Welcome to Skyline Support 

Welcome to the Skyline support forum. If you have a question about using Skyline, or if you encounter a problem, you can post your questions here.

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Requests 
Showing: limited to 100 requests
Can not import all peptides from library into Skyline dokument
(8 responses) fraenze 2017-02-02
Dear Support Team and Skyline users,

I am working with Skyline version 3.6 on a Windows 10 system and have following issue: If I explore my library and want to import all peptides which are in my library, than 50% of my peptides will not be imported into my Skyline document since it has no icon in front. I checked the forum and there are already a lot of similar questions around but no solution posted there helped me. I really appreciate any help at this point!

description in more detail:
We are working with cross-linked peptides and do a linearization of those with an artificial lysine in between as cross-linker modification. We use a script to transform peptides in there linearized form and create an .ssl file afterwards according to the Skyline website, with recomandat columns. If we use this .ssl file and our mxML files(converted from raw using MSconvert), we are able to build a library in Skyline. Afterwards we explore our library and do see not many icon assigned to our peptides. If I hover over the peptide I can see all modification masses and they are all defined correctly in peptide settings. There are still unassined intense fragments but they are likely from the cross-linking area and can not be assigned due to the fact that our cross-links where linearized. Additionally I also saw spectra with unassigned intense fragments with an icon which could be imported so I am not sure where are the difference in this cases. I checked all peptide and transition settings, played with it but nothing changed. I will still loose half of my peptides. I also changed the cut-off score while building the library but also no changes. I tried to subdivide my library into smaller lists to separate peptides with similar sequences because I thought Skyline may not be able to distinguish between peptides with simlilar fragments (without specific fragments for each peptide) but still no changes.

I hope you have suggestions. Thank you for your time and solutions you can provide me.
See Complete zip file attached.

Best wishes
Fränze

PS: After updating to Skyline 3.6 my Library explorer crashes quite often although all storage places for files are the remained the same. And one last question: How does Skyline calculate the CV values over replica? Is it the standard diviation within replica divided by the mean?
 not importable cross-linked peptides.sky.zip 
view request
Problem with custom report after updating skyline
(2 responses) tyler w bradshaw 2017-10-17
Hi Skyline group,

I updated Skyline daily today, and unfortunately I'm having an issue with a custom report (!RatioOnly.skyr) that I've made. Previously I didn't have any problems with this data export. Other reports seem unaffected.

When I try to preview the report I receive an error message:
"An error occured while displaying the data rows:
Object must implement |Convertible.
Do you want to continue to see these error messages?

If I go ahead and save the report anyway the .csv file is empty.

Thank you for any help in advance,
Tyler
 !RatioOnly.skyr 
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Not all fragments shown in Skyline that are found in library
(14 responses) Helian 2017-10-03
Hi,

I have run two shotgun runs for some PRM development and built a library from those shotgun runs after making a MaxQuant search. For some peptides I see more fragments in the library than in the shotgun run the library was built from (added a picture from Skyline). Do you know how that can happen or how to fix this problem.

In the example shown, y11 fragment has been found in MaxQuant search (msms file also added here, saved as Excel for keeping the formatting) and is also included in the library. However y11 fragment has not been found by Skyline while importing the same shotgun runs which the library was built from. In some of the peptides with the same problem the missing fragment has rank 1, so it would be a pity to miss it. I have ticked the integrate all setting and the fragment was there while importing the results.

Hope I have enough information included and waiting for your reply,
Helian
 Example peptides with problem.sky.zip  Peak areas with library and results.png  msms used for library.xlsx 
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Cant see the chromatograms
(12 responses) mostafa khan 2017-10-16
Hi,

For some reason I am not able to see the chromatogram for any of the samples for one of the compounds, while I see them clearly with the rest of the compounds. I changed the product ions in the transition list, yet I cant see them, although I see them clearly in xcalibur. What am i doing wrong?

Thank you in advance.
 Sky1.jpg  Sky2.jpg 
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Original Peak Skyline Daily
(1 response) trisha almazi7688 2017-10-16
Hi,

I have just started using skyline daily with the latest update.

I have noticed this new function called 'Original Peak'

How is the original peaks picked? What factors?

How sure can I be that this is my peptide peak?

Cheers,

Trisha
view request
loading sky doc genrated in daily on older version
(2 responses) eer55 2017-10-16
Dear Skyline,

We have an offline LC-MS system running 3.7.0.11317.

On our processing workstation (PC) I am running 3.72 (Skyline daily).

I would like to be able to load methods generated on my PC onto our instrument PC, but the methods are incompatible. Should I install the non-daily version of my PC to allow this? I would like to continue using daily but need to be able to transfer methods.

Best regards,

Elden
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process of IDA (information dependent acquisition) ABSciex Qtrap 6500
(1 response) shulei lei 2017-09-30
Hello,

I have a problem to open IDA data which was acquired on AB Sciex Qtrap 6500. The method for the IDA data is a scheduled MRM in experiment 1 followed by EPI full scan in experiment 2 (MRM-IDA-EPI). The purpose of IDA is to confirm the peptide sequence of precursor ions in the MRM list.

When I opened with Skyline, I can not see any chromatography and MS spectra. In the transit list, I only got a few precursor ions which have a red dot and a few product ions associated with those precursor ions, but half of precursor ions in my transit list do not even have a red dot.

Probably I did not set up right. Do you have a manual for me to follow in this specific case?

Thank you,

Shulei
view request
Suggestion - Multiple libraries use
(1 response) vdelcourt 2017-10-12
Hi,

As it's my first post on Skyline support, I'd like to thank the Skyline team for providing such amazing program and detailed tutorials to the community
This a suggestion rather than a bug, so I'll go through each steps.

We are beginning a new targeted proteomics project (using PRM) and we decided to generate a new spectral library (AP-MS experiments on target proteins to generate many high-quality PSMs).

After samples were submitted to the MS, RAW files were identified using MaxQuant and msms.txt files were imported into Skyline. In order to build the m/z selection list for targeted experiments on target proteins, I went through every peptides of targeted proteins and took a look at each MS/MS spectra stored in Skyline. I've been quite surprised to see some low quality MS/MS spectra by clicking on different peptides (left bar of skyline). After looking them up in the MaxQuant viewer, I noticed that these particular peptides had better MS/MS spectra than the one Skyline showed me.

After looking around, I noticed that old libraries (from previous projects but generated on the same instrument) were still used (check box checked). After unchecking those boxes (and leaving only the one we just generated), Skyline now shows me peptides with high-quality MS/MS spectra.

I guess that if I didn't uncheck those "old" spectral libraries, confidence on spectral matching (dot products) would have been really low and thus might have been wrongly discarded.

My suggestion would be that Skyline could show best MS/MS spectra (highest score) between multiple loaded spectral libraries. (I think that would be fixed by generating new spectral library with every msms.txt files but this would be also time consuming when importing large spectral libraries)

Best regards,
Vivian
view request
Request for Synthetic Biology Applications
(1 response) Peter Jackson 2017-10-07
Hi Skyline Team,

I have a request that I suspect may be unique to working in a synthetic biology environment.

We frequently examine groups of engineered strains with lots of different heterologous enzymes expressed. When working in a single document, I frequently have the need to perform 'remove peak' from lots of different samples because they simply don't have the proteins that other samples do. What would be very useful to me is a way to go through either the peak area graph, or the retention time graph and ctrl+click many different peptides and remove them all in bulk. Could this functionality be added?

Thanks,

Peter
view request
Strange peak quantification behavior
(1 response) gabe 2017-10-12
I'm encountering a strange behavior lately that I hope someone could comment on. The automatically picked peak will show a surprisingly low AUC and when I look at the chromatograph the dashed lines (showing the area for quantitation) are in the right place. But the little black arrow showing which peak is picked is pointing at one of the less-abundant transition ions. If I manually re-select the RT-range, the black arrow jumps to the top transition and the AUC goes up to normal. Why is this happening?

Here's an example

Thanks
view request
concatenating Skyline documents via Skylinerunner?
YangZhang 2017-10-11
Dear Skyline team,

Is there a way to use Skylinerunner to combine multiple Skyline files?
In the GUI, this is easily doable by File->Import->Document, but according the runner document, import seems only work for results/replicates.

Thanks,

Yang
view request
Semi-Cleavage
(4 responses) Vera 2017-10-10
Hi Skyline team,

I know you have already update an "allow Semi-Cleavage" section for enzyme in Version 3.7. However, it doesn't really work because although I clicked the option in enzyme edit, nothing changed and if I click the enzyme I just edit, the option was shown not checked at all. It looks like skyline can't remember or recognize the setting. Could you help figure out what happened?

Thanks a lot.
Vera
view request
transitions not showing
(3 responses) tannous 2017-10-10
Hi,
Is anyone facing this? Skyline daily is not showing any transitions when I paste a peptide list or import a FASTA.
I tried that in Public skyline using the same peptides and transitions settings and it is working fine (ie showing all the transitions).
Is there something I am missing?
I have always used these peptides and transitions setting before in daily and it seems to be fine.
Is there some settings elsewhere other than peptides and transitions settings that I may have inadvertently used that is preventing transitions from showing up?
Thanks
Abla
view request
Spectrum Library with specific modifications
(7 responses) sophie lagache 2017-09-25
Hi Skyline team,
I have a quick question regarding the spectrum library ... how the modifications are coded by mass difference or by mass adduct ?
I would like to look at the citrullination modifications on the Arginine which were identified on my sptxt file library but after adding in Skyline it seems to be disappeared !!! Is it because it is not a modification existing in your list, or do I have to do something special to see it ...
Thanks in advance,
Sophie
view request
Conflict with transition precursor++ in ion for peak area calculation
(11 responses) lparsons 2017-09-28
I do not understand how to address this error. I have loaded a list of transitions into a new skyline document and am attempting to load some Thermo MS1-only spectra into the document. These transitions were loaded with assigned charges (assigned by DeconTools) leading to skyline reporting 3,068 precursors / transitions. When I then try to import 4 raw files (File -> Import -> Results) I see errors such as this. I eventually want to import 30 files but am stuck trying to troubleshoot this error.
The 4 files that I am trying to load are related in file name such that they end as "A01, A02, A03, B01" (later files pick up at B02 and go on through J03). If I load only A01, A02, A03 I generally do not get this error though any file I load after does give the error.
Here is the detailed error:

