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.

It is likely that your question has already been asked and answered.  Please use the search box in the upper right corner of this screen before posting a new question.

Support is provided by the creators of the software, as time allows, though we hope others will share their experience as the user community is now quite large.

If your question is about an External Tool, please contact that tool's developers directly. Contact information can usually be found on Skyline's Tools | Tool Store... menu.  

In order to post to the forum, you'll need to sign-in or if you don't yet have an account sign up. Forgot your password? You can reset it using the "(forgot password)" link on the sign-in page.

You can also follow the Skyline support board through email updates after you sign up.

When you post a question, please include the following information:

  • A detailed description of your problem or question, including instructions for re-creating any problem that you are encountering. Screenshots are often helpful.
  • Your operating system, and the version of the software that you are using.
  • Any other information that may help us to answer your question, including whether you are working with proteomics or small molecule data.

If you are including text output from a tool, please attach files to your message, rather than pasting in long text.

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that .sky.zip file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
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Showing: limited to 100 requests
Waters Synapt G2-Si - multiple issues to access CCS values
(6 responses) nicolas macorps 2022-08-31
Dear Skyline support,

I have been trying to use Skyline to analyse data obtained with a Water Synapt G2-Si with ion mobility (IM) experiments. Experiments are performed with the HDMS mode (ion mobility, elution time and full scan MS only). The ion mobility technique is TWIMS that use a calibration curve (mob_cal.csv file in the sample root) that the proprietary software uses to correlate measured drift times to CCS values.
I want to use Skyline.ms to have a solid workflow with this software to quantify PFAS (small targeted molecules) in various samples. To confirm that I am indeed looking at a PFAS, I need to access CCS values (which include the need to create an IM library) so I can then have it when exporting a "report'.

I encountered some issues when using Skyline features which is why I am opening this support feed. You can find uploaded in the "File Sharing Folder for Skyline Support" my folder under the name "Synapt_CCS_PFAS_SkylineSupport.zip". This file contains *.raw data from the Water Synapt, *.sky file (with the trageted list molecules in a *.csv in case it is needed) and snapshots of some error message I got along my tests.

1) Skyline crashing when importing *.raw files :
When trying to import Synapt *.raw files (220621008.raw and 220621009.raw in the *.zip folder), skyline crashes at the importation step (software closes without error message). This issue was encountered and reported in another support feed. The proposed solution in this feed was to convert *.raw into *.mzML files, but it seems that this approach doesn't allow for CCS values when creating IM library.

I went looking into the *.raw folders and found that when changing _FUNC002.DAT into _FUNC002.temp (in other words I "remove" this function from the *.raw file) I was now able to import without crashing the *.raw file and was able to obtain chromatograms with full scan spectrum and drift time (ms) distribution.
I initially thought that this "Function 2" contained "lockmass" data which can cause some issues when converting into *.mzML so I thought that removing this function could fix the problem. However I cannot be sure that this function is indeed the "lockmass" when looking in the "_extern.inf" file nor in the "_HEADER.TXT". The only information I have on these files are for the function 1 : "MOBILITY MS FUNCTION". So function 2 may also contain IM data from the HDMS experiments but I cannot be sure.

In any case, I kept going with modified *.raw files with the change of _FUNC002.temp (220621008_modify.raw and 220621009_modify.raw) to try to obtain CCS values

2) Negatives CCS values when creating IM library with "use results"

Now having something to work with, I tried creating the IM library for my molecules. I was happy to see that I could obtain some CCS values by doing so. Unfortunatly they are all with a negative sign and as such completely incorrect. I had two thought on that :
- Conversion of drift time into CCS values using the mob_cal.csv file (as done by the proprietary software) is not performed in Skyline in this manner or Skyline needs another type of file to do so.
- We need the function 2 to be active to have CCS values

For the latter, I tried to reactivate the function 2 in the *.raw file and reimport them. Doing so led to no crash so I followed with the creation of an IM library. I ended up with an error message "Failed using results to populate ion mobility library: [SpectrumList_Waters:spectrum()] Bad index: 18446744073709551615" (you can find the snapshot in the *.zip file and the extended error message in a *.TXT file in the *.zip).
Further trying with reimportation led to a crash of the software as mentionned in my first point.

I hope you have enough elements with this description to help me ?
- Do you know why Waters HDMS experiment *.raw files would make the software crash ?
- Do you know why I have negative CCS values when creating the IM library with the "use results" feature?

Let me know if you need anything else from me.

Best regards,
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Past Releases?
(2 responses) nate 2022-11-23

The docker image of Skyline is a bit behind - can anyone help me find previous releases for windows? See below.

Thanks! Nathaniel

"
The document format version 22.2 is newer than the version 22.13 supported by Skyline-daily (64-bit : automated build) 22.1.9.244 (804ca65).
Exiting...
"

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Problem with Import or Search of TMT Labeled Peptides
(6 responses) philip remes 2022-11-22

Hello,

I'm trying to build a library of Yeast peptides labeled with 11-Plex TMT, using Eclipse DDA MS2. I've tried doing this in several ways:

  1. Search the data in Proteome Discoverer, import the .pdResult files. There are 17k peptides with q-value < 0.01, however I'm not able to successfully import the results. We get the old, "Importing the FASTA did not create any protein targets".

  2. Search the data in Skyline. I tried using MSAmanda, and MS-GF+. There appears to be an error parsing the TMT modification string. I'm pretty sure that this is the string that Skyline supplied for that modification.

Based on this thread I made sure that there were a number of good targets in PD. I've had no trouble importing standard, unlabeled HeLa results from PD or searching them in Skyline, so it seems like the TMT modifications must be the issue. https://skyline.ms/announcements/home/support/thread.view?entityId=5a28316e-8430-1035-b2a7-e465a393b02f&_docid=thread%3A5a28316e-8430-1035-b2a7-e465a393b02f

I've uploaded the pdResult files, raw files, and Skyline files to the file sharing server with the name ForSkyline.zip. Could you have a look and see if the problem is on my end, or the Skyline end?

Thanks
Philip

 221122_skyline_tmt_import.pptx 
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MS2 Quantitation with Orbitrap ddMS2 Data
(1 response) johnny perez28236 2022-11-21

Good Morning, I hope this email finds you well.

I am using Skyline to interpret ThermoFisher Exploris data. My method performs SIM analysis followed with MS2 analysis if about a certain ion threshold. What I have noticed is that Skyline extrapolates data for the entire LC run time. Therefore, if the ddMS2 data was only collected for 30 seconds and the run is 5 minutes, it will extrapolate the baseline from 0 - 5 minutes. See attached picture. This causes a problem when attempting integration. Is there any way to prevent skyline from doing this?

I've also attached what ThermoFisher's qualitative analysis software shows for the same MS2 experiments.

Thanks again!

