I am seeking help for setting site specific modification. I have been fixed Peptide setting - Modification - structural modification with all the respective targets which focused for. However I am facing issues to getting all the targets meanwhile, I have received comments as follows,
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Skyline
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The following modifications could not be interpreted.
N[+3007.05796] N[+3082.11514] N[+3227.15265] N[2059.73493]
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OK More Info
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System.FormatException: The following modifications could not be interpreted.
N[+3007.05796] N[+3082.11514] N[+3227.15265] N[2059.73493]
at pwiz.Skyline.Model.ModificationMatcher.CreateMatches(SrmSettings settings, IEnumerable`1 sequences, MappedList`2 defSetStatic, MappedList`2 defSetHeavy, IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 65
at pwiz.Skyline.SkylineWindow.ImportFasta(TextReader reader, Int64 lineCount, Boolean peptideList, String description, ImportFastaInfo importInfo) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 1647
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Please help me to get all the structural modifications.
Thanks
VC |
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| Nick Shulman responded: |
2024-06-27 11:57 |
In order for Skyline to be able to understand a modification mass like "[+3007.05796]" in a peptide sequence, you usually need to tell Skyline ahead of time about the modification by adding it to one of the lists on the Modifications tab at "Settings > Peptide Settings".
This is the webinar that we recommend for learning about working with modifications in Skyline:
http://skyline.ms/webinar10.url
If you are still having trouble you could send us your Skyline document as well as whatever text you are trying to paste into Skyline.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.
Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick |
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| cvadival responded: |
2024-06-27 12:33 |
Many thanks, Nick Kindly include all the changed sites that I have mentioned below, and refer to the Skyline file that is provided.
MVSHHN[+2204.77244]LTTGATLINE
MVSHHN[+2204.77244]LTTGATLINE
MVSHHN[+2351.85075]LTTGATLINE
MVSHHN[+2351.85075]LTTGATLINE
MVSHHN[+2350.83035]LTTGATLINE
MVSHHN[+2716.98295]LTTGATLINE
MVSHHN[+2716.98295]LTTGATLINE
MVSHHN[+3008.07836]LTTGATLINE
MVSHHN[+3008.07836]LTTGATLINE
MVSHHN[+2863.04086]LTTGATLINE
MVSHHN[+3154.13627]LTTGATLINE
K.NLFLN[+2059.73493]HSE.N
K.NLFLN[+2350.83035]HSE.N
K.NLFLN[+2424.86713]HSE.N
K.NLFLN[+2715.96255]HSE.N
K.NLFLN[+2715.96255]HSE.N
K.NLFLN[+3007.05796]HSE.N
K.NLFLN[+3082.11514]HSE.N
K.NLFLN[+3082.11514]HSE.N
K.NLFLN[+3227.15265]HSE.N
K.VVLHPN[+2350.83035]YSQVD.I
K.VVLHPN[+2350.83035]YSQVD.I
K.VVLHPN[+2424.86713]YSQVD.I
K.VVLHPN[+2715.96255]YSQVD.I
K.VVLHPN[+3008.07836]YSQVD.I
K.VVLHPN[+3008.07836]YSQVD.I
K.VVLHPN[+2862.02046]YSQVD.I
K.VVLHPN[+2862.02046]YSQVD.I
K.VVLHPN[+3154.13627]YSQVD.I
K.VVLHPN[+3154.13627]YSQVD.I
K.VVLHPN[+3373.21056]YSQVD.I
K.VVLHPN[+3372.19016]YSQVD.I
K.VVLHPN[+3664.30598]YSQVD.I
K.VVLHPN[+3664.30598]YSQVD.I
K.VVLHPN[+3809.34348]YSQVD.I
K.VVLHPN[+3809.34348]YSQVD.I
K.VVLHPN[+3008.07836]YSQVDIGLIK.L
K.VVLHPN[+3008.07836]YSQVDIGLIK.L
K.VVLHPN[+1913.67702]YSQVDIGLIK.L
K.VVLHPN[+2935.03683]YSQVD.I
Thanks
VC |
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| Nick Shulman responded: |
2024-06-27 18:29 |
When you are specifying the modification mass in a peptide sequence, you only need to specify one digit of precision after the decimal.
