How to open a spectra in skyline? qwa227  2021-04-23

Hi, there:

I am new to skyline and have problem opening spectra that were collected elsewhere. Just wondering if you have a step-by-step instruction? Do I need any other file besides the spectrum RAW data file? I watched your educational online videos but still could not get it to work.



Nick Shulman responded:  2021-04-23
Skyline is a tool for quantifying mass spectrometry data by integrating chromatogram peak areas. Skyline is not a very good tool for looking at individual spectra. If you want to look at spectra in a raw file, we recommend using our tool called "SeeMS.exe", which gets installed when you install ProteoWizard.

What type of mass spectrometer did you use? Do you know what sort of data you collected?

A typical first experiment with a mass spectrometer would be Data Dependent Acquisition (DDA) where the mass spectrometer was told to collect MS1 scans and then select the largest peaks in those MS1 scans for fragmentation and identification with MS2.
If you want to get started with DDA data, I would recommend taking a look at the DDA Search tutorial:

If that tutorial does not seem helpful, you should tell us more about what type of mass spectrometer you have and what sort of data you collected. If you want to send us one of your raw files, you can upload it here:

-- Nick
qwa227 responded:  2021-04-23
Thanks, Nick! I am doing PRM with heavy peptides spiked in. I was told that the spectra were collected on Q Exactive. I knew heavy peptides showing up well. I would like to see heavy and endogenous peptides simultaneously first before future heavy peptide titration experiment. However, both the person who generates the data and me are not familiar with skyline. I cannot load it correctly. I tried file/open, it asked for spectra library. I tried to type in all transitions manually, or import etc. Nothing seems to work. Just wondering if you got a step-by-step instruction. Thanks. QJ
Nick Shulman responded:  2021-04-23
I would recommend that you do the following steps:
1. Use "Edit > Insert > Peptides" to tell Skyline which peptides you are interested in.
2. Go to:
Settings > Peptide Settings > Modifications
and use the "Edit List" button that's in the lower half of that dialog to tell Skyline which isotope modification should be applied to your heavy peptides.
3. Go to:
Settings > Transition Settings > Full Scan
and tell Skyline how to extract chromatograms from your MS1 and MS2 spectra.
You can choose either "Count" or "Percent" for "Isotope peaks included" (or you can leave it as "None" if you do not want MS1 chromatograms)
You should choose "Targeted" for "MS/MS filtering acquisition method"
We recommend choosing "Centroided" for both the "Precursor mass analyzer" and "Product mass analyzer".

After you done all that, you are ready to extract chromatograms from your raw file.
The menu item to extract chromatograms from a raw file is:
File > Import > Results

-- Nick
qwa227 responded:  2021-04-25
Thanks, Nick! This is really helpful. I am able to open RAW files this way! So you are saying that even for Orbitrap instrument, you still choose "Centroided" for both the "Precursor mass analyzer" and "Product mass analyzer"? Do you need to open other Transition Settings info such as Instrument etc? - QJ
Nick Shulman responded:  2021-04-25
Yes, we recommend "centroided" for all high resolution Thermo instruments.
When you choose "centroided" on the Transition Settings Full Scan page, you are telling Skyline to use the centroiding algorithm that Thermo has implemented. Their algorithm does a better job of figuring out how much of which m/z was observed.

You might also want to look at the "Settings > Transition Settings > Filter" page in order to tell Skyline which precursor charges and product ion types and charges you are interested in.
-- Nick