Johnny

 MS2 Data - Skyline - Orbitrap Exploris.png  MS2 Data - FreeStyle - Orbitrap Exploris.png 
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Gas-phase-fractionation and iRT
(3 responses) Joerg 2022-11-18

Hi,
I have a problem with iRT alignment with my gas-phase fractionation-SWATH-runs. As I am doing GPF for the SWATH-analyses some of the iRT peptides (Pierce C18) are fragmented only in fraction while others are fragmented in fraction 2 and so on, but none of the peptides are fragmented in the different gas-phase-fractions. I have full-MS-spectra for the entire precursor region in every run, though. Is there a possibility to align the runs according to the precursor signals or do I have to fragment each peptide in each run?
Many thanks!
Joerg

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Issue with loading transition list with deuterium as H'1 in formula for newer Skyline version
(4 responses) j3 menzel 2022-11-12

Hi Skyline team,

I'd like to ask, whether the notation for heavy atoms has changed recently?
I used to enter deuterium into transition lists as H' (one deuterium being H'1), which worked fine for Skyline (64-bit) 21.1.0.278.
I can still use this older Skyline version without problems, but the newest one shows an error when trying to load such transition lists.
The error message I get is:

Failed reading the file C:... .csv. The given key was not present in the dictionary.

The same transition list without any H' is loading fine in the latest Skyline version.
Is there a way I can generate transition lists with deuterium containing precursors and products that will work in all versions of Skyline?

Thanks,

Phil

 D problem snip.PNG 
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Cannot read Bruker .d without TIMS enable
(4 responses) ho-tak lau 2022-11-15

Hello Skyline Team,

I have a set of TimsTOF pro2 files (.d) that Skyline sees them as normal folders rather than spectra files. These data are recorded as MS1 or MRM mode without TIMS enabled. Is there a way to force Skyline to read them?

MSConvert also see them as normal folder, so I cannot convert them manually.

I can upload a few of them if that will help.

Thanks
Ho-Tak

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Unaligned retention time issue
(3 responses) dominik kopczynski28488 2022-11-14

We have an issue with Skyline 22.2.0.312 which looks like a potential bug. Maybe you could have a look at it, here is the description:

  • We set up a scheduled targeted method for our lipidomics measurements with (among others) a retention time window from 13.9 to 15.9 min.
  • An example is shown in the attached Image Analyst-correct-retention-time.png. You can see that both chromatograms are aligned.
  • When importing the data (minimal example in: Test.zip), we get a shift in retention times as visible in Image Skyline-incorrect-retention-time.png.
  • However, this shift should not be there, it should be aligned.

Thank you for your support.

 Analyst-correct-retention-time.png  Skyline-incorrect-retention-time.png  Test.zip 
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Error during import DIANN .speclib as library
(1 response) ziyue wang 2022-11-14
Hello,
I was trying to import the .speclib file generated by DIANN to skyline for building library, however, it failed several times.
My skyline is up to date and I tried .speclib with both .wiff and .wiff2.dia as suffix of File.Name column, both networking.
please find the error and my .speclib file in attachment.
Thanks,
ZW
 skyline error.JPG  report-lib.tsv.speclib 
view request
Exploris PRM data import issue
(4 responses) wal mair 2022-11-10

Dear Skyline team,

I am trying to setup a PRM method on our new Exploris 240 and to analyze the data using Skyline. There is no "PRM" mode per se on the Exploris and the engineers have told me to use "product ion scan" mode which should result in equivalent data. The resulting raw file, opened in FreeStyle, contains no ms1 spectra, but ms2 spectra continuously acquired for the precursor mass added in the inclusion list during the time window specified. I am new to PRM so I have no comparison but it's exactly what I expected. I imported the file into my Skyline project and it says "Chromatogram information unavailable". I figured my Skyline document might have wrong settings, so I manually added a BSA peptide and fragment masses from the Skyline PRM tutorial and imported one of the associated Agilent QTOF data files (3-BSA-1fmol). The file imports without problem and the precursor and fragment ions are displayed.

Another user had submitted a PRM&Exploris question in 2020 so I assumed it should be possible to analyze data from that instrument with Skyline, generally. However, not sure if relevant, I noticed that user used a different "MS/MS filtering" - "Acquisition method" --> "Targeted (obsolete)" which is not available in my Skyline (see his post).

Some info that might be relevant:
I am working with small molecules, in particular I am targeting derivatized fatty acids. I am transferring info from a MRM method which is why I only have 2 product ions at this point. Data was acquired in positive mode. I'm using Skyline 64-bit 22.2.0.255, Windows 10. I will upload the skyline document with the name "2022-09-29_NPH-OnlyButyricAcid.sky".

+Is there an issue with the way I acquired the data?
+Is the data ok but Skyline is confused by the derivatization adduct?
+Is there a mistake in the way I setup the transitions?

Any help is appreciated! Thanks!

 MethodProductIonScan.PNG  RawFilePrecursorPlusProducts.PNG  Skyline.PNG 
view request
Partial missing fragment ion exracted from FAIMS-DIA file by skyline
(5 responses) Winnie 2022-11-08

Hi skyline team,
I used skyline to extract the interested peptide precursors from the FAIMS-DIA files. The interested peptide precursors were almost extracted from files acquired from Q Exactive HF instruments or TripleTOF instruments. But there are some weird things about those peptide precursors extraction from FAIMS-DIA files. 1. Some of the peptide precursors show good MS1 extraction but without fragment ions. 2. Some of the peptide precursors could extract fragment ions from A files but could not extract from B files. The A and B files are technical replicates.
I do not know whether there are some issues with skyline settings, so I upload the skyline documents if you can check it would be better. Many thanks.

I have uploaded the file to the link. http://skyline.ms/files.url
Best regards,
Winnie

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Library Build for SILAC labelled PTM spectra from MetaMorephus mzID and mzML files ERROR No spectra were found for the new library.
(5 responses) gwitek 2022-09-21

Hello,

I am trying to build a library from my spectra files that contain peptides digested with Pepsin, grown in SILAC media, and that have phosphorylation on S/T/Y (PTM search).
I get mzID and mzML files from MetaMorpheus but I get an error that No spectra were found for the new library.
I tried changing files to lowercase letters mzid and mzml both or each and it does not work.

I did read another request that said PTM searches can cause library build issues and wanted to reach out to you if this might be another issues with PTM or SILAC.

Thank You,
Gabriela

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Issue with Protter Tool
(1 response) dilip singh 2022-11-10

Hi, I want to use Protter tool to show the unique peptide selected in skyline in the protein structure (Skyline--Tools--Protter)

But I am getting error, attached herewith.

My operation system is windows 10 enterprise with 11th Gen Intel ® Core (TM) i7-1165G7 @2.80GHz 1.69 GHz; RAM 16 GB, 64-bit operating system
Skyline (64-bit) 22.2.0.255 (c99206313)

Could you take a look at this problem? Thank you very much!