System.IO.InvalidDataException: Conflict with transition precursor+++ in Ion [1147.581097/1147.581097] for peak area calculation. Did you mean to use a different precursor charge state or label? ---> System.IO.InvalidDataException: Conflict with transition precursor+++ in Ion [1147.581097/1147.581097] for peak area calculation. Did you mean to use a different precursor charge state or label? ---> System.ArgumentException: An item with the same key has already been added.
   at System.ThrowHelper.ThrowArgumentException(ExceptionResource resource)
   at System.Collections.Generic.Dictionary`2.Insert(TKey key, TValue value, Boolean add)
   at pwiz.Skyline.Model.PeptideDocNode.PeptideChromInfoCalculator.AddChromInfo(TransitionGroupDocNode nodeGroup, TransitionDocNode nodeTran, TransitionChromInfo info) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:line 1578
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.PeptideDocNode.PeptideChromInfoCalculator.AddChromInfo(TransitionGroupDocNode nodeGroup, TransitionDocNode nodeTran, TransitionChromInfo info) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:line 1588
   at pwiz.Skyline.Model.PeptideDocNode.PeptideChromInfoListCalculator.AddChromInfoList(TransitionGroupDocNode nodeGroup, TransitionDocNode nodeTran) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:line 1333
   at pwiz.Skyline.Model.PeptideDocNode.PeptideResultsCalculator.AddGroupChromInfo(TransitionGroupDocNode nodeGroup) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:line 1139
   at pwiz.Skyline.Model.PeptideDocNode.UpdateResults(SrmSettings settingsNew) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:line 1051
   at pwiz.Skyline.Model.PeptideDocNode.ChangeSettings(SrmSettings settingsNew, SrmSettingsDiff diff, Boolean recurse) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\PeptideDocNode.cs:line 864
   at pwiz.Skyline.Model.SrmDocument.<>c__DisplayClass4a.<ChangeSettingsInternal>b__39(Int32 i) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 938
   at System.Threading.Tasks.Parallel.<>c__DisplayClass17_0`1.<ForWorker>b__1()
   at System.Threading.Tasks.Task.InnerInvokeWithArg(Task childTask)
   at System.Threading.Tasks.Task.<>c__DisplayClass176_0.<ExecuteSelfReplicating>b__0(Object )
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Util\Util.cs:line 1936
   at pwiz.Skyline.Util.ParallelEx.LoopWithExceptionHandling(Action loop, Action`1 catchClause) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Util\Util.cs:line 2129
   at pwiz.Skyline.Model.SrmDocument.ChangeSettingsInternal(SrmSettings settingsNew, SrmSettingsChangeMonitor progressMonitor) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 926
   at pwiz.Skyline.Model.Results.ChromatogramManager.Loader.FinishLoad(String documentPath, MeasuredResults resultsLoad, MeasuredResults resultsPrevious) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\Chromatogram.cs:line 281
   at pwiz.Skyline.Model.Results.ChromCacheWriter.Complete(Exception x) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheWriter.cs:line 171

I have previously been able to load all 30 these files into skyline at once with a different transition list without seeing this error at all. Any suggestions on how to address this error would be helpful. Also, the identity (mass) of the ion is not the same for all 4 of these files.
view request
Unsupported score type error when building library from mzIdentMLfiles
(4 responses) michael hall11191 2017-09-21
Dear Skyline team,
We are performing comparative DIA analysis using a Waters Synapt G2Si instrument and I have a short question regarding DDA data which we are trying to use for a Skyline library.

We are trying to integrate a workflow where we build a library for Skyline using multiple DDA datasets which have been processed/identified using the Waters software PLGS.
While PLGS outputs final_fragment.csv files for MSe identification workflows, it does not do so for fragment ion DDA searches. Instead it can be set to output mzIdentML files from such identification searches. I thought we would simply be able to use these mzIdentML files to build a Skyline library, but when I try to build I get the error message ‘Unsupported score type’ and it fails to build the library. We are currently using Skyline daily v. 3.7.1.11357.

Do you know of any simple workaround to this problem?

We have instead used Mascot for the DDA identification and the resulting .dat files for building a Skyline library and this works fine. We would however prefer a workflow directly from PLGS if that was possible.

Best regards,

Michael Hall
view request
Importing data slow?
(1 response) nschuld 2017-10-05
Hello,

I've been having issues with the skyline program import function on my computer. Specifically, when I use the File_Import_Results function the program responds very slowly. The Import Results window pops up no problem, but when I click on 'OK' to search my directory for the files it takes about 1-2 mins to load this directory, and anytime I try to click on a new folder it takes 1-2 mins. This only happens on my desktop (which is the newest one in the lab). This function works fast on our older model computers in the lab. I have had my IT guy look at my computer specifically and he found no apparent issues with why this would be occurring. Also, I've tried with both Skyline.daily and the Skyline (64-bit) 3.7.0.11317.

I was wondering if this might be a simple fix based on something I can switch around in the settings or if anyone has a good idea on how to fix this.

Thanks,

Nathan
nschuld@mcw.edu
view request
Peak picking models for MS1 data from DIA runs
(12 responses) k valgepea 2017-09-19
Hello!

I have DIA data from Thermo HF runs and I would like to use MS1 data for quantification. Regardless of whether I use only MS1 data (precursors) or also fragment ions (y- and b-ions), both the Skyline default and mProphet peak picking models yield q-values>0.01 for all peptides regardless of which combination of feature scores I use (and whether decoys or second best peaks are used). So no peptides/proteins pass the 1% FDR cut when MS1 data is included. Also the feature scores and p-value distributions look strange for both cases (see attachments). Things look okay when using only fragment ions.

Brendan might have answered my question in a previous reply to another question (see below), but does my observation mean that Skyline is currently not suitable for analysis of DIA data at MS1 level, as the peak picking models have been built based on MS/MS data?

Brendan's previous reply - https://skyline.ms/announcements/home/support/thread.view?rowId=26706
"Anyway, I can't personally vouch for the mProphet models produced for MS1 filtering of DDA data. The mProphet approach itself has only publications validating its use on SRM and DIA data, from which we might guess that it should also work on PRM data, all approaches that yield MS/MS chromatogram peaks."

Thanks a lot for your help as always!
 p values (precursors+fragment ions).png  p values (precursors).png 
view request
MaxQuant Peak Boundaries
(3 responses) <1803> 2014-03-20
Hey Brendan,
I am trying to import MaxQuant peak boundaries to be used in Skyline to view SILAC labelled heavy and light peaks. Is there a specific format that Skyline requires to load such data? It mentions that it needs PeptideModifiedSequence, FileName, MinStartTime, MaxEndTime. Should there also be a 'precursor charge' column?

Thanks!

-Chris McKennan
view request
Unique peptides column limit
(2 responses) Max Karlsson 2017-10-03

Hi,

Hope all is well with you guys. I tried to use the unique peptides tool (probably on far too many peptides) and got the following error:

Skyline version: 3.6.0.10493 (64-bit)
Installation ID: d044130d-6124-49e5-88d3-36c1bbddf29a
Exception type: InvalidOperationException
Error message: Sum of the columns' FillWeight values cannot exceed 65535.

--------------------

Stack trace:

   at System.Windows.Forms.DataGridView.OnAddingColumn(DataGridViewColumn dataGridViewColumn)
   at System.Windows.Forms.DataGridViewColumnCollection.Add(DataGridViewColumn dataGridViewColumn)
   at pwiz.Skyline.EditUI.UniquePeptidesDlg.LaunchPeptideProteinsQuery() in c:\proj\skyline_3_6_x64\pwiz_tools\Skyline\EditUI\UniquePeptidesDlg.cs:line 220
   at pwiz.Skyline.EditUI.UniquePeptidesDlg.OnShown(EventArgs e) in c:\proj\skyline_3_6_x64\pwiz_tools\Skyline\EditUI\UniquePeptidesDlg.cs:line 186
   at System.Windows.Forms.Control.InvokeMarshaledCallbackHelper(Object obj)
   at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
   at System.Windows.Forms.Control.InvokeMarshaledCallback(ThreadMethodEntry tme)
   at System.Windows.Forms.Control.InvokeMarshaledCallbacks()

Exception caught at:
   at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
   at System.Windows.Forms.Control.InvokeMarshaledCallbacks()
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.Form.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
   at pwiz.Skyline.SkylineWindow.ShowUniquePeptidesDlg() in c:\proj\skyline_3_6_x64\pwiz_tools\Skyline\Skyline.cs:line 2200
   at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
   at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
   at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ToolStrip.WndProc(Message& m)
   at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at pwiz.Skyline.Program.Main(String[] args) in c:\proj\skyline_3_6_x64\pwiz_tools\Skyline\Program.cs:line 299


Does the unique peptide tool work as well if I do it on subsets of the peptides one at a time, or is there some workaround?

Thanks,
Max
view request
mProphet models, % contribution and min. number of groups
(1 response) mvillal1 2017-10-03
Hello Skyline team, I hope you are doing fine.

I am using different datasets to train three MRM mProphet models that I then apply to some other MRM raw files from samples with more complex backgrounds and get q-values of the peak groups that were detected. However, after I train a model and then use it to re-integrate the results from the other more complex samples, I am noticing that the % contributions of the same model change, before and after I re-integrate the results... Why is this happening? Is the scoring changing? I am attaching a document with one of these cases.

Finally, according to your experience, do you suggest a minimum number of decoys/target transition group records for training (or using) mProphet models?

Many thanks,
Ivan.
 mProphet_Model_Example.docx 
view request
Skyline support PRM method importation
(1 response) William 2017-10-03
We are trying to do a SRM vs PRM comparison but have a question of how a method generated from Skyline be imported to QTOF (or Q Exactive)?
view request
Updating after changing Full-Scan filters
(3 responses) gwilson9 2017-10-03
Hi All,

Thanks for a great tool and a lot of technical resources, as well.

I have been having an issue, that when I change the retention time setting in 'Transition setting'->'Full-scan' (Use only scans within ... minutes of MS/MS IDs) it does not change the results. And when I try to force the data to update by Re-importing in 'Manage Results', the process does not go through. All that happens is a '*' appears in front of each raw file's name.

My only work around has been to start from scratch with the new retention time window. Is there an easier way to do this?