You can specify more digits of precision if you need to disambiguate between modifications with similar masses.
When you are pasting peptide sequences into Skyline you are also allowed to refer to the modification by name, for example:
MVSHHN[Mod_2204_8]LTTGATLINE
The only place where you are not allowed to refer to modifications by name is in spectral libraries (i.e. .blib files).
I think some of the modifications that are defined in your Skyline document might have different masses than you are expecting.
These are the modifications that are in your document:
| Name |
Formula |
Mass |
| Mod_1400_8 |
C124H204N8O90 |
3245.163216 |
| Mod_1352_2 |
C119H197N7O86 |
3100.125708 |
| Mod_1014_4 |
C119H197N7O86 |
3100.125708 |
| Mod_1327_5 |
C115H188N8O84 |
3025.068528 |
| Mod_1230_4 |
C104H171N7O76 |
2733.973111 |
| Mod_923_1 |
C104H171N7O76 |
2733.973111 |
| Mod_1133_4 |
C93H154N6O68 |
2442.877694 |
| Mod_1108_7 |
C90H148N6O66 |
2368.840915 |
| Mod_1011_7 |
C79H131N5O58 |
2077.745498 |
| Mod_2350_8 |
C90H146N6O65 |
2350.83035 |
| Mod_2424_8 |
C93H152N6O67 |
2424.86713 |
| Mod_2715_9 |
C104H169N7O75 |
2715.962546 |
| Mod_2862_0 |
C110H179N7O79 |
2862.020455 |
| Mod_3154_1 |
C122H199N7O87 |
3154.136273 |
| Mod_3373_2_N8 |
C130H212N8O93 |
3373.21056 |
| Mod_3372_2_N9 |
C129H209N9O93 |
3372.190159 |
| Mod_3664_3 |
C141H229N9O101 |
3664.305977 |
| Mod_3809_3 |
C146H236N10O105 |
3809.343484 |
| Mod_3008_1 |
C116H189N7O83 |
3008.078364 |
| Mod_1913_7 |
C73H119N5O53 |
1913.677025 |
| Mod_2939_1 |
C112H182N8O81 |
2935.036833 |
| Mod_2221_8 |
C85H139N5O62 |
2221.787757 |
| Mod_2512_9 |
C96H156N6O70 |
2512.883174 |
| Mod_2204_8 |
C84H136N6O61 |
2204.772441 |
-- Nick
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| cvadival responded: |
2024-06-28 07:24 |
Hello Nick,
I attempted to include the modified sequences as per your recommendation, but it was unsuccessful.
Kindly refer to the image and skyline document for a better understanding.
Help us in injecting each of the modified sequences that the settings mentioned.
Thanks
VC
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| Nick Shulman responded: |
2024-06-28 07:45 |
When you see the message "The following peptides did not meet the current filter criteria" you should push the "Keep" button so that Skyline will add those peptides to your document anyway.
The "filter criteria" that Skyline is referring to could be the "Max missed cleavage" settings on the "Digestion" tab at "Settings > Peptide Settings", or any of the settings on the "Filter" tab. If that's the case, then when you press "Keep" you will end up with peptides in your document that are fine.
It also might be that when Skyline used charge state and other settings from the "Filter" tab at "Settings > Transition Settings" Skyline could not find any precursors or transitions that would be appropriate for that particular peptide. In that case, when you press "Keep", the peptide will be added to your document but it will not have any Precursor or Transition nodes below it in the Targets tree.
If you end up with empty Peptide nodes in the Targets tree, you should right-click on one of the Peptides and choose "Pick children".
In the child picker window that comes up, it will probably show you an empty list of choices. You should click on the filter button in the child picker window (it looks like an upside-down triangle) and then Skyline will show you some precursor charge states that you can choose from and you will be able to add some precursors for that peptide to your document.
Then, you will might also need to click on each of the Precursors that you have added to the document and choose "Pick children" and add some Transitions.
-- Nick |
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| cvadival responded: |
2024-06-28 08:29 |
Hello Nick,
I appreciate your detailed explanation.
Thanks
VC |
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test.sky.zip
Skyline.jpg