Regards
Dilip

 Protter Error.docx 
view request
ERROR: Failed to find required column named 'file'.
(28 responses) zzhu248 2022-05-26

Dear Developers and all researchers,

When I was trying to build the library via peptide setting, I upload my .ssl file and it pops up warning as title.

For the specific workflow, I am following a paper from nature protocol: https://doi.org/10.1038/s41596-018-0089-3
I was working on the crosslinking setting and stuck on the step 39.

I would be very appreciated for your help!

Best Regards,
Peter

 Screenshot 2022-05-26 143119.png 
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SSL_ERROR
(6 responses) ebru9679 2022-11-08

Dear Skyline time,

I am trying to download encyclopedia-1.12.31 but I get an error "SSL_ERROR_RX_RECORD_TOO_LONG". could you please check your ssl record?
Thank you
Ebru

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Unable to use 'External Tool' feature
(2 responses) dilip singh 2022-11-07

Dear Skyline Team,

I am trying to use Protter from tool store. To do so, I need to add "Protter" in external tool option.

I am getting error, which is attached for your reference.

Kindly suggest solution.

Regards
Dilip

 External Tool_Error.docx 
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export protein-peptide list from skyline
(1 response) kmsd 2022-11-07

Hello, I am new to skyline so I am still learning new things. I have followed MS1filtering tutorial to import my short listed protein target into skyline along with 8 orbitrap pdresult files. After cleaning them now I need to export out the protein-peptide list from skyline to hard drive for future use. Is there any supporting tutorial available?

thanks,
kmsd

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Latest CIRT library
(2 responses) Brett Phinney 2022-11-03

Hi All, does anyone have the latest list of CIRT peptides skyline looks for? Preferably in a bilb library with predicted ion mobilities ? Just curios , I looked and couldn't find it

Cheers

Brett

view request
Exporting scheduled method for collision energy optimisation
(13 responses) matthew daly 2022-10-17

Hi,

I am having difficulty exporting a scheduled method for collision energy optimisation.

My colleagues ran a panel of peptides and I have the RT for these peptides, however I don't have the .raw files. I have pasted the results into the iRT calculator.

I am trying to run these same peptides using the same chromatography on a different platform, so I need to optimise the CE and as I am sample limited would like to do it in as few injections as possible. When I try to export a scheduled method optimising CE I get the error "Retention time predictor is unable to autocalculate a regression". Is it possible to export a method for peptides that you've used to train the iRT calculator?

Many thanks
Matt

view request
export dot p result between MIDAS vs Library
heyang 2022-11-02

Hi Support team,

Could I export dot p value of MS2 spectra comparison between MIDAS vs library?

thanks,
Heyi

view request
Error retrieving chromatogram "UV 1"
(1 response) licky lck 2022-11-01

Dear Skyline team,

The samples run from Our Q Eaxctive had a hard time recording the UV data, so we could not retrieve the UV data after the sequence run. So when I am trying to import the raw file into skyline, this error message pops out "Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type". (The full error message can be found below)

Do you have any suggestions to recover the data from my raw file, or can skyline ignores this UV signal but import my MS data?

Full error message:
Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'XXXX.raw'.
[ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type ---> System.Exception: [ChromatogramList_Thermo::chromatogram()] Error retrieving chromatogram "UV 1": [RawFileImpl::getChromatogramData()] Unknown UV/PDA packet type
at pwiz.XX.msdata.ChromatogramList.chromatogram(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetQcTraces() in C:\proj\skyline_22_2\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 846
at pwiz.Skyline.Model.Results.GlobalChromatogramExtractor..ctor(MsDataFileImpl dataFile) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromDataProvider.cs:line 129
at pwiz.Skyline.Model.Results.SpectraChromDataProvider..ctor(MsDataFileImpl dataFile, ChromFileInfo fileInfo, SrmDocument document, IRetentionTimePredictor retentionTimePredictor, String cachePath, IProgressStatus status, Int32 startPercent, Int32 endPercent, IProgressMonitor loader) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 87
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1306
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 244

view request
Rugged extracted ion chromogram of targeted peptide precursors from FAIMS-DIA file by Skyline.
(5 responses) Winnie 2022-10-30

Hi, skyline,

I extracted chromotogram of targeted peptide precursors from FAIMS-DIA files by skyline, the xic show rugged profiling. The screenshot is attached. I was wondering there are issue on ion moblity library because i did not set ion mobility sepctral library. The sepctral library was imported by the following strategy. File>import>assay library. I found the column named precursor ion mobility in this spectral library is NA. The spectral library was build by Fragpipe with files acquired by FAIMS-DDA. I first communicated with Fragpipe team, they told me it is normal the column named precursor ion mobility is NA, because they this the FAIMS is low resolution device for ion moblity. So they set NA of precursor ion mobility for the library genetated by FAIMS-DDA.

So, did you have suggestion on the rugged xic and how to spectral library generated by FAIMS-DDA

 Screenshot.png 
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Can't connect to prosit server
(7 responses) sp807 2022-10-27

Everytime I try to conncet to the prosit server, it says "server unavailable".
I have updated my software to Skyline (64-bit) 22.2.0.255 (c99206313) but the same things happens.

Any way to fix this?

view request
Skyine-Daily failing due to application error thrown on Windows 2016
(7 responses) hchandler 2022-10-22

Dear Skyline support team:

When launching Skyline-Daily or even trying to run the program, we are getting the same error that we have attached to this trouble ticket. We first thought that RAM was problematic as another product had taken so much, but even when the other program is not running and the server is rebooted to clear resident memory and caches, the error occurs. It states that the system did not have enough resources to complete the operation. The server is pretty beefy with 128GB of RAM, plus 2 Xeon 3.2GHz CPUs in an enterprise level Dell server running Windows 2016. The drives all have plenty of disk space (500GB or more) available, plus the NAS drives not only have plenty of free space, but the Network load does not reach above 10% utilization on the 1TB network interface. So not sure of the resources needed, other than we thought that the browser level was still using the old deprecated IE 11 ( we just installed Edge, but IE 11 is still on the system) and maybe the .NET needs to be updated (it is v4.8). So rather than guess at the issue, we are asking if you could read the error and determine the possible issue at hand.

Thank you for any help or assistance on this matter, in advance.

Sincerely,

Henry Chandler

 PRC_Post_10222022.txt 
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Using an earlier version of MSstats
(2 responses) susannah hallal 2022-10-27

Hello,

I hope you're well.

Is it possible to install and use an earlier version of MSstats in Skyline? I currently have the most recent version installed, but would like to use an earlier version to compare my data to an analysis I had done a couple of years ago

Kind regards,
Susannah

view request
Determination of LOD and LOQ using the height of the peak and the background signal amplitude
(1 response) gomesrja 2022-10-25

Hi, good afternoon

Is there a way of getting the peak intensity and background signal amplitude to determine the LOD and LOQ (being LOD 3x and LOQ 10x)? And is this the best way to determine these parameters using DIA MRM-like data?