Thanks!
view request
retention time restriction for targeted EIC determination
(2 responses) Jens 2017-09-28
Dear Skyline-Team

I am currently looking for a software, which is able to semi-automatically extract EICs for certain defined peptide species (e.g. non-deamidated and deamidated peptides and/or non-oxidized and oxidized peptides etc.) only in defined retention time windows, just based on MS1 (Precursor+corresponding isotopes) and without a previous identification e.g. using a search engine. In Skyline (v3.5.0.9319) I was able the create a transition peptide list for different charge states of such peptides and use it to get EICs. But, unfortunately I was not able to define retention time limits for gaining those EICs. When I tried to put in explicit retention times and windows (e.g. in the document grid under peptide view) it was ignored by the software (or I did it wrong?).
The retention time restrictions would be necessary to separately determine EICs for e.g. iso-aspartate and deamidations of a peptide, which have exactly the same mass (and isotopes), but different elution times/windows. The goal would be to get those EICs without correcting retention times or reassigning peaks.
Is it possible to define retention time limits/restrictions within Skyline in which the EIC for a certain species is determined and use it to get EICs directly from Thermo-raw-files?

Thanks a lot in advance!

Best regards,
Jens
view request
Waters Unifi .uep file to mzML
(4 responses) gegoe 2017-09-28
Hi, i saw this question has already been asked a few months ago but there was no solution, i would like to convert Unifi .uep files (oracle database format) to mzML files, is this possible already? I want to share my data that was acquired on a Vion Qtof with other people, and use the data in other applications such as Skyline, but Waters does not offer any other export type but .uep files, and n software except Progenesis QI can read this. Any suggestions?

Cheers, Geert
view request
specify score type used for blib library from pepXML
(5 responses) gabe 2017-09-26
Hi,
I'm creating .blib libraries from pep.xml files. It's using the peptideProphet probability scores to choose the best spectra, but I would prefer (I think) for it to use XCorrs, which are also in the pepXML files:

   <search_result search_id="1">
    <search_hit hit_rank="1" peptide="SVCHVPGLKK" peptide_prev_aa="R" peptide_next_aa="M" protein="sp|P19474|RO52_HUMAN" num_tot_proteins="1" num_matched_ions="13" calc_neutral_pep_mass="1390.8117335332" massdiff="-0.001656" num_missed_cleavages="1" is_rejected="0">
     <modification_info>
      <mod_aminoacid_mass position="3" mass="427.225184479"/>
     </modification_info>
     <search_score name="peptide_xcorr" value="2.7737"/>
     <search_score name="peptide_deltacorr" value="0.5253"/>
     <search_score name="peptide_diff_seq_deltacorr" value="0.5253"/>
     <search_score name="peptide_initial_score" value="251.5"/>
     <search_score name="peptide_initial_rank" value="1"/>
     <search_score name="expect" value="0"/>
     <analysis_result analysis="peptideprophet">
      <peptideprophet_result lda_probability="0.0022943213649662" probability="0.99770567863503"/>
     </analysis_result>
    </search_hit>
   </search_result>


Is there any way to force BiblioSpec to use XCorrs instead of PeptideProphet probabilities? (Score type 12 rather than 2)

Thanks

Gabe
view request
Trouble with upgrade 3.7.0.11317
(1 response) Sadr 2017-09-26
Hi,

I am trying to upgrade skyline to 3.7.0.11317 but facing problem.

Please check the attached text files for full details.

can you guide me on this?

Regards,

Sadr
 KONUVV74.log  File 2 of error.txt 
view request
Trouble importing peptide search ("Importing the FASTA did not create any target proteins")
(9 responses) whitsonj 2017-09-25
Hi, I'm a new Skyline user and I'm having trouble getting some global phospho proteomics data into Skyline.

I ran our .RAW phospho data through ProteoWizard and the Crux-pipeline in order to produce the peptide search files in .SQT format as well as a .perc.xml file. I've been trying to import the search into Skyline using the import peptide search wizard, following the instructions in the MS1 filtering tutorial. When I get to the last step Skyline tells me that "Importing the FASTA did not create any target proteins" and will not proceed with the import. I'm just using the most up to date Uniprot FASTA of all mouse proteins. I also have the associated .MS1, .MS2, and .RAW files in the same directory with the .SQT and .perc.xml files and the names all match up.

Any help would be greatly appreciated!
view request
Questions about iRT calculators
(9 responses) gabe 2017-09-11
Hi Brendan,

I'm trying to understand iRT predictions: we routinely generate DDA data to populate reference spectral libraries for use in PRM experiments. We use 12 ubiquitous, endogenous "standard" peptides that span the retention-time range that we're interested in, rather than commercial/exogenous standard peptides. I create spectral libraries by exporting filtered SEQUEST results from these DDA runs and use Skyline to Build .blib libraries. Skyline gives the option of specifying "iRT standard peptides" in the 'Build Library' tool, but only allows you to choose from a pre-defined list of options (Biognosys-10, Biognosys-11, etc). This brings me to my first question:

Are iRT values stored in .blib files? If so, how can these be calculated if the pre-defined lists of standard peptides are not used? If the iRT values aren't stored in .blib files then what is the point of selecting a group of iRT standard peptides during the blib build process? How is that information used?

Next question is when I combine reference spectral libraries (either from primary data or by combining .blib files) how are the iRT data stored? If I generate a spectral library from 30-minute runs, and combine it (without keeping redundant spectra) with a spectral library from 180-minute runs, and add the *combined* .blib file to an iRT calculator - how will it find the correct iRT values? Is it necessary to keep the redundant spectra if you want to be able to do this sort of thing?

Thanks

Gabe
view request
Skyline daily saved file error when trying to open
(2 responses) azad 2017-09-15
Hello,

A skyline daily file that was previously working will not open. I've attached a screenshot of the error message. Thanks in advance for any assistance.

Best,
Azad
 Skyline file error.pdf 
view request
a question of labelfree quantitation
(3 responses) 1009114398 2017-09-21
Hi,skyline team,
I have a question about using MS1 extracted ion chromatograms in Skyline for label-free quantitation.
When conducting Large-scale data analysis,What is the criterion for judging the XIC chromatograms is right or not?
view request
More Keyboard Shortcuts
(4 responses) zane 2017-06-21
Hi guys,

I go through LARGE data sets on Skyline routinely. RSI is a real problem in these cases. Currently, the only way I know how to remove peaks, is to do a mouse-click, another mouse-click, and a final mouse-click going through the standard way. Is there an option for peak removal that's a hot key? (example: ctrl+r removes peak on current chromatogram).

The only short-cuts I know of for keyboards are to scroll through the chromatograms :)

Thanks in advance!
Jess
view request
Choose specific reference spectrum
(3 responses) gabe 2017-09-23
If I have several different reference spectra available for a given peptide, is there a way to specify a particular reference spectrum to use just for that peptide? I know how to switch between different spectral libraries, but that changes the library for the whole document. What I'd like to be able to do is specify a reference spectrum from library A for peptide 1, and from library B for peptide 2, etc. Is there a way to do this? Sometimes the options of 'picked intensity' or 'spectrum count' don't do a perfect job of choosing the best reference spectrum and I'm not sure how to get around this...

Thanks

Gabe

view request
Filtering DIA Data
(4 responses) Brett Phinney 2015-11-24
Is there a filter for filtering away peaks whose mass error is above a certain threshold? For example this peptide is not in one of my samples, but I can't figure out how to filter out the incorrect peak area (see .png file) besides manually inspecting it. This is DIA data btw on an engineered organism where I know this peptide is not in one of the samples. Any advice? I'm looking for an automated filter like the idotp filter. Sorry if this is in a tutorial somewhere and I have not found it.

thanks!

Brett
 skyline1.png  skyline2.png 
view request
how to build a library by .msf file
(2 responses) hanmeng 2017-09-14
Hi,I am a new user of skyline, but recently I have a question when I was used. I hope you can give me an answer.
    I want to no why the result of Proteome discovery (Thermo fisher) can not import in skyline as a library and there is a error appeared .just as the follows,

ERROR:this file dose not contain q-values. You can set a cut-off score of 0 in order to build a library from it, but this may cause your library to include a lot of fasle-positives.

ERROR
ERROR: reading file BSA.msf

I look forward to receiving your reply.

Thank you

meng han
view request
spectral library as reference
(3 responses) sarah 2013-02-01
Hi Brendan,

I have a quick question concerning use of spectral libraries for results evaluation. If I have several libraries loaded and am looking at an SRM experiment, Skyline will give me a dot product score next to the precursor as well as the order of fragments in both chromatographic peak (from my experiment) and spectrum. Now, when I switch between different libraries (which all contain a spectrum for the peptide of interest), the dotp and order of fragments stays the same although sometimes it's obvious that the spectra are quite different. So I have a feeling that Skyline doesnt update the values here....But then my question would be: Which of the libraries is used when calculating the dotp (and order of fragments)?

Thanks in advance!
Sarah
view request
Skyline Regular unable to process SWATH data set
(9 responses) mmidha 2017-09-21
Hi Brendan,

I have a SWATH data set comprising of 45 Wiff files (15 conditions X 3 replicates each). I am trying to import many swath files simultaneously to speed up the analysis. At Data analysis step, importing results window got stuck at 40% forever, which I later cancel all imports. I tried the same data set by importing several files simultaneously, though it completes the analysis but takes days for me to fetch the report.

During any time in analysis, Skyline did not use more than 10% CPU. It seems Skyline was unable to use maximum potential of the system. Here are the details of the system on which I am running Skyline regular.

Intel Xenon CPU E5-2683 v4 @2.10Ghz
Logical Processores: 64
Memory: 256GB
DISK 0(C):4TB
DISK 1(D):10TB

My question is can I use Skyline regular for analyzing this size of SWATH data set? If yes, how can I speed up the analysis (it would be great if skyline can utilize the full potential of the system)? If not, then what is the alternative (Skyline runner)?

Let me know if you need any other details.

cheers

Mukul
view request
Import peptides AND charge states
(5 responses) gabe 2017-09-22
Is there a way to import a list of peptides AND charge-states into Skyline? I know I can import peptide sequences and have the charge-states auto-populated based on spectra in the reference library, but I can't figure-out a way to import specific charge-states into a document.

Thanks

Gabe
view request
Reorder Tabs
(1 response) dawn.r.dufield 2017-09-22
Is there a way to reorder tabs? If I sort the "managed" files they show up in the table and lists in the correct order, but the tabs do NOT. Is there a way to change this so when I scroll through the tabs that are in the correct order?

