| Brian Pratt responded: |
2017-07-18 12:08 |
Hi Lee,
If you only care about a single mz (that is, you aren't worrying about multiple charge states for the same molecule) then you can just claim charge +1.
Thanks for using the Skyline support board!
Brian Pratt |
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| Brendan MacLean responded: |
2017-07-19 21:02 |
Also note that once you have entered the single precursor with "charge 1", you can click on the chromatogram in the chromatogram graph and potentially get some insight into the real charge state, if you are using a high-resolution mass spectrometer. Clicking on the chromatogram will show you the MS1 spectrum from which the chromatogram point you clicked on was extracted, and then you would be able to tell by the M+1, M+2, etc. the charge of the molecule the produced the monoisotopic peak.
Then you could go back and reconstruct your transition list with proper charge states, if you wanted to. |
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| lparsons responded: |
2017-07-28 11:01 |
Thank you both for the quick replies, that worked great!
I have a follow-up question pertaining to how to deal with a very large number of ions that were compared this way. My initial list was around 1,800 ions and now I am looking for a way to trim the list in a semi-automated manner. Is there a way to do this in SkyLine?
For the purpose of this work I would particularly like to filter based on a combination of intensity and the time dimension; for example drop all ions where <95% of the total signal occurred within the 5 minute time window. Is there a way to do this?
thank you!
Lee |
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| Brian Pratt responded: |
2017-07-28 11:28 |
You might try looking at the Document Grid, and sorting by peak area etc - you can copy and paste from there into Excel (or notepad, as you please) for further processing, or just select and delete the transitions that don't interest you right there in Skyline.
Brian |
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| Brendan MacLean responded: |
2017-07-28 15:53 |
I should note that only the most recent version of Skyline-daily has the ability to delete elements from the Targets view in the Document Grid. But, once you have that, then Brian is right that it will be your best option, since you will be able to set a filter and then click the delete button to get rid of targets that match the filter.
For peptides we also have Edit > Refine > Accept Peptides, which provides a great deal of power in allowing you to export report information from Skyline and then use any other tools you like to refine your targets to the list you want to keep. Then you return to Skyline and use Edit > Refine > Accept Peptides to narrow your Targets list to only the set you chose elsewhere. I don't think we yet have this working for small molecules, though.
Glad to hear you are making progress.
--Brendan |
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| lparsons responded: |
2017-08-07 09:34 |
Thank you both again for your help. I have another question in the same vein here, this time relating to how Skyline handles my ion list.
The list I imported has ions with m/z reported only to the fourth digit after the decimal (I truncated it to this while building the list). However once I have imported the list and then imported the raw files, Skyline lists the ions to slightly different m/z values than what was on the list. For example one ion was imported as 1024.5928 but on the small molecule list it shows up as 1024.593349 (specifically listed as "ion[1024.593349/1024.593349]").
Can you explain what is going on here? I don't understand why Skyline changed this on its own.
thank you |
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| Brian Pratt responded: |
2017-08-07 11:17 |
May I have a copy of the list you are importing?
Thanks,
Brian |
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| lparsons responded: |
2017-08-07 11:24 |
I'd be happy to send it to you; would you like me to email it? The list is over 10,000 lines long currently (though once it is imported via Edit -> Insert -> Transition List, where it is specified as small molecules, Skyline then lists less than 2,000 ions). Alternately if you would like I would be happy to post just a subset of the m/z values or lines here - my list goes in as a tab-delimited list where each line is "m/z{tab}1". |
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| Brian Pratt responded: |
2017-08-07 11:35 |
Please do - I'll contact you off-list.
Brian |
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| Brian Pratt responded: |
2017-08-07 13:21 |
What you're seeing is the neutral mass value, which is slightly different than the ion mass value. If you expand the precursors in the Targets tree you'll see that the mz values are actually correct.
The word "ion" here is a misnomer which exists for arcane historical reasons, when I import your list in the upcoming Skyline-Daily this says "molecule" instead.
Sorry for the confusion!
Brian |
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| lparsons responded: |
2017-08-08 07:29 |
Thank you for your help Brian!
I have one more question pertaining to the population of ions on the list. The list I imported was ~10,000 ions while it shows up in Skyline as <2,000 (I already removed some and don't recall the exact starting number). Was the reduction due to the transition settings in Skyline (ie, some ions in the list were too close together and were treated as being the same if their m/z was sufficiently close)? Or did something else happen here?
thank you
Lee |
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| Brian Pratt responded: |
2017-08-08 08:41 |
I'd have to see your document settings to speak precisely, but the ion list you sent me is at least 1/3 composed of lines which are exact copies of others, which Skyline will certainly thin out, and your Transition Settings are doubtless also causing very large, very small, or very similar values to be ignored. The particular settings to look at are the Instrument min/max and method match tolerance m/z values. Though you should make sure that these values match the reality of your equipment - there's no point to looking for ions at .0001 resolution if your mass spec can only resolve .001, it will only cause problems.
You might want to work through the appropriate tutorials if you haven't already - even though they're largely in terms of peptides, most of the basic principles apply.
Onward!
Brian |
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