Many thanks,

Ricardo Gomes

view request
Non-Canonical Amino Acid
(6 responses) lulmer 2022-10-24
Hello,

I have a unique use case working with the non-canonical amino acid, benzoylphenelaylanine, and I'm having trouble importing my results into skyline. I'm trying to import comet/peptideprophet search results that have the benzoylphenelaylanine non-canonical amino acid defined (attached interact.pep.xml). When I import these results there is a suggested modification that makes the peptide with the non-canonical amino acid viewable in skyline (attached jpegs 1 and 2 described the modification). However, when I try to reopen the skyline document with that modification I get the error "Variable modifications must specify amino acid or terminus" (attached error.jpeg). I've attached the skyline document that gives this error as well. Any suggestions on how to make these comet/peptideprophet search results with the non-canonical amino acid compatible with skyline would be greatly appreciated.

Thanks!
Lindsey Ulmer
 suggestedmod_01.JPG  suggestedmod_settings_02.JPG  210727_hspb5_b9_nouv_t_mon_interact.pep.xml  T_noUV_dim_BPA.sky  error.JPG 
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High Energy DT Offset
(5 responses) sxu374 2022-10-20

Hi Skyline support team,

I have some data acquired from Agilent 6560 IM-QTOF. I noticed that for some analytes, the IM drift times of the fragments are lower than their precursors, leading to unsuccessful alignment. In a literature, I found a function called "HighEnergyDTOffset", which could set in skyline transition list. This function indicates how much the fragment drift times could be lower than their precursor. I added the "HighEnergyDTOffset" column to my transition lists of different batches of data. Something interesting is that this function only works for certain transition lists. In other transition lists I loaded, the IM drift time window applied in MS/MS spectra was the same as the precursor, not having any drift time offset at all. I wonder if there is a guidance on how to set "HighEnergyDTOffset" correctly, or if there is any restriction on how the data is acquired by MS (AIF, PRM, etc.) in order to enable this "HighEnergyDTOffset" function.

Thanks,
Anne

view request
Sciex SWATH data we see precursor response but not fragments
(1 response) jlaycock 2022-10-21

Dear Skyline Team:

We have 5 replicates of predigested BSA injected with fixed window SWATH on our Sciex a 7600 ZenoTOF. The BSA Fasta was imported and the 5 .wiff data files were imported. I have tried a several different settings in the Transitions form but still do not see the fragments.

I have confirmed by viewing the data in OS Explorer that there is response for at least some of the fragments.

Thanks,
John

Data files available to upload if helpful.

 SkyLine SWATH no fragments.docx  BSA Digested Standard MRM-HR_080222.sky.zip 
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Using software for MacBook user
mhossai9 2022-10-21

I am using MacBook. I can’t able to use the software from my MacBook. Is there any solution?

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Target peptide monitoring using DDA Orbi-Lumos LFQ data
(1 response) kmsd 2022-10-19

Hello, I have a list of peptides that I want to monitor against lumos LFQ (DDA) raw data file. I know there are lot of good articles and Ques/Answer available in skyline but I am new in this platform and need some guidance. Can someone direct me how to do this using skyline weblinks/tutorials etc?

Thanks
kmsd

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I can't choose my quantification ion
(10 responses) antonin padioleau 2022-10-11

Hi all,

I have a problem with my transitions. I work with GC-(EI+)-HRMS and LC-(ESI+/-)-HRMS data acquired in full-scan mode and I use the small molecule part of Skyline.

I created my excel sheat with 1 quantifier ion and 1 or 2 confirmation ions, the quantifier in the upper line of my sheat. When I imported this sheat with copy/paste, I selected all ions as precursor (it didn't work with precursor and product ions, no data in the chromatograms). Next, in the left side, for each molecule, my precursors were rearranged from the smaller mass to the highter.
My problem is that for all the molecules, the quantifier ion is automatically the first precursor ion, the smaller one, even if it's not the best choice. I tried to select the "Quantitative" choice only for my quantifier ion but, when it's not the smaller ion, the message "Error: All of the external standards are missing one or more peaks" appears in the calibration curve window. I can only quantify with the smaller ion...

I tried to change the monoisotopic and average masses of my molecules, which are always the mass of the smallest ion, to my quantifier ion mass but Skyline automatically adds the difference between the former masses...

How can I choose my quantification ion and confirmation ion in another way?

Thanks!

view request
Problems importing Bruker MRMS data
(3 responses) dennisjakob 2022-03-07

I've been running samples on a Bruker Scimax MRMS. When I import the data I get this error message:

At 3:47 PM:
Failed importing results file 'D:\Users\Data\2020-11-24-Aminoacid mix\Amino acid_1_2_1_123.d'.
unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'D:\Users\Data\2020-11-24-Aminoacid mix\Amino acid_1_2_1_123.d'.
unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7 ---> System.Exception: unknown compressor id: 6bb2e64a-27a0-4575-a66a-4e312c8b9ad7
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, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 194
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 291
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 188
--- End of inner exception stack trace ---

Do you have an idea what the issue could be here?

Cheers,
Dennis

view request
Peptide Identification using DIA SWATH Data
(1 response) gomesrja 2022-10-17

Good morning

I have a question...How to I know each peptide and proteins are considered identified and present in a sample using DIA SWATH data?

I am doing an untargeted global analysis of all possible proteins in the library. Is the library match peptide score?

Many Thanks,

Ricardo Gomes

view request
Q Exactive method export
(1 response) ad6628 2022-10-14

Hi,

I was at the training on October 10 and 11 this week.Now I am trying to use a Skyline for the lab's needs. I was wondering if you could, please, give me some directions on a couple of problems. I was able to create a transition list for the peptide we are studying. However I would like to export a method for the Q Exactive mass spectrometer. When I go to file-export-method, I do not see Q Exactive in the list "instrument type". Also, when I tried to export one of them, the program is asking for a template. I would appreciate if you could direct me how the template should be for the Q Ex method and how to find the Q Ex in the drop down list. I tried to export the transition list method from a Fusion mass spectrometer since it is showing in the list of instrument just to practice, but I get an error message that I am attaching to this request.

Thank you very much
Rita

 Skyline-Error.jpg 
view request
Mobility filtering and data importing issues
(3 responses) mmgadz 2022-10-11

I am working on developing PRM-PASEF methods. I have encountered some issues with importing data and have some questions about the mobility filtering/method exporting.

Importing: Importing the same raw file with the same peptide/transition settings into separate skyline files produces A) peak area, RT, and spectral data and confident identifications or B) no peak areas or RT data displayed, unconfident identifications. I cannot figure out why this is happening.

Mobility filtering: The filtering window is frequently offset from observed maximum - is there a way to mediate this in skyline? When exporting a bruker method, how does skyline incorporate the mobility filtering? Is it necessary to refilter the data with the same library used in the method/data acquisition or should I use "Use Data" and refilter for a given dataset?

Attached is a powerpoint with screen grabs demonstrating the above challenges, as well as descriptions. The slides are segmented into "Issue 1" and "Issue 2".