It seems like they are listed in alpha numberic order...
 Tab Order.png 
view request
AutoQC-conflict in Skyline version
(8 responses) tannous 2017-09-20
Hi,
I am trying to set up AutoQC with Skyline daily. I downloaded AutoQC for daily.
It worked on one computer where I have both daily and regular skyline. When I tried to set up again on a different computer also with both versions, once I ran AutoQC, an error message was telling me that skyline was created with a newer version (which corresponds to daily) than the version that is available (which corresponds to regular), even though everything was created in daily.
Do I have to have only one Skyline version for Auto QC to work? it seemed to be OK to have both when I first tried that on a different computer.
Another question, are the skyline daily updates going to create an issue for the automation process? is it better to set up AutoQC in regular Skyline?
Thanks
Abla
view request
How does SkylineCmd.exe calculate chromatographic area?
(1 response) lparsons 2017-09-21
If I import raw data into SkylineCmd.exe and use the --chromatogram-file option to export chromatograms to a tab delimited file, how are the peak areas calculated?

I had - perhaps erroneously - expected that the peak area would be the sum of the intensities for the ion in question. I am using a transition list that is already in the template file, which is looking only at MS1 data. However I found that if I take the intensities from the exported chromatogram file and sum up the intensities of just the window that is +/- 60 seconds of the most intense peak for the ion m/z in question, I often get a sum that far exceeds the area reported for the chromatogram.

Is the area calculated differently? I may have missed a tutorial that better describes how this is done; please feel free to refer me to the one I should review for this if so.

Thank you
Lee
view request
Supporting generic peptide-level q-value in mzIdentML
(3 responses) rmillikin 2017-09-06
Hello,

Our lab is working on search software that extends Morpheus, called MetaMorpheus ( https://github.com/smith-chem-wisc/MetaMorpheus ). One format that we output is mzIdentML, which I would like to be able to import into Skyline. However, only some scoring mechanisms are supported by Skyline; we have a q-value output that we would like to use. MS-GF outputs q-values and these are supported by Skyline, so it should not be much of a stretch to import our q-values. We don't have CV terms yet, but would it be possible for Skyline to support a generic q-value (already listed by PSI-MS)?

[Term]
id: MS:1002354
name: PSM-level q-value
def: "Estimation of the q-value for peptide spectrum matches." [PSI:PI]
xref: value-type:xsd\:double "The allowed value-type for this CV term."
is_a: MS:1002347 ! PSM-level identification statistic
relationship: has_domain MS:1002305 ! value between 0 and 1 inclusive

from https://raw.githubusercontent.com/HUPO-PSI/psi-ms-CV/master/psi-ms.obo

An example mzML and mzID from MetaMorpheus (yeast lysate) can be found at:
https://uwmadison.box.com/s/6uaasr4foe3vf5eyjz4mko3wjy3x6iog

If you simply find+replace the following, Skyline will import correctly, otherwise it will give an unsupported score error upon import:

'accession="MS:1002354"' replace with 'accession="MS:1002054"'
'name="PSM-level q-value"' replace with 'name="MS-GF:QValue"'

Thank you for your time,
Rob Millikin
UW-Madison
 Capture.PNG 
view request
Discrepancies in Heavy and Light Peptide Chromatogram Import Ranges
(2 responses) robwsprung 2017-09-21
Hi Brendan,

This message is in response to issue #544

I am extracting fragment ion information as well (3 fragment ions per peptide). The file in question was a DDA run in which our heavy and light peptide standards are spiked at 100fmol each (on column) into a complex matrix (1ug tumor tryptic digest on column).

The issue appears to be specific to Skyline-daily (64-bit) 3.7.1.11357.

When I import the file into Skyline (64-bit) 3.7.0.11317, I see the entire chromatogram as expected.

Let me know if you need additional information or files.

Robert
view request
how to do the retention time alignment
cjchen 2017-09-20
HI skyline team,
We are confused about the protocol to do the retention time alignment between run-to-run of DIA data.
We searched methods in the skyline website, but we can only find the iRT calibration protocol.
We follow the iRT calibration steps as illustrated in the attached file. However, we think that the iRT calibration is used for time prediction of MRM. Is it right?
If we want to do retention time alignment in DIA data by using our added peptides or endogenous protein digests, is there any detail protocol for us to follow step-by-step?



Best,

Chen
 Skyline iRT retention.pptx 
view request
Problems generating background proteome
(3 responses) chiva cristina 2017-09-18
Dear Brendan,
We have a random error when trying to generate a background proteome. Sometimes after clicking "Add", then "Create", defining the path, and clicking "Save" we are unable to add a fasta file because the "Add File" option is inactive (in grey). Any clue?
Thanks a lot and see you soon!
Cristina Chiva
view request
Can I get more Verbose output from SkylineCmd.exe ?
(6 responses) lparsons 2017-09-11
I don't see a verbosity argument in the documentation I have for Skyline; is there a way to get more verbose output? I have been trying to get it to build a file using the results of a large number (30) of raw files and once the last one reaches 96% there is no longer any sign of anything happening. If I check Windows Task Manager the most CPU activity I see from a SkylineCmd.exe" process is less than 10% (only 4 or 5 are listed the rest are 0% CPU). None of them are using more than ~120MB RAM either (on a system with 64GB).
I have a SkylineCmd.exe process that seems to have been stalled now for well over a week. It doesn't seem to have crashed, but it doesn't seem to be making any progress either. No new files have been written or started since starting the run.
I plan to try this again with far fewer files - when I run this same command with just one Thermo RAW file everything is fine - but I was wondering if there is any way to get more information from a CLI run.

thank you
Lee
view request
Feature request: show predicted retention times on replicate comparison plot
(1 response) gabe 2017-09-13
Hi, forgive me if this is possible or has been requested before, but it would be very useful to be able to see the predicted retention time (and range) on the replicate comparison plots of Retention Times. See attached mock-up. Note that the quantified peak is at 12.9 minutes but the predicted RT is at 9.6 minutes. It would be useful to be-able to see this discrepancy in the replicate comparison plot.

Thanks

Gabe
 Untitled.png 
view request
Program does not start after installing a patch to v3.7
(2 responses) nikki 2017-09-13
I tried to install v3.7.0.11317 over my v3.7 and now the program will not start. Could you help? I have attached the error log with this message.
 BSCQ73UK.log 
view request
Separating heavy and light during data analysis
(1 response) anamikab 2017-09-13
Hi
We have a question regarding data analysis in Skyline. We use heavy labeled standard along with endogenous peptides. Thus when we open our raw data we get to see both heavy and light peptides and after choosing the peaks we export the data of both heavy and light peptides including their ratios. Our question is: is there a way skyline enables us to separate the heavy from the light? What we are trying to get is two xl files one having quantitative info of light and one having that of heavy. We know we can do it manually selecting heavy and light separately; but we are dealing with 600 transitions in sets of 40-50 samples or more, thus manually doing it is impossible.
Will greatly appreciate if you can send us your feedback.
view request
DIA_overlapmz_import
(10 responses) tannous 2017-09-08
Hi Brendan,
Is there a script available/other ways of importing DIA data with overlapping mz windows into Skyline?
It took a long time to import one run (about 2 hours)?
I have a script I was using for importing DDA data. Should the same script work for DIA data with overlap? I tried it once and it failed.
Thank you
Abla
view request
remove missing results
(2 responses) chia-lin 2017-09-12
hello,

 I recently meet some problems, when I try to refine-remove missing results any change does not happen. Because I have use mprophet adjust my peptide with q value, there are many red dots. These dots does not match library well and they are still there even I do the remove missing results. Look forward to your reply.
view request
Retention time prediction without running any DDA
(1 response) Samuel Weng 2017-08-31
Hi all,

I have setup my retention time predictor in my peptide settings (I use Pierce iRT standards,60 min gradient). Right now I want to predict the retention time with a set of peptides that I am interested (I don't have any spectral library). Is that possible if I can predict their retention time without running any DDA? I want to directly run a PRM run because I don't have alot of sample. I have attached the protein I am interested for your information.

Thank you so much!

Samuel Weng
 LCL SPROUTY_60 min.csv 
view request
int32 overflow during mProphet Model Training Failure on 3.7.1.11357
(1 response) wbarshop 2017-09-11
Hello Skyline team,

After updating to the newest Skyline-Daily release, I'm having trouble training mProphet peak picking models on some of my DIA data. The resultant error only leaves me more puzzled, but in the end looks to be an int32 overflow.

First, the error:
[2017/09/11 13:14:51] Calculating peak group scores
...
... (truncated for space)
[2017/09/11 13:16:28] 100%
[2017/09/11 13:16:31] Error: Failed to create scoring model.
[2017/09/11 13:16:31] System.IO.InvalidDataException: Insufficient target peaks (240199 with 281448 decoys) detected at 15% FDR to c
ontinue training.
   at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.CalculateWeights(String documentPath, ScoredGroupPeaksSet targetTran
sitionGroups, ScoredGroupPeaksSet decoyTransitionGroups, Boolean includeSecondBest, Boolean nonParametricPValues, Double qValueCutoff,
 Double[] weights, Double& decoyMean, Double& decoyStdev, Boolean& colinearWarning) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Resul
ts\Scoring\MProphetScoringModel.cs:line 405
   at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.<>c__DisplayClass7.<Train>b__4(MProphetPeakScoringModel im) in c:\pr
oj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 255
   at pwiz.Common.SystemUtil.Immutable.ChangeProp[TIm](TIm immutable, SetLambda`1 set) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\Sy
stemUtil\Immutable.cs:line 201
   at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.Train(IList`1 targetsIn, IList`1 decoysIn, LinearModelParams initPar
ameters, Nullable`1 iterations, Boolean includeSecondBest, Boolean preTrain, IProgressMonitor progressMonitor, String documentPath) in
 c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 196
   at pwiz.Skyline.CommandLine.CreateScoringModel(String modelName, Boolean decoys, Boolean secondBest, Boolean log, Nullable`1 modelI
terationCount) in c:\proj\pwiz_x64\pwiz_tools\Skyline\CommandLine.cs:line 1461

=========================================================================
=========================================================================

After seeing this, I was curious what this sanity check was in pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.CalculateWeights .

So, I checked it out on the sourceforce repo (maybe out of date? Not sure if this has the most recent -Daily updates):

// Better to let a really poor model through for the user to see than to give an error message here
if (truePeaks.Count*10*1000 < decoyPeaks.Count) // Targets must be at least 0.01% of decoys (still rejects zero)
    throw new InvalidDataException(string.Format(Resources.MProphetPeakScoringModel_CalculateWeights_Insufficient_target_peaks___0__with__1__decoys__detected_at__2___FDR_to_continue_training_, truePeaks.Count, decoyPeaks.Count, qValueCutoff*100));
if (decoyPeaks.Count*1000 < truePeaks.Count) // Decoys must be at least 0.1% of targets
    throw new InvalidDataException(string.Format(Resources.MProphetPeakScoringModel_CalculateWeights_Insufficient_decoy_peaks___0__with__1__targets__to_continue_training_, decoyPeaks.Count, truePeaks.Count));




From this, it seems that it must be the case that truePeaks.Count*10*1000 is evaluating to be less than decoyPeaks.Count -- curious as the output of the same line from the error at runtime shows the values of truePeaks.Count and decoyPeaks.Count to be 240199 and 281448, respectively.


