ion Current of all of the MS1 scans.

Right next to the Total Ion Current Area column is the "Explicit Global Standard Area" column.
If you want to normalize to an arbitrary number, then you can type that number into the Explicit Global Standard Area column. Then, whenever you ask Skyline to normalize to Global Standards, Skyline will use that number instead.

A common thing to do is to copy the values from the Total Ion Current Area column and paste them into the Explicit Global Standard Area column.

I hope this makes sense.

Regarding the question that you asked: when you export chromatograms, the "TotalArea" column is the integral of the intensities in the particular chromatogram. This integral is different than the sum of the intensities, since each intensity is multiplied by the amount of time between the points.