Any help will be greatly appreciated - thank you!!

 SkylineInquiry.pptx 
view request
Custom fragment ion for ECD
(2 responses) ankittjain89 2022-10-12

Hi

I was trying to look for a custom fragment ion from ECD and ETDhcd spectra for the isoAspartate residue of the peptide that gives off either c+57 or z-57 ions. I am able to add c and z ions in transition settings but unable to add any other loss/ gain to it. One workaround I had to this problem was to use a peptide modification of 57 gain/ loss and then select the c ions with a 57 loss or z ions with 57 gain. So is there any other simpler way to do this custom fragment ion search?

Thanks
Ankit

 SkylineECD settings.pptx 
view request
Q executive method
ad6628 2022-10-13

Hi,

I was wondering if you could, please, give me some directions on a couple of problems. I was able to create a transition list for the peptide we are studying. However I would like to export a method for the Q Executive mass spectrometer. When I go to file-export-method, I do not see Q executive in the list "instrument type". Also, when I tried to export one of them, the program is asking for a template. I would appreciate if you could direct me where to get a template for the Q Ex method and how to find the Q Ex in the drop down list.

Thank you
Rita

view request
Collision Energy for Thermo Altis
(4 responses) Gao 2022-10-12

I noticed the collision energy calculated by Skyline Daily for Thermo Altis were dramatically changed in recent updates. They are much higher than the previous versions. Would it be possible to have the conversion of CE between current version and previous version? The collision energy of previous versions work better for me on my Altis. Thank you!

view request
MRM assay development
(3 responses) ad6628 2022-10-12

Hi,

I have attended the introductory training on October 10 and 11 but I am not sure how to apply the training to my immediate problem. My boss wants me to establish MRM conditions for a QExecutive mass spectrometer to check on a synthetic peptide. I will attach the slides I have. The peptide has modifications as oxidation, acetylation and has an amide attached to the n-terminal. It can be analyzed as heavy versus light. I was wondering which tutorial I should follow to create the assay. Any advice would be greatly appreciated.

Thank you
Rita

 MRM assay development.pdf 
view request
Command line to set the peptide as a global standard
(3 responses) jianqiaoshen 2022-10-08

Dear Skyline team, I am working on the automation for Skyline. I am wondering is there any command lines or ways to set one peptide as the global standard?

Regards,

Jianqiao Shen

view request
Multiple precursors library match
(1 response) Juan C. Rojas E. 2022-10-11

Hi all,

I could have sworn that a recent version of Skyline-daily was displaying at least one of the PSMs for one of the charge states if the selection was focused on the peptide. But on Skyline-daily 22.2.1.278 this useful feature seems to be gone (attached pic).

Would it be possible to get it back? :)
Sincerely,
Juan C.

 Library_match_display.PNG 
view request
Ghost peak in Skylin
(3 responses) ychiu2 2022-10-07

Hello Skyline team,
I have been using Skyline to do the quantification for small molecule and recently we found a ghost peak integration issue. In Skyline, you can see that the software integrate a peak at m/z 1055.5057, but when I plot m/z 1055 in Freestyle (Thermo software), no peak was found. I really want to know if there is a way to fix this as we use Skyline heavily for quantification. If the peak integration was not good, then the data validation will be very difficult. I have attached the skyline file and two graphs demonstrating the peak
Thank you

 221007_Skyline_GhostPeak.PNG  221007_GhostPeak_Thermo.PNG  Sep2022_MC-YR_test.sky.zip 
view request
unify raw file can't be imported to sklyline.
(1 response) juan xu918 2022-10-09

Dear Skyline team,

I am trying to import 2 Waters Xevo G2 raw files into skyline. The skyline document has only 1 protein and the raw files being imported were collected using MSe method. I began importing the data and didn't see any chromatograms exported. After a few mins, the program skyline closed down by itself.

I am using Sklyine (64 bit) 22.0.255 (C99206313) on Windows 11 Pro. The processor is an 12th Gen Intel(R) Core(TM) i7-12700H 2.30 GHz and 40 Gb of Ram.

Many thanks,

Juan

 unify raw data import issue.pdf 
view request
Concentration Multiplier in Molecules
(1 response) franzone 2022-10-07

I am doing quantification on amino acids so I am using the Molecules layout for my analysis. I read previous posts about using Concentration Multiplier in the Peptides layout. Is this function available in Molecules and, if not, will it be added in the near future?

view request
Trouble in export method to MassLynx
(3 responses) guioliveirareis 2022-10-05
Dear Brandon and Skyline Team,

I've downloaded the new Skyline version to run a SRM experiment in Waters Xevo TQS-micro, however when I try to export the method this message pop-up:

---------------------------
Skyline
---------------------------
An error occurred attempting to export.
ERROR: Saving the method was unsuccessful.

Command-line: Method\Waters\BuildWatersMethod -w 5 -m "C:\MassLynx\Default.pro\Acqudb\template_skyline.exp" "C:\MassLynx\Default.pro\Acqudb\t5oo4e1q.bxc\transitions.txt"
Working directory: C:\MassLynx\Default.pro\Acqudb
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: Saving the method was unsuccessful.

Command-line: Method\Waters\BuildWatersMethod -w 5 -m "C:\MassLynx\Default.pro\Acqudb\template_skyline.exp" "C:\MassLynx\Default.pro\Acqudb\t5oo4e1q.bxc\transitions.txt"
Working directory: C:\MassLynx\Default.pro\Acqudb ---> System.IO.IOException: ERROR: Saving the method was unsuccessful.

Command-line: Method\Waters\BuildWatersMethod -w 5 -m "C:\MassLynx\Default.pro\Acqudb\template_skyline.exp" "C:\MassLynx\Default.pro\Acqudb\t5oo4e1q.bxc\transitions.txt"
Working directory: C:\MassLynx\Default.pro\Acqudb
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_22_2\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 161
   at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
   at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List`1 argv, String fileName, String templateName, Dictionary`2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Export.cs:line 4674
   at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Export.cs:line 4466
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Util\Util.cs:line 1963
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action`1 performExport) in C:\proj\skyline_22_2\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2240
---------------------------


Here, we use MassLynx 4.2 SCN 977 (with TargetLynx installed).

I've tried reinstall and uninstall several times, with several ways. I have no ideia what is happening, and then I was wondering if you have seen this message before in Waters equipaments?

Thank you very much for your help,

Guilherme
view request
peptide absolute quantification from PRM data sanity check
(2 responses) manguy jean 2022-09-23

Hi,

I learned how to use Skyline on the job for a peptide absolute quantification project. I read a lot of the tutorials, some of the posts I found here from different google searches, and looked at the methods sections of diverse papers. I think what I did makes sense I just want to make sure I didn't make a mistake because of some blindspots, so any help or advice is welcome.