Certainly 240119 is not less than 1/10000th of 281448. This looks like an integer overflow, as 240119*10*1000 is (2,401,190,000) greater than the int32 max of 2,147,483,647.

Would be happy to hear your thoughts!

Cheers,
William
view request
Question about SProCop
(1 response) fazeliniah 2017-09-11
Hi Skyline team,
I have been trying to use SProCop for a PRM project. Unfortunately I get the following error. Any thoughts? I have attached the .sky file.
Thanks
Hossein

1>Error in dim(PA) = c(L, L2) :
1> dims [product 170] do not match the length of object [79]
1>Calls: tryCatch -> tryCatchList -> parse.cmdline
1>Execution halted
1>
1>Finished!
 HI_PRM_4samples.sky 
view request
Skyline support PD 2.1 result files?
(7 responses) xicao 2016-03-05
Hi,

I am wondering if Skyline support PD 2.1 result files? When I tried building a peptide library, if I loaded pdResult file, I got the error message:
System.IO.IOException: ERROR: Ziv_3_02232016-(01).pdResult
ERROR:

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, ProgressStatus& status, TextWriter writer) in c:\proj\skyline_3_5_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 109
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, ProgressStatus& status) in c:\proj\skyline_3_5_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 53
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, ProgressStatus& status, String[]& ambiguous) in c:\proj\skyline_3_5_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 166
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\skyline_3_5_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 128

If I loaded msf file, eventually I got a message to tell me no peptide matched the filter settings, even though I set the cutoff score as 0 (see attachment).

Thanks,

Xing-Jun
 PD.PNG 
view request
DIA extraction window
(3 responses) joanna bons 2017-08-21
Hello,

I am analyzing DIA data using Skyline.
iRT peptides have been spiked into the samples for retention time calibration. I have reintegrated peaks for some iRT precursors, which resulted in a new regression. Afterwards, I have re-imported the results.
But, for some peptides of interest, the extraction window is not centered around the predicted RT, as it is specified in the Transition Settings".
Should I thus delete the iRT peptides which are not well integrated from the Calculator +/- the target list?

Thank you for your help,
Joanna Bons.
view request
CE Optimization Method/Transition List Export does not seem to be working
(2 responses) Will Thompson 2017-08-30
Hi Brendan and Brian,

See attached document. When I attempt to export transition lists or methods with CE Optimization, the methods only seem to contain a single transition CE value. Additionally, and this may be a feature change request instead of a bug, but it appears that Skyline does not support transition-level CE optimization for small molecules; when you export a method/transition list the CE is the same for every precursor, even if you have inserted different CEs for different transitions using Edit->insert->transition list functionality. This is a little dangerous because it is 'hidden' that you're not using the correct CE for those transitions unless you dig into the exported transition list or method. CEs should be allowed to be optimized at the transition level for small molecules, just like they are for peptides.

I also noticed that the CE optimization import does not seem to work for small molecules (CE Optimize methods generated manually but tried to analyze in skyline as such).

ALso, there seems to be a little display bug when you right click on peak area view and choose Graph-> CV histogram, it permenantly stays checked, even with you try to go back to replicate comparison view. (screenshot attached)

Thanks, and let me know if you have any questions.

Will
 SmallMol_CEOPtimizationExport_Report.sky.zip  DisplayBug_CVHistogram_083017.pptx 
view request
Chromatogram unavailable after import of results
(2 responses) natbat 2017-09-08
Hello!
Yesterday I had a problem with Skyline and this problem remains today.
I imported the results of the alignment run but the info about iRT peptides was not seen: "chromatogram unavailable". The run itself was OK, I could open it in Excalibur and all peptides were at place. Moreover, it seems that the Skyline had SOME info about the run because it allowed export transition lists based on THIS particular alignment run. But I don't see iRT peptides.
Today I imported results of the real run, import was OK since I have seen the chromatogram import process. Moreover,the chromatogram originally was seen! However, when I clicked another peptides in the target pane, again the chromatogram picture disappeared, and I see nothing. The pane Targets shows green spots but no picture available.
Skyline or computer re-starts did not help.
Please advice what to do.
Technical info:
I recently updated to Skyline (64-bit) 3.7.0.10940
The SRM runs were performed with TSQ Vantage (Thermo)
Whatever else info you need, inform, I will act ASAP

Best regards
Natalia
 Problem with Skyline-Chromatogram unavailable.png 
view request
Importing error "Index was outside the bounds of the array"
(5 responses) rschoenh 2017-08-29
Hi,

I have a set of wiff files that I'm trying to import into Skyline, and some are imported fine, while others give me an error message (pasted partially below). I used the same Analyst scheduled acquisition method for all files, and I can open up the wiff files in Analyst without a problem. I'm using Skyline version 3.7.1.11271.

Thanks so much in advance for your help!

Best,
Regine


At 7:13 AM:
Failed importing results file '(...path and file name...)'.
Index was outside the bounds of the array.
view request
Failed importing Thermo QE-HF PRM Raw File
(4 responses) lbeer 2017-06-22
Hello,

I received the following error while importing Thermo PRM Raw files...Please advise.

Specified argument was out of the range of valid values.
Parameter name: The value 66277 must be between 0 and 65535.
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.PostChromDataSet(PeptideChromDataSets chromDataSet) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1273
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 454
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 270

Inner exceptions:
Exception type: System.ArgumentOutOfRangeException
Error message: Specified argument was out of the range of valid values.
Parameter name: The value 66277 must be between 0 and 65535.
Specified argument was out of the range of valid values.
Parameter name: The value 66277 must be between 0 and 65535.
   at pwiz.Skyline.Model.Results.ChromGroupHeaderInfo.CheckValue(Int32 value, Int32 min, Int32 max, Boolean allowNegativeOne) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:line 314
   at pwiz.Skyline.Model.Results.ChromGroupHeaderInfo..ctor(SignedMz precursor, Int32 textIdIndex, Int32 textIdLen, Int32 fileIndex, Int32 numTransitions, Int32 startTransitionIndex, Int32 numPeaks, Int32 startPeakIndex, Int32 startScoreIndex, Int32 maxPeakIndex, Int32 numPoints, Int32 compressedSize, Int32 uncompressedSize, Int64 location, FlagValues flags, Int32 statusId, Int32 statusRank, Nullable`1 startTime, Nullable`1 endTime, Nullable`1 collisionalCrossSection) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:line 250
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.WriteChromDataSet(ChromDataSet chromDataSet, Dictionary`2 dictScoresToIndex, Boolean saveRawTimes, Boolean isProcessedScans) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1373
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.WriteChromDataSets(PeptideChromDataSets chromDataSets) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1287
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.ScoreWriteChromDataSets(PeptideChromDataSets chromDataSets, Int32 threadIndex) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 101
   at pwiz.Common.SystemUtil.QueueWorker`1.Consume(Object threadIndex) in c:\proj\skyline_3_7_x64\pwiz_tools\Shared\Common\SystemUtil\QueueWorker.cs:line 165


Thanks,
Lynn Beer
view request
Pecan/walnut
(3 responses) tannous 2017-09-06
Hi,
I have been trying to work with some DIA data using the Walnut/Pecan interface.
However, the output is giving me no peptide matches with 1% FDR. I am testing with DIA data obtained with standard 20mz isolation windows and not DIA from gaz phase fractionnation (GPF) type of acquisition. Does Walnut/Pecan work only with DIA obtained with small isolation windows using GPF or is there something else going on that is causing me to have no peptide matches with 1% FDR?
-Thanks
Abla
view request
Does Skyline combine isotopes (deisotope) when extracting fragment ion traces
(3 responses) danielz 2017-09-05
Hi,

I have looked through the documentation and this board and found nothing, therefore:

Does Skyline take into account other isotope peaks than the mono-isotopic when extracting fragment ion traces from PRM runs? E.g. de-isotope the fragments and add-up the intensities?

I have a case when opening up the isolation width for PRM (stepwise from 0.4th to 2 th) increases the intensity of my targets by up to a factor of 2 in a complex mixture. This is a bit counterintuitive, as larger windows lead to co-isolation of other species that would "fill-up" my AGC quicker.

The only explanation that I can come up with, is that when I use a isolation window large enough that the +1 Da isotope (or even +2 Da isotope) is included when collecting ions for the MS2 scan, that this intensity is added to the mono isotopic fragment ion peak when extracting the data.

Thanks for clarifying this
Best
Dan
view request
SkylineProcessRunner.exe error: "Error: could not connect to Skyline"
(12 responses) lparsons 2017-08-25
I am starting to work with SkylineProcessRunner to try to make a more standardized workflow for a project I"m working on. Right now I am trying to import a (small molecule) transition list "transition.txt" and then save out a skyline file "output-25August2017.sky". Every time I try this I get the error above ("Error: could not connect to Skyline").
An older thread here seems to suggest it could be a Skyline version mismatch; I used Windows Task Manager while Skyline was running to verify that I was running SkylineProcessRunner from the same directory that I was running Skyline itself from. My command therefore looks like
SkylineProcessRunner.exe --out=output-25August2017.sky --import-transition-list=transition.txt
After about 10 seconds or so I am given the error. I tried running it with only the output switch and the error is the same. If I try to run it with no switches at all it throws an exception and dies.
view request
chromatogram information unavailable
(4 responses) bin fang 2017-08-30
Hello,

I have Pierce PRTC peptide mixture and 60 other peptides in my transition list. I ran a batch of 58 samples on Thermo Quantiva using the same instrument method. When I import the data files into Skyline (ver. 3.7), I can see PRTC from all samples but got "chromatogram information unavailable" for the rest of the peptides in some files, while some files had no problem at all.
All files had the same instrument method. I couldn't figure out the reason.

I checked the raw data files and they looked normal. Could you please help me on this issue?

Thank you very much!

bin
view request
Duplicate/repeated peptides
(12 responses) sandra maass 2015-07-03
Hi,

I'm currently working on a project where the "refine"-options really helps me a lot. Regarding this I have a question: What exactly is the difference between duplicate and repeated peptides?

