Hi there,
Is there a way for me import a transition list with precursor ion, precursor isotopic ions, retention time, and ranking, here is what I have:
835.451488,835.117178,34,VSDFGLTK[+16]EASSTQDTGKLPVK,CSK,precursor,7
835.451488,835.451488,34,VSDFGLTK[+16]EASSTQDTGKLPVK,CSK,precursor,2
835.451488,835.785798,34,VSDFGLTK[+16]EASSTQDTGKLPVK,CSK,precursor,1
835.451488,836.120059,34,VSDFGLTK[+16]EASSTQDTGKLPVK,CSK,precursor,3
835.451488,836.454291,34,VSDFGLTK[+16]EASSTQDTGKLPVK,CSK,precursor,4
835.451488,836.788503,34,VSDFGLTK[+16]EASSTQDTGKLPVK,CSK,precursor,5
835.451488,837.122701,34,VSDFGLTK[+16]EASSTQDTGKLPVK,CSK,precursor,6
but I keep on getting this error:
"The product m/z 34 is out of range for the instrument settings, in the peptide sequence VSDFGLTKEASSTQDTGKLPVK. Check the instrument tab in the Transition Settings."
I take the format is a little off, but I'm using the format from an exported transition list from previous experiment.
Thanks!
David
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| Brendan MacLean responded: |
2019-04-09 14:51 |
Could you just insert the peptide instead, using Edit > Insert > Peptides?
You can use modifications there and even specify a precursor charge state... Hmmm, but I just pasted this peptide into Skyline, and I can't see a charge state with a precursor m/z 835.451
VSDFGLTK[+16]EASSTQDTGKLPVK (missed 2)
1162.6193++
775.4153+++
581.8133++++
I just cannot work out what you are trying to achieve with the m/z values in the first two columns of your transition list.
Can you explain?
--Brendan
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| davidz responded: |
2019-04-09 14:58 |
Oh, the modification is [+196.13683], and what I'd like to do is somehow ask Skyline to only look for this peptide with this isotopic pattern within a certain retention time range.
Thanks,
David
835.451488,835.117178,34,VSDFGLTK[+196.13683]EASSTQDTGKLPVK,CSK,precursor,7
835.451488,835.451488,34,VSDFGLTK[+196.13683]EASSTQDTGKLPVK,CSK,precursor,2
835.451488,835.785798,34,VSDFGLTK[+196.13683]EASSTQDTGKLPVK,CSK,precursor,1
835.451488,836.120059,34,VSDFGLTK[+196.13683]EASSTQDTGKLPVK,CSK,precursor,3
835.451488,836.454291,34,VSDFGLTK[+196.13683]EASSTQDTGKLPVK,CSK,precursor,4
835.451488,836.788503,34,VSDFGLTK[+196.13683]EASSTQDTGKLPVK,CSK,precursor,5
835.451488,837.122701,34,VSDFGLTK[+196.13683]EASSTQDTGKLPVK,CSK,precursor,6
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| Brendan MacLean responded: |
2019-04-09 15:25 |
I don't think there is an easy way to achieve what you are looking for. I guess the best I can come up with is:
- Change your settings to use "p" and all the precursor charge states you want to allow in the Transition Settings - Filter tab.
- Change your settings to 1% of Base peak in the Transition Settings - Full-Scan tab for MS1filtering.
- Insert your peptide of interest through Edit > Insert > Peptides
- If you get more precursors than you want, then use Edit > Refine (or just Refine in Skyline-daily) > Accept Peptides, and paste in the peptide charge states you want to keep like this "VSDFGLTK[+196.13683]EASSTQDTGKLPVK+++"
- In View > Document Grid create a report with "Explicit Retention Time" in it and past in the retention time values you want
Not quite as straightforward as your transition list, but at least it doesn't require editing each element one at a time.
Hope this is helpful. Sorry, I don't have a simpler solution.
--Brendan
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| davidz responded: |
2019-04-09 15:30 |
Thanks, Brendan, I will give your suggestion a try.
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| davidz responded: |
2019-04-10 09:54 |
Hi Brendan,
One quick question: there is one column called: Explicit Retention Time and another one: Explicit Retention Time Window, which is also very helpful, where I can specify an explicit retention time value and a time window range, but what value should I put in there if the acceptable time window is +/- 3 minutes, would it just 3?
Thanks,
David
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| Nick Shulman responded: |
2019-04-10 10:38 |
The Explicit Retention Time Window is the full window width.
Therefore, you would set it to 6 in order to specify a window which extends 3 minutes on either side of the Explicit Retention.
-- Nick |
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| davidz responded: |
2019-04-10 11:16 |
Thanks! But specifying the explicit time and time window doesn't really force Skyline to find the peaks within that range, does it?
David |
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| davidz responded: |
2019-04-10 17:17 |
Would it be possible for me to set up a RT predictor to force Skyline only look for peaks for a given peptides within a given time window?
Thanks,
David |
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| Brendan MacLean responded: |
2019-04-12 13:25 |
Hi David,
Remind me the source of your chromatograms (i.e. SRM, PRM, DIA, DDA). For SRM and PRM the time range is determined at method generation time by creating a scheduled method. For DIA and DDA (MS1-only) the time range is determined at extraction time when you use File > Import > Results. We don't currently have a way to instruct Skyline to limit its peak picking to a range of time that is shorter than the range of the chromatograms themselves.
But, if you give us a bit more detail, we may be able to help you set up your Skyline document and acquisition strategy to limit your chromatograms to just the range of time you are willing to consider. Or maybe you can explain why you want chromatograms longer than you are willing to consider your target molecule showing up in.
Thanks for your feedback.
--Brendan |
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| davidz responded: |
2019-04-15 12:00 |
Hi Brendon, these are PRM runs, and we are certain that peptides will elute at the specified time windows, that's why we'd like to look for the peptide peaks only in those RT windows. In this exercise, I instruct Skyline to do MS1 filtering using precursor isotopic patterns. There are many cases that perfectly matching peaks around the explicit RT are found by Skyline, but somehow it picked other peaks somewhere else. I hope this explains my intent well.
Thanks,
David |
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