We want to quantify some peptides from a hydrolysate (not for protein quantification but just for peptide quantification so we didn't have the choice of which peptides would be best). We have heavy variants of each of them. We built an internal calibration curve for each peptide using varying concentrations of heavy peptides. The first series of runs (PRM on a QE) uses a wide scale of concentrations to find the approximate concentration of light peptide, then another series of runs to adjust the calibration curve for each peptide to quantify and then replicated that again.

I used Skyline to analyze these data. I used the retention time of these peptides in previous DDA runs using the same LC parameters and the presence of peaks for the heavy and light products to select the correct peaks. Removed the product ions interfering with the peak. I then extracted the ratio of heavy/light for each analytical replicate and for each concentration. I used these ratios to make a calibration curve (linear regression) and I looked for the ratio heavy / light == 1 to find out the concentration of light. I had to go outside Skyline for this step as all my samples are marked as Standard, no sample being just Unknown, but being both at once, and as far as I know I cannot ask Skyline to look for a ratio of 1.

I hope someone can tell me if I am completely off the tracks or not.

Thank you

Jean

view request
Analysis of uncharacterized peptide-modifications
laura boffel 2022-10-07
Dear Skyline team,

I am currently investigating drug-protein adduct formation of reactive chemicals at Cys34 of human serum albumin. More specifically, I am investigating whether a specific drug becomes reactive after in vitro metabolization and whether this reactive metabolite is captured by the thiol group of Cys34 and consequently, a covalent bond is formed between the reactive metabolite and amino acid.

So my question is if I can use Skyline to detect such protein/peptide modifications, by comparing IDA data obtained from a blank sample and a sample incubated with the drug, to be able to detect differences in the fragmentation pattern of the peptide? Since I don't know the chemical composition of the reactive metabolite on beforehand, an untargeted modification approach should be used.

Thank you in advance!

Kindest regards,
Laura
view request
Feature request: Maximum pressure value for reports
(2 responses) cashwood27088 2022-10-06

Dear Skyline team,

I hope you're doing well. I'm loving the new Skyline-daily update featuring the pressure traces, but currently the chromatogram visual alone makes automatic QC difficult. Could you please add a parameter in the report section that provides the maximum height observed in the pressure trace? I imagine it would be similar to the "Total Ion Current Area" parameter except only returning the maximum pressure. It would also need to ignore the automatic peak picking of the pressure trace since the maximum height would be the maximum y-axis value for the entire retention time range.

I've attached a mock-up of what I think it should look like.

Cheers,
Chris

 PressureReportRequest.png 
view request
Signal to noise in Skyline
(1 response) kh 2022-10-06

Dear Brandon and Skyline Team,

I would have a question regarding S/N, is it somehow possible to access it in Skyline?
We currently run an instrument benchmarking experiment (small molecules, neg and pos mode, high resolution) and would like to compare also the signal-to-noise values for several metabolites in a fast and easy way (if possible). Would you have any recommendations for us?

Thank you for your help!

All the best,
Kathi

view request
exporting Prosit dotp scores
cabarnescabarnes 2022-10-04

Hi all,

I've seen this requested a few times, but is there any update on the ability to export the Prosit dotp scores for each peptide in a mirrored database match-based spectral library?

Best,

chris

view request
highlighting peptides that map to multiple proteins
(2 responses) cabarnescabarnes 2022-09-30

Hi all,

Simple question, but is there a way to highlight which peptides are matching to multiple proteins? I have a proteogenomics type of effort going on and there is utiility in me finding protein evidence for multiple isoforms that might all individually have the same tryptic peptide, but that tryptic peptide may only map to that novel isoform group and nothing else, which would be helpful in and of itself for me as I want to keep all of the isoforms in there.

Best,

chris

view request
Spectral library quality control data
(4 responses) cervenka 2022-09-29

Dear Brendan,
I would like to ask you if there is any way how to get data about spectral library and its quality for QC, e.g. how many y ions/b ions/PMS/peptides/shared peptides/peptides with PTM(s)/proteins the library contains, what is the average mass error and spectra quality etc.? Basically something as the DIALib-QC tool does (http://www.swathatlas.org/DIALibQC.php). I tried to export library in format for Spectronaut (Skyline Schema for exporting Spectronaut compatible libraries - https://biognosys.com/resources/spectronaut-further-user-resources/) and used the DIALib-QC tool, but I was not able to get any results.

I found slightly similar question in the forum - https://skyline.ms/announcements/home/support/thread.view?entityId=e2b45ef9-e45b-1035-96fc-e465a39343b4&_docid=thread%3Ae2b45ef9-e45b-1035-96fc-e465a39343b4 Do you think that such apporach would also work for me (Skyline 22.2), please?

Thank you very much for your help!

Jakub

view request
Unable to generate calibration curve for small molecules using "Ratio to Heavy"
(4 responses) ben ahiadu 2022-10-03

Dear Skyline team,
I have been trying to create and use a document for small molecule quantitation. I created the skyline document and imported the raw files for quantitation but skyline does not recognize the light and heavy forms of my molecules. I tried to set the transition list as shown in the small molecule tutorial but to no avail. The error message skyline I get is: the isotope modification is not found in document settings. I tried to add the Light and heavy modifications in the document settings but this did not work.
Please I need help.
Thank you

 Oxytocin-fibrino peptide transision list_Skyline team.xlsx  9-6-22 Oxy-Fib calcurve as small molecuels-Team.skyd  Missing peaks_Skyline team.PNG 
view request
Addition of a deamination modification on an N-ter cysteine
(1 response) laurie josset 2022-09-30
Hello to all,

I am working on the quantification of desmopressin, when I add the peptide sequence in skyline I end up with arginine vasopressin (that is normal). I tried to manually create the deamination modification to apply on the N-ter cysteine, only I don't end up with the right m/z for the precursors (I want 535.35 ++ and Skyline predicts me 545.7196 ++). For the chemical formula I indicated "O-HC", maybe this is not correct. Would it be possible to help me?

Thanks in advance,

Sincerely,

Laurie Josset

Translated with www.DeepL.com/Translator (free version)
view request
Shimadzu GC-MS
(1 response) victoria dorrer 2022-09-29

Hi,
I am currently working with a Shimadzu GC-MS, hence the GCMSsolution Version 4.52. This software creates GC/MS Data File (.qgd) files (see attachement) which I cannot use in Skyline. Even if I try to convert it to mzXML (see attachement) in the software Skyline does not recognize the peaks within the data file. It still tells me chromatogram information unavailable. Did someone have similar problems with the combination Shimadzu and Skyline? If yes how was it solved?