Thank you in advance
Sandra
view request
spectral library from pep.XML and mzXML
(11 responses) gabe 2017-08-18
Hi, I'm trying to import pep.XML/mzXML data to generate a spectral library and I'm getting this error:
System.IO.IOException: ERROR: No spectra were found for the new library.

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\skyline_3_7_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in c:\proj\skyline_3_7_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 122

A representative pep.XML file is attached (containing only a few spectra). I could figure-out how to share one of my mzXML files, too, if it would be helpful. I'm just not sure what this error means and why it can't find the spectra since the pep.XML and mzXML files are in the same folder. Does anything appear to be wrong with the pep.XML file? Thanks
 320_THP1_small.pep.xml 
view request
Co-elution count
(6 responses) mvillal1 2017-08-14
Hello Skyline team,

A quick question, in the MProphet Features export option, how is exactly the "co-elution count" feature being calculated? Does it take into account the # of co-eluting peaks having the same or similar apexes (falling within certain delta RT) or is it based on the "co-elution" score (cross-correlation)? In some cases, after doing an automatic selection of the top 5 transitions per peptides, the number of co-eluting transitions goes up (i.e., from 2 to 4) even when the non-refined list included those transitions. Also, the feature just seems to report a max. of 4 co-eluting transitions.

Many thanks!
Ivan.
view request
Skyline-Daily v. 3.7.1.11208 CLI mProphet NullPointerException
(5 responses) wbarshop 2017-08-24
Hello Skyline Team!
The aforementioned version of Skyline-Daily fails to correctly apply mProphet model to the data, returning a NullPointerException (as shown in the partial output below). The same command set//data works just fine under the stable v. 3.7 release. This is only the case when the analysis is done through the CLI (SkylineCmd).

For reference, the command is included here:
SkylineCmd --timestamp --in=combined_analysis.sky --full-scan-rt-filter-tolerance=2.0 --add-library-path=filtered.blib --import-fasta=C:\pulsar\files\staging\1953\inputs\dataset_3491.dat_corrected.fasta --decoys-add=reverse --import-all=. --reintegrate-model-name=REVERSEcombinedanalysis180434961000 --reintegrate-create-model --reintegrate-overwrite-peaks --report-add=WOHL_MSSTATS_REPORT.skyr --report-conflict-resolution=overwrite --report-name=Wohl_MSstats_Input --report-file=MSstats_input.csv --chromatogram-file=C:\pulsar\files\staging\1953\outputs\dataset_3732.dat --chromatogram-precursors --chromatogram-products --chromatogram-base-peaks --chromatogram-tics --out=docker_protection_temporary.sky


Please let me know if you would like me to upload my data directly to the Skyline team to use as a test case.
==========================================================================


[2017/08/24 19:49:43]    0% - Updating peak statistics

[2017/08/24 19:49:45]    6%

[2017/08/24 19:49:48]    12%

[2017/08/24 19:49:50]    18%

[2017/08/24 19:49:52]    24%

[2017/08/24 19:49:54]    30%

[2017/08/24 19:49:57]    36%

[2017/08/24 19:49:59]    43%

[2017/08/24 19:50:01]    49%

[2017/08/24 19:50:03]    55%

[2017/08/24 19:50:05]    61%

[2017/08/24 19:50:07]    67%

[2017/08/24 19:50:09]    73%

[2017/08/24 19:50:12]    79%

[2017/08/24 19:50:14]    84%

[2017/08/24 19:50:16]    90%

[2017/08/24 19:50:18]    95%

[2017/08/24 19:50:49]    100%

[2017/08/24 19:50:49]    Creating scoring model REVERSEcombinedanalysis180434961000

[2017/08/24 19:50:51]    Calculating peak group scores

[2017/08/24 19:50:54]    5%

[2017/08/24 19:50:56]    7%

[2017/08/24 19:50:58]    9%

[2017/08/24 19:51:01]    12%

[2017/08/24 19:51:03]    14%

[2017/08/24 19:51:05]    16%

[2017/08/24 19:51:08]    19%

[2017/08/24 19:51:11]    22%

[2017/08/24 19:51:14]    25%

[2017/08/24 19:51:16]    28%

[2017/08/24 19:51:19]    31%

[2017/08/24 19:51:21]    34%

[2017/08/24 19:51:24]    37%

[2017/08/24 19:51:26]    40%

[2017/08/24 19:51:28]    43%

[2017/08/24 19:51:31]    46%

[2017/08/24 19:51:34]    49%

[2017/08/24 19:51:37]    52%

[2017/08/24 19:51:40]    55%

[2017/08/24 19:51:43]    58%

[2017/08/24 19:51:46]    61%

[2017/08/24 19:51:49]    64%

[2017/08/24 19:51:51]    67%

[2017/08/24 19:51:54]    69%

[2017/08/24 19:51:56]    71%

[2017/08/24 19:51:58]    73%

[2017/08/24 19:52:00]    75%

[2017/08/24 19:52:02]    77%

[2017/08/24 19:52:04]    79%

[2017/08/24 19:52:06]    81%

[2017/08/24 19:52:09]    84%

[2017/08/24 19:52:11]    87%

[2017/08/24 19:52:14]    90%

[2017/08/24 19:52:16]    92%

[2017/08/24 19:52:18]    94%

[2017/08/24 19:52:20]    96%

[2017/08/24 19:52:22]    98%

[2017/08/24 19:52:23]    100%

[2017/08/24 19:52:26]    0% - Training peak scoring model

[2017/08/24 19:52:30]    9% - Training scoring model (iteration 1 of 10)

[2017/08/24 19:52:33]    18% - Training scoring model (iteration 2 of 10 - 242,066 peaks at 2% FDR)

[2017/08/24 19:52:36]    27% - Training scoring model (iteration 3 of 10 - 245,356 peaks at 2% FDR)

[2017/08/24 19:52:39]    36% - Training scoring model (iteration 4 of 10 - 245,739 peaks at 2% FDR)

[2017/08/24 19:52:42]    45% - Training scoring model (iteration 5 of 10 - 245,791 peaks at 2% FDR)

[2017/08/24 19:52:45]    54% - Training scoring model (iteration 6 of 10 - 245,824 peaks at 2% FDR)

[2017/08/24 19:52:48]    63% - Training scoring model (iteration 7 of 10 - 245,828 peaks at 2% FDR)

[2017/08/24 19:52:51]    72% - Training scoring model (iteration 8 of 10 - 245,831 peaks at 2% FDR)

[2017/08/24 19:52:54]    81% - Training scoring model (iteration 9 of 10 - 245,831 peaks at 2% FDR)

[2017/08/24 19:52:57]    90% - Training scoring model (iteration 10 of 10 - 245,832 peaks at 2% FDR)

[2017/08/24 19:52:57]    100%

[2017/08/24 19:53:05]    7% - Calculating score contributions

[2017/08/24 19:53:05]    100%

[2017/08/24 19:53:05]    Intensity: -0.3061 (-11.6 %)

[2017/08/24 19:53:05]    Retention time difference: -0.3580 (5.6 %)

[2017/08/24 19:53:05]    Retention time difference squared: 0.0057 (-0.1 %)

[2017/08/24 19:53:05]    Library intensity dot-product: 5.1910 (49.3 %)

[2017/08/24 19:53:05]    Shape (weighted): 1.7624 (20.8 %)

[2017/08/24 19:53:05]    Co-elution (weighted): -0.0448 (1.3 %)

[2017/08/24 19:53:05]    Co-elution count: -0.0136 (-0.6 %)

[2017/08/24 19:53:05]    Signal to noise: 0.1551 (5.1 %)

[2017/08/24 19:53:05]    Product mass error: -0.0513 (-0.4 %)

[2017/08/24 19:53:05]    Precursor-product shape score: -0.0254 (-0.3 %)

[2017/08/24 19:53:05]    Precursor mass error: -0.1419 (-0.4 %)

[2017/08/24 19:53:05]    Precursor isotope dot product: 2.7389 (26.1 %)

[2017/08/24 19:53:05]    Identified count: 0.5870 (5.2 %)

[2017/08/24 19:53:09]    Adjusting peak boundaries

[2017/08/24 19:53:11]    6%

[2017/08/24 19:53:13]    12%

[2017/08/24 19:53:15]    18%

[2017/08/24 19:53:17]    24%

[2017/08/24 19:53:20]    30%

[2017/08/24 19:53:22]    36%

[2017/08/24 19:53:24]    42%

[2017/08/24 19:53:26]    48%

[2017/08/24 19:53:28]    55%

[2017/08/24 19:53:31]    62%

[2017/08/24 19:53:33]    68%

[2017/08/24 19:53:35]    74%

[2017/08/24 19:53:37]    80%

[2017/08/24 19:53:40]    85%

[2017/08/24 19:53:42]    90%

[2017/08/24 19:53:44]    95%

[2017/08/24 19:53:45]    100%

[2017/08/24 19:53:45]    Error: Failed to reintegrate peaks successfully.

[2017/08/24 19:53:46]    System.NullReferenceException: Object reference not set to an instance of an object.

   at pwiz.Skyline.Model.DocNodeChildren.IsOrderSame(DocNodeChildren previousChildren) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DocNodeChildren.cs:line 45

   at pwiz.Skyline.Model.DocNodeChildren..ctor(IEnumerable`1 items, IList`1 previous) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DocNodeChildren.cs:line 20

   at pwiz.Skyline.Model.DocNodeParent.set_Children(IList`1 value) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DocNode.cs:line 384

   at pwiz.Common.SystemUtil.Immutable.ChangeProp[TIm](TIm immutable, SetLambda`1 set) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\Immutable.cs:line 192

   at pwiz.Skyline.Model.DocNodeParent.ChangeChildren(IList`1 children, IList`1 counts, Int32 indexReplaced) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DocNode.cs:line 0

   at pwiz.Skyline.Model.DocNodeParent.ChangeChildrenChecked(IList`1 children, Boolean changeAutoManage) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\DocNode.cs:line 1170

   at pwiz.Skyline.Model.SrmDocument.RegroupMolecules(IList`1 children, PeptideDocNode[] moleculeNodes, Func`3 rankChildren) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 803

   at pwiz.Skyline.Model.SrmDocument.ChangeSettingsInternal(SrmSettings settingsNew, SrmSettingsChangeMonitor progressMonitor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 995

   at pwiz.Skyline.Model.MProphetResultsHandler.ChangePeaks(IProgressMonitor progressMonitor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\MProphetResultsHandler.cs:line 177

   at pwiz.Skyline.CommandLine.Reintegrate(ModelAndFeatures modelAndFeatures, Boolean isOverwritePeaks, Boolean logTraining) in c:\proj\pwiz_x64\pwiz_tools\Skyline\CommandLine.cs:line 1511
view request
Skyline targeted method editing tutorial
(1 response) hamishk 2017-08-28
Just had a question in regards to the targeted method editing tutorial concerning the construction of the background proteome file.
On pages 4 and 5 of the manual it says to appears that we are required to upload a Yeast.protdb file from the FASTA folder although there are not .protdb files present in the whole method edit parent folder. Is this Yeast.protbd file something that we should be able to generate or is there a file missing from the method edit FAST folder?