Cheers,
Vici

PS: I work with Skyline regularly with files from Bruker and there I have no problems.

view request
NEEDLE IN THE HAYSTACK
(1 response) Victor 2022-09-27

Hi,
Is it possible to clarify and briefly outline current state-of-the-art methodology for detection of single low abundant protein ( 1000-10000 copies per cell ) with known sequence using tools available in Skyline arsenal. I have taken a look into all currently available proteomics repositories with regards to quality and the overall number of peptides detected so far and is rather low so relying on publicly available data is not option. We have taken care of sample prep so now the question is about most optimal way to set up data acquisition and data processing part that would maximize our chances for success. As discussed due to low abundance going DDA would be hardly beneficial. DIA even more so. So I am thinking to go targeted from the get go. To the best of my knowledge the best chances would be to pursue either MS1, SRM or PRM monitoring. Setting up and performing targeted MS1 scans with or without dissociation is pretty straightforward. Though I am a bit uncertain how to approach SRM and PRM workflows. In particular I am a bit unclear which software tools may be used to create targeted transitions to increase sensitivity even further, in case targeted MS1 data would not yield any result. Of particular interest which peptides to choose, and which transition offers best chances for detection and of cause which Skyline plug-ins to use for that. With regards to data processing the jury is still out whether classical data base search from fasta file or using in-silico created spectral libraries would be preferred way to set it up . May be even both. I have researched applicable Skyline tutorials and while some of them offer some guidance still would be quite usefull to get up-to-date guidance in one place. Thank you.
Victor

view request
Command Line Enhancement Request : Include MS/MS Filtering Options
(3 responses) philip remes 2022-09-21

Hi guys,

The command line documentation lists some options for MS1 filtering that can be changed, under the Document Settings. What do you think about adding the MS/MS filtering options? These are: Acquisition Method, Product mass analyzer, Resolution, and even Isolation Scheme for the DIA acquisition method. A work around could be for an application to store Skyline file templates with the desired settings, but this is not as flexible as programmatic access.

Thanks
Philip

view request
SureQuant with two FAIMS CV voltages on Orbitrap Fusion Lumos - ZigZag shaped peaks on Skyline
(11 responses) aheinone 2022-08-11

Dear Skyline Team,

We are starting to do SureQuant on Orbitrap Fusion Lumos with FAIMS by using two CV voltages (-50 and -70). We are dealing in total with about 2000 peptides, but < 500 peptides at a time in one Skyline document. We have imported the raw files to Skyline for further processing. We have built Ion Mobility Libraries by using the results. Some peaks are drawn correctly, but in many cases product ion peaks are jagged (zigzag shaped in MS2 level). But still precursor peaks (MS1 level) look ok. It seems that this happens with CV -50 peaks. Probably Skyline takes data points from the both CV values for some peaks? We have tried many different settings for MS/MS filtering, but we haven't found any solution. We tested to change the CV values manually in the Ion Mobility library, but it doesn't solve the problem. And the CV values seem to be correct in the library. If we split the original raw files to two separate files according to the CV voltage, and then import them to Skyline, the peaks are fine. But if we build the Ion Mobility Library by using the two separately imported files, it didn't solve the problem. Is there any other way to handle multiple CV values than split the files according to the CV voltage. We would appreciate for any help with this, thanks.

Regards,
Arttu

 Skyline_issue_FAIMS_two_CV_voltages.pdf 
view request
Peak symmetry, asymmetry or tailing factor
(2 responses) o heravizadeh 2022-09-25

Hi,

Is there any way to have the information regarding peak asymmetry, symmetry, or tailing factor in the "Document grid" table?
I currently tried to use the SProCop_1.3 tool, However, it gives me a bunch of errors and the result is not clearly reported in a table format for all the compounds' peaks.
As the mentioned parameters are important for peak quality assessment and I am not interested to use two separate software for analyzing my data, I hope somebody can help me regarding this matter.

Thank you so much,
Omid

view request
Spectral Library Build - error
(1 response) ana normando 2022-09-23

Hi,

I am trying to build a spectral library with .msf files but it keeps showing an error:
"This file does not contain q-values. You can set a cut-off score of 0 in order to build a library from it"

However I cannot find where I can set this cut-off score. Could you please help me find?!
I am using Skyline 22.2.0.255; in older versions there was a small window where we could set the cut-off score...

Thanks

view request
DDA search hangs on "Running percolater..." with a specific FASTA file
(3 responses) Remco van Soest 2022-09-20

Skyline (64 bit) 22.2.0.255 (c99206313)

When using the "Import DDA Peptide Search" workflow Skyline hangs on "Running percolator..." after the search has successfully finished. I have to manually end the "percolator.exe" task in order to make the software respond again.

The issue seems related to the FASTA file (attached; downloaded from NCBI; sequence is correct). If I manually remove line 11 and 12 everything works just fine. It is not the length, as everything works fine as well if I replace line 11 and 12 with A's.

Why does the percolator task hang when using a specific FASTA file? I don't see anything wrong with the file, and obviously want to be able to use the full sequence for my search.

Thanks,
-Remco

 sequence (2).fasta 
view request
Issue with edit >insert>transition
(1 response) bkr 2022-09-22

Hi Skyline team,

I have been trying to insert the custom transition but the edit insert transition opens to a blank tab instead of the tabular layout that used to be in previous version. How can i insert transition in the Skyline 22 (newest veersion)

cheers
bharath

view request
Issue with "Edit -> Insert -> Peptides"
(2 responses) pavel 2022-09-12

Hi,

I have a small issue when pasting peptides via "Edit -> Insert -> Peptides". My system is Windows 10, Skyline-daily 64-bit, 22.1.9.252.
When I try to copy a few peptides with descriptions from excel into the Skyline document, description is not included. The excel table looks like this:
EAGHLVFDR protein 1 description 1
IEAGHLVFDR protein 2 description 2
LIEAGHLVFDR protein 3 description 3
Just taking these three peptides from the Excel file into the clipboard and going to Edit -> Insert -> Peptides results in the third column empty in the table (see the attachement). I would expect to paste also the “description…” part.
Pasting only the description column into the third column of the grid did not work as well.

Thank you for your help.

Best regards,
Pavel

 Edit_Insert_Peptides_from_clipboard_issue.jpg 
view request
Path to SkylineCmd as External Tool Macro?
philip remes 2022-09-21

In reading about the SkylineCmd, you mention various ways of knowing where it is, which require the user to do a special kind of install, either as an Administrator or with the Unplugged installer. Would it make sense to provide Tool creators a macro that resolves to the SkylineCmd path? This way a Tool would have easy access to SkylineCmd without relying on the user to download SkylineRunner and put it in a particular place, or for the tool to package up the Skyline runner in its installation zip file.

Thanks
Philip

view request
spectral library _ Error
(4 responses) sudhir pr 2022-09-20

Hi All,

I tried to build spectral library using MaxQuant msms.txt file and modifications.xml files. But I got the following error message. Would you please help to solve the problem? Please suggest next steps. Thank you.

More info (Error message):

System.IO.IOException: Error occurred running process.