Any assistance would be greatly appreciated :)
view request
error train mprophet model
(4 responses) chia-lin 2017-08-24
Dear
    I want to train a model use mprophet with dcoys. This error message arise and it arise many times. Is the targets in my data not enough or the bug of skyline? Look forward to your reply.
Skyline version: 3.7.1.11208 (64-bit)
Installation ID: fc4baaa2-65be-48f9-b088-0a5dbfbedaeb
Exception type: TargetInvocationException
Error message: Insufficient targets detectect to continue training.

--------------------

Stack trace:

   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1964
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 176
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 131
   at pwiz.Skyline.SettingsUI.EditPeakScoringModelDlg.TrainModel(Boolean suppressWeights) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\EditPeakScoringModelDlg.cs:line 285
   at pwiz.Skyline.SettingsUI.EditPeakScoringModelDlg.TrainModelClick() in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\EditPeakScoringModelDlg.cs:line 204
   at System.Windows.Forms.Control.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnClick(EventArgs e)
   at System.Windows.Forms.Button.OnMouseUp(MouseEventArgs mevent)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ButtonBase.WndProc(Message& m)
   at System.Windows.Forms.Button.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)

Inner exceptions:
Exception type: System.Exception
Error message: Insufficient targets detectect to continue training.
Insufficient targets detectect to continue training.
   at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.CalculateWeights(String documentPath, ScoredGroupPeaksSet targetTransitionGroups, ScoredGroupPeaksSet decoyTransitionGroups, Boolean includeSecondBest, Boolean nonParametricPValues, Double qValueCutoff, Double[] weights, Double& decoyMean, Double& decoyStdev, Boolean& colinearWarning) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 404
   at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.<>c__DisplayClass7.<Train>b__4(MProphetPeakScoringModel im) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 255
   at pwiz.Common.SystemUtil.Immutable.ChangeProp[TIm](TIm immutable, SetLambda`1 set) in c:\proj\pwiz_x64\pwiz_tools\Shared\Common\SystemUtil\Immutable.cs:line 193
   at pwiz.Skyline.Model.Results.Scoring.MProphetPeakScoringModel.Train(IList`1 targetsIn, IList`1 decoysIn, LinearModelParams initParameters, Nullable`1 iterations, Boolean includeSecondBest, Boolean preTrain, IProgressMonitor progressMonitor, String documentPath) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\Scoring\MProphetScoringModel.cs:line 196
   at pwiz.Skyline.SettingsUI.EditPeakScoringModelDlg.<>c__DisplayClass13.<TrainModel>b__b(IProgressMonitor progressMonitor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\EditPeakScoringModelDlg.cs:line 282
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 228
Exception caught at:
   at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
   at System.Windows.Forms.Control.WndProcException(Exception e)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
   at pwiz.Skyline.Properties.PeakScoringModelList.EditItem(Control owner, PeakScoringModelSpec item, IEnumerable`1 existing, Object tag) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Properties\Settings.cs:line 2082
   at pwiz.Skyline.SettingsUI.SettingsListComboDriver`1.EditItem(TItem itemEdit, IItemEditor`1 itemEditor) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\SettingsListComboDriver.cs:line 217
   at pwiz.Skyline.SettingsUI.SettingsListComboDriver`1.EditCurrent() in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\SettingsListComboDriver.cs:line 193
   at pwiz.Skyline.SettingsUI.SettingsListComboDriver`1.SelectedIndexChangedEvent(Object sender, EventArgs e) in c:\proj\pwiz_x64\pwiz_tools\Skyline\SettingsUI\SettingsListComboDriver.cs:line 139
   at System.Windows.Forms.ComboBox.OnSelectedIndexChanged(EventArgs e)
   at System.Windows.Forms.ComboBox.WmReflectCommand(Message& m)
   at System.Windows.Forms.ComboBox.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.SendMessage(HandleRef hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.UnsafeNativeMethods.SendMessage(HandleRef hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.Control.SendMessage(Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.Control.ReflectMessageInternal(IntPtr hWnd, Message& m)
   at System.Windows.Forms.Control.WmCommand(Message& m)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.Application.ParkingWindow.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.UnsafeNativeMethods.CallWindowProc(IntPtr wndProc, IntPtr hWnd, Int32 msg, IntPtr wParam, IntPtr lParam)
   at System.Windows.Forms.NativeWindow.DefWndProc(Message& m)
   at System.Windows.Forms.Control.WmCommand(Message& m)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ComboBox.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
   at pwiz.Skyline.SkylineWindow.ShowReintegrateDialog() in c:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1436
   at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
   at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
   at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
   at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
   at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
   at System.Windows.Forms.Control.WndProc(Message& m)
   at System.Windows.Forms.ToolStrip.WndProc(Message& m)
   at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
   at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
   at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
   at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
   at pwiz.Skyline.Program.Main(String[] args) in c:\proj\pwiz_x64\pwiz_tools\Skyline\Program.cs:line 306
view request
Adding heavy/ISTD area to results/document grid
(1 response) dawn.r.dufield 2017-08-17
We would like to be able to add a column to the current document grid or results grid which contains the heavy or ISTD area (not just the ratio). We like to be able to look at the ISTD response for a given sample/replicate without having to backcalculate from the ratio of light to heavy. Seems like skyline knows this area since it shows up for the heavy transition area, but would be more efficient to just have it as a choice in a column with the light or precursor analyte. This is useful for both small and peptide quant.
view request
Help getting mods to show up on peptides
(9 responses) Theresa 2017-08-14
Hi,

I have a list of peptides of interest. I've put CAM as a fixed mod and variable mods including S/T/Y, oxidation, acetylated N', and N/Q deamidation. Only the CAM is showing up on the peptides and the exported transition lists. I've made sure that variable box is checked for all variable mods. I'm not sure what else to try.

Thanks for any help you can offer,
Theresa
 08142017 – requesting help with getting variable mods.pptx 
view request
Internal Curve issues
(1 response) hbettenhausen 2017-08-24
Hi,

I have been off/on using skyline for metabolomics analysis. Recently, we took some beer samples and instead of doing the external curve, we did an internal curve. The amounts we added of S-adenosylmethionine are attached(just for reference).
The issue is…I don’t know if Skyline likes my internal curves. I did not do an external WITH the internal, so it has nothing to reference as the curve…and since the samples are not “standards,” I’m not sure what to categorize them as.
We are looking to quantify the SAM in these samples so that we can go further with some more experiments.
 
I’m not sure if it makes sense, but if you have any insight, I’d love to hear!
Thank you,

Harmonie
 a.png 
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iRT Spectral Library Calibration
(4 responses) esabido 2017-08-22
Dear Skyline Team,

I have problems calibrating the iRT values of a peptide library.

I defined a set of heavy-peptides as iRT standards and then created a library from shotgun data containing the same set of peptides. Skyline assigns the spectra to 7/8 of the defined iRT standards (symbol next to the peptide) regardless whether the identification corresponded to the light or heavy form. So far so good.

Nevertheless, when I build an RT Calculator and select "add Spectral Library" then it only finds spectra for 3 peptides. Manual inspection of the library indicates that the mapped spectra correspond to the light-version of the peptides ignoring the heavy-version spectra. Is this a normal behaviour? Should I modify the "Modified Sequence" entry somehow to make the heavy peptides also recognized in the Calculator?

Best regards,

Eduard
 2017-08-22 14_39_20-Greenshot.jpg 
view request
SMILES input for small molecules
(3 responses) meowcat 2017-08-23
Currently small molecule transitions can be entered as a chemical formula with adduct, or as a mass. It would be great if there was a column where one could enter the SMILES code, from which the formula and mass would be automatically calculated.

I would also be interested in using the SMILES code in external tools (of course the value can be added already as a custom annotation, but a "real" field would be much nicer.)
view request
Waters .raw files
(1 response) simonedowning 2017-08-24
Hi, I am trying to viewing MRM Waters .raw files. Can you help on how to view these files?
view request
error when input .group.xml file
(1 response) shulei lei 2017-08-23

Hi,
I need to build up a library from proteinpilot search results. I converted .group into .xml, and then renamed into .group.xml. But, when I put .group.xml into skyline, giving me the error below.

-------------------------------------------------------------------------------------
ERROR: 14Oct16_Sam_rat_library.group.xml(line 197220): Unclosed token error parsing XML.
------------------------------------------------------------------------------------

I attached .group.xml file. Would you please check where is the problem.?

Thank you,

Shulei
 14Oct16_Sam_rat_library.group.xml 
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no peaks in chromatogram
(3 responses) amy nguyen 2017-08-22
I added my peptides and their transitions. Imported my MS data from my sciex 5500 ms and nothing showed up.
view request
Interference or not?
(1 response) aulke 2017-08-22
Dear Skyline users.
We are having a hot discussion in the lab about data acquired by a previous lab member (see attached).
The researcher ran MRM on a peptide from partially purified protein samples (human serum). The transitions of the signal from the endogenously detected peptide look slightly different compared to the equivalent spiked-in heavy peptide.
We can detect the same peptide from the recombinant protein in a separate run.
Some more information:
The method did not contain additional transitions then the 4 ones shown.
The method did not contain additional charge stages of the same peptide.
The method did not contain additional peptides from the same protein.
The gradient was a short step-gradient. (0-30% organic in 1 minute, then 30-40 % organic over 20 mins, followed by washing)
Question to you: What do you think, interference or not? What experiments would you suggest to verify? And mostly, do you think the data is salvageable?

thanks
Anne
 Presentation1.pptx 
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How to import orbitrap results?
(1 response) Aljona Saleh 2017-08-21
Hello,
I was trying to import .raw data (from import results) but receive following message:

At 21:12:
Failed importing results file 'C:\Users\AljonaS\Desktop\Orbitrap data\DP_SL_048_hBSSL_digest_tryp_170817.raw'.
[read_file_header()] Unable to open file C:\Users\AljonaS\Desktop\Orbitrap data\DP_SL_048_hBSSL_digest_tryp_170817.raw (invalid permission or file locked)


Inner exceptions:
Exception type: System.Exception
Error message: [read_file_header()] Unable to open file C:\Users\AljonaS\Desktop\Orbitrap data\DP_SL_048_hBSSL_digest_tryp_170817.raw (invalid permission or file locked)
[read_file_header()] Unable to open file C:\Users\AljonaS\Desktop\Orbitrap data\DP_SL_048_hBSSL_digest_tryp_170817.raw (invalid permission or file locked)
   at pwiz.CLI.msdata.ReaderList.read(String filename, MSData result, Int32 runIndex, ReaderConfig config)
   at pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints) in c:\proj\skyline_3_7_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 138
   at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 199




Our.raw data files are not locked or invalid permission in any way, that we are aware of.
How can I import orbitrap files?

Thank you very much for help!
With kind regards,
Aljona
view request
CCS calculations ?
(5 responses) blaine roberts 2017-08-17
Is there a way to convert the drift times from ion mobility spectra to calculated CCS within skyline?
view request
Reintegration of analyte (light) affects ISTD integration (heavy)
(10 responses) dawn.r.dufield 2017-08-18
Many times for a low level analyte (peptide or small molecule) quant, you have to adjust the integration parameters to get a good peak. Currently I only see a way to "manually" integrate a new peak, which although is not ideal as hard to reproduce etc is reasonable, however I have found that when I manually integrate the light or analyte peak, it also changes my heavy or ISTD peak which in most cases since its typically a larger more easily integrated peak we do not want to do that. Can you please comment on if there is a way around this. I will attach a screen shot of what I'm talking about.
 DRD Integrations.xlsx 
view request
Need to batch-extract the EICs or XICs
(2 responses) juliec0211 2017-08-16
Hi

Is there any way to batch-extract the extracted ion chromatograms (EICs or XICs) for all quantified peptides (quantifier transition of light and heavy). I didn't find the option for related output in Skyline.
Thanks a lot.

Best,
Julie
view request
Disappearance of neutral loss ions when changing the Instrument Max m/z value
(1 response) rschoenh 2017-08-18
Hi,

I have a Skyline file that includes neutral loss ions (e.g. [b13 -98]) and I initially had the Max m/z value in the Transition Settings > Instrument tab set to 2000 m/z. I then changed the Max m/z value to 1250 m/z and I noticed that all neutral loss transitions disappeared from the Targets list, even the ones with m/z values below 1250 m/z! Is there a checkbox that I'm not aware of that I need to check to not have this happen?

Thanks so much in advance for your help!

Best,
Regine
view request
Building DDA spectral library
(3 responses) mrmiller 2017-08-18
I'm very familiar with MS but have just started looking at proteins so my problem is probably my unfamiliarity.

I'm struggling with figuring out how to adapt the DDA MS1 filtering to my LC-MS file because of the first steps in the DDA import. I'm not sure how to go about getting the files equivalent to the .group and .wiff files in the tutorial.

I tried using the XML outputs from Protein Prospect to see if it would accept any of those, including the DIG, PRODUCT, and BRIDGE programs.

Thank you,
view request
Peptide ID Times
(1 response) ecanessa 2017-08-18
Hi,

I was wondering if it was possible to turn of peptide ID times in skyline. I just ran a PRM exp, and the lines are causing a lot of background noise that makes it hard to work with the file. Including a picture of it below, thanks for the help.
 Capture2.PNG 
view request
Keep on the same Replicate
(9 responses) mh167 2014-01-17
Dear Brendan,

I was trying to check the result of all replicates. For example, Peptide A to C and Replicate 1 to 3. Skyline always stick on Replicate 1 ( first one in document)when I check different peptide. Everytime I select different peptide, Skyline jump back to Replicate 1 no matter which Replicate I am looking at. Did I select any setting?

I am using Skyline-daily 2.1.1.5463 on Windows 7/64bit machine.
Thank you for your help.

MF
view request
questions about "MZXML" format data
(12 responses) chia-lin 2017-08-16
dear

    I tried to convert my raw file to MZXML format. But when I analysis my data with skyline and it can not get chromatography. So I want to know if skyline can analysis format of MZXML or mzml data. Look forward to your reply.
view request
To sum or not Sum ions in quantitation
(3 responses) dawn.r.dufield 2017-06-29
It appears that skyline sums transitions within a precursor for calculating concentration, accuracy and regression. However, it looks like if you click on the individual fragment ion, the area that is displayed is just from that ion. When you click on the precursor it shows total area of all transitions. Can skyline be modified so that if you are on the transition you see the regression, accuracy, etc for non-summed ions and if you click on the precursor, you get the summed ion? This would allow you to quickly assess if summing helps or not in your quantification method.

thanks
Dawn
view request
NIST spectral library for small molecule
(2 responses) YangZhang 2017-08-17
Dear Brian and Skyline team,

Thanks a lot for your effort on improving Skyline support for small molecule.
I have a naive question, may I ask where to get NIST library for small molecule(msf)?
I can't seem to find a link online, is there a price tag for that?
Thanks again!

Yang
view request
adding peptides to Skyline
(1 response) rolandworth 2017-08-17
Hello,

I've added a peptide list to Skyline using the Insert peptide option but once the peptides are into the peptide tree they do not have any reporter masses ("children"). The peptide and transition settings are set to MS1 filtering so expect to see the 2+ and 3+ precursors for each peptide.
view request
Error reading .msf files
(2 responses) irinatcher 2017-08-15
I am trying to import search results from Proteome Discoverer (both 1.4 and 2.01) and this error comes up (see the attached screen shot)

System.IO.IOException: ERROR: reading file '1hour-1231724Lib-GrpB-T0-Fraction-01.msf'

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\skyline_3_7_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 59
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in c:\proj\skyline_3_7_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 171
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\skyline_3_7_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 122
 PD msf error.PNG 
view request
Cannot open files made with previous Skyline daily
(5 responses) Will Thompson 2017-08-15
Hi Brendan and team. Upgraded to the latest daily and don't appear to be able to open skyline documents from the previous daily, example attached. Help! I guess I need to 'downgrade' to previous daily.

Cheers

W
 4723_HCs_061617.sky.zip 
view request
Skyline installation without admin privileges?
(3 responses) lparsons 2017-08-15
It seems that at least in my experience Skyline requires administrator access to install. While I understand that some of the requisite packages (particularly the vendor libraries to read proprietary file formats) need administrator access to install, is there any chance that there could be a future version of Skyline that could be installed by users who do not have administrator access?

This could be particularly valuable for helping users who are customers of core facilities but have their PCs managed by yet another department; they might not have admin privileges on the PCs in their labs for this reason. If they could just ask their IT managers to install the vendor libraries and then do Skyline installation and updates themselves that could be beneficial.

thank you
view request
missing a detected dominant fragment in our PRM data
(3 responses) ru wei 2017-08-11
Dear Skyline team,

We have been using Skyline for years now, thanks for great work!! Also appreciate it very much for addding quantitation module. We recently start PRM assay on our QE HF platform and use Skyline for quantifying proteins using external calibration curves.

One issue we encountered when processing our PRM data is missing a detected and dominant fragment ion (y15++) for one of our assayed peptides. Please see details in the attached pptx file.

Thanks in advance,

Ru
 Ru_Skyline_MissingFragment_170811.pptx 
view request
incresing the number of decimal in report?
(1 response) Antoine 2017-08-14
Hello,

Is there a way to increase the number decimals of the time related values(e.g. RT and FWHM values) in reports?

So far it seems to be limited to 2 decimals and that can be problematic with UHPLC applications where a typical peak width is about 2 seconds.

In fact, I can extract non rounded to the second decimal RT/FWHM values if I "copy data" from the retention time graph. However,it is a quite tedious process with large data sets.

I would be grateful if someone as a solution to share.

Thank you

Antoine
view request
isolation window setting
(4 responses) chia-lin 2017-08-07
Dear,

  If I use different RT with different mass range, how should I set my isolation window. That means I use 300-1000 M/Z in a period of 20 mins, 500-1200 M/Z in another period of 20 mins. The "results only" can work for my data? look forward to your reply.
 微信图片_20170807151841.png 
view request
error in updated skyline
(1 response) chunxiang yao 2017-08-09
Hi,

Recently I installed an updated version of skyline 3.7. The installation was smoothly completed, and the front page was successfully open. However, when I uploaded a sequest output .msf, it couldn't extract the peak properly. I also tried to install the unplugged version of skyline, as suggested in your support forum. Same problem. Could you help me fix this problem? I would like to use your sofeware for DDA MS1 peak extraction analysis. Thanks a lot.

Here are the error message popped out:

System.IO.IOException: ERROR: reading file '###############.msf'

   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in c:\proj\skyline_3_7\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 139
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in c:\proj\skyline_3_7\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 170
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in c:\proj\skyline_3_7\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 121
view request
issue with skyline and instrument communication
(1 response) bernd roschitzki 2017-08-08
Hello Brendan, we have an issue with the instrument communication after we installed Skyline 3.7 at our instrument PC. (that is what the service engineer said). I am not quite sure about that statement. Did you face any problems like that before?
our instrumentation is:
Qtrap 5500:
Analyst: V.1.6.3
Eksigent: V.4.2 build 151019-1321
OS . Windows 7, 64 bits.

Thanks for your help!
Greetings, Bernd
view request
Calculate peak areas based only on MS1 m/z ?
(12 responses) lparsons 2017-07-18
I have a number of spectra for which I would like to use Skyline to calculate information about parent ions of specific m/z. The data was acquired in MS1 only, which makes it at least very difficult to calculate the charge of the parent ion from only the spectra. I would like to be able to have Skyline show me how these ions compared across the spectra; for example for a peak of m/z value X what is the area of the peak and how far off in chromatographic time is it on sample 1 from sample 2.

I tried to do this through Edit -> Insert -> Transition list, entering these as small molecules. However in order to do this I am told by the software that I need "any two of Formula, m/z, or Charge". I have m/z though I do not have formula or charge. Is there a way to tell Skyline to go only on m/z? I could potentially make a list where I enter each of the ions with charge ++1, +2, +3, +4, etc but that seems like it might be a less than ideal solution.

thank you
Lee
view request