Command-line: C:\Users\reddys49\AppData\Local\Apps\2.0\580TP24P.9OP\H3L3QW2E.RPJ\skyl..tion_e4141a2a22107248_0016.0002_1c0fe1ba9c975222\BlibBuild -s -A -H -o -i Urine_QEHF -S "C:\Users\reddys49\AppData\Local\Temp\tmp2031.tmp" "C:\Users\reddys49\OneDrive - Bristol Myers Squibb\Desktop\SPR_BMS\Sudhir Reddy\Database\From_XQ-AD_MQtxtfiles_UrineQEdata\txt\txt\Urine_QEHF.redundant.blib"
Working directory: C:\Users\reddys49\OneDrive - Bristol Myers Squibb\Desktop\SPR_BMS\Sudhir Reddy\Database\From_XQ-AD_MQtxtfiles_UrineQEdata\txt\txt
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_22_2\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 161
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_22_2\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 412
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161

view request
Library Build for SILAC labelled PTM spectra from MetaMorephus mzID and mzML files ERROR No spectra were found for the new library.
gwitek 2022-09-20

Hello,

I am trying to build a library from my spectra files that contain peptides digested with Pepsin, grown in SILAC media, and that have phosphorylation on S/T/Y (PTM search).
I get mzID and mzML files from MetaMorpheus but I get an error that No spectra were found for the new library.
I tried changing files to lowercase letters mzid and mzml both or each and it does not work.

I did read another request that said PTM searches can cause library build issues and wanted to reach out to you if this might be another issues with PTM or SILAC.

Thank You,
Gabriela

view request
Configure External Tool Custom Report to Use .tsv?
(4 responses) philip remes 2022-09-19

I have an external tool configured according to the nice documentation in https://skyline.ms/_webdav/home/software/Skyline/@files/docs/Skyline External Tools-2_1.pdf. The custom report has the Raw Times and Raw Intensities columns defined, which use comma delimited lists. It is more convenient to parse this report with tab delimiting, allowing to parse the Raw Times lists easily: the alternative I've found is to use regular expressions to tell the difference between the commas for columns and the commas in the Raw Times, and this is orders of magnitude slower than a simple line.Trim().Split('\t'). Therefore I'd like to request some way to specify the delimiting of these external reports, either in the tool.properties files, or else in the custom report .skyr file itself.

Thanks
Philip

view request
PRM search
(4 responses) xhuang 2018-10-08

Hi, I'm a new user for skyline. I have a more general question as I browse previous post and can't find the answer. I'm running targeted msms using Q-exactive. When I try to set up those transitions, for example, I can't find the instrument listing as QE. Shall I choose orbitrap instead? Also how do you define those values (such as library-ion matching tolerance, instrument-method matching tolerance)? Where to define the dot color (red/yellow/green) next to the peptide fragment? Thanks!

view request
No able to do Grouping based on condition
(8 responses) sstoychev23513 2022-09-15
Hi guys,

I'm getting the following error when trying to group samples by condition... any suggestions?

Skyline version: 22.2.0.255-c99206313 (64-bit)
Installation ID: bdb00579-df5f-442a-82cc-2f7e2f5b8602
Exception type: AssumptionException
Error message: chromInfoData.ChromFileInfo is null

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

pwiz.Skyline.Util.AssumptionException: chromInfoData.ChromFileInfo is null
   at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Util\Util.cs:line 2080
   at pwiz.Skyline.Util.Assume.IsNotNull(Object o, String parameterName) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Util\Util.cs:line 2019
   at pwiz.Skyline.Controls.Graphs.RTReplicateGraphPane.RTGraphData.IsMissingAlignment(ChromInfoData chromInfoData) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\RTReplicateGraphPane.cs:line 366
   at pwiz.Skyline.Controls.Graphs.RTReplicateGraphPane.RTGraphData.IsMissingValue(TransitionGroupChromInfoData chromInfoData) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\RTReplicateGraphPane.cs:line 450
   at pwiz.Skyline.Controls.Graphs.SummaryReplicateGraphPane.GraphData.<>c__DisplayClass49_0`1.<MakePointPairLists>b__3(TChromInfoData c) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\SummaryReplicateGraphPane.cs:line 704
   at System.Linq.Enumerable.WhereListIterator`1.MoveNext()
   at System.Collections.Generic.List`1.InsertRange(Int32 index, IEnumerable`1 collection)
   at pwiz.Skyline.Controls.Graphs.SummaryReplicateGraphPane.GraphData.MakePointPairLists[TChromInfoData](DisplayTypeChrom displayType, IList`1 chromInfoResults, Func`2 isMissingValue, Func`3 createPointPair) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\SummaryReplicateGraphPane.cs:line 686
   at pwiz.Skyline.Controls.Graphs.SummaryReplicateGraphPane.GraphData.GetPointPairLists(PeptideDocNode peptideDocNode, TransitionGroupDocNode nodeGroup, DisplayTypeChrom displayType) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\SummaryReplicateGraphPane.cs:line 767
   at pwiz.Skyline.Controls.Graphs.SummaryReplicateGraphPane.GraphData.GetPeptidePointPairLists(PeptideGroupDocNode peptideGroup, PeptideDocNode nodePep, Boolean multiplePeptides) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\SummaryReplicateGraphPane.cs:line 519
   at pwiz.Skyline.Controls.Graphs.SummaryReplicateGraphPane.GraphData.InitData() in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\SummaryReplicateGraphPane.cs:line 347
   at pwiz.Skyline.Controls.Graphs.RTReplicateGraphPane.UpdateGraph(Boolean selectionChanged) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\RTReplicateGraphPane.cs:line 145
   at pwiz.Skyline.Controls.Graphs.GraphSummary.UpdateGraph(Boolean selectionChanged) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\GraphSummary.cs:line 370
   at pwiz.Skyline.Controls.Graphs.GraphSummary.UpdateUI(Boolean selectionChanged) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Controls\Graphs\GraphSummary.cs:line 299
   at pwiz.Skyline.SkylineWindow.<UpdateRetentionTimeGraph>b__1131_0(GraphSummary g) in C:\proj\skyline_22_2\pwiz_tools\Skyline\SkylineGraphs.cs:line 3476
   at System.Collections.Generic.List`1.ForEach(Action`1 action)
   at pwiz.Skyline.SkylineWindow.UpdateSummaryGraphs() in C:\proj\skyline_22_2\pwiz_tools\Skyline\SkylineGraphs.cs:line 4582
   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)
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 pwiz.Skyline.Program.Main(String[] args) in C:\proj\skyline_22_2\pwiz_tools\Skyline\Program.cs:line 312
view request
DDA data question
(3 responses) chen qian 2022-09-20

Hi Skyline team,

I am trying to use Skyline to track the performance of our System Suitability (DDA run, 15 peptide standards mix). The goal is to monitor MS1 and MS2 intensity change across the time.

Below is what I did: I searched the raw data in PMI and generated the mzid file that can be imported into Skyline. I used this mzid file to build the spectrum library. Then I set the peptide setting and transition setting based on the method edit tutorial. Then I imported all 15 peptides that I would like to monitor. Once everything is set up, I used File>Import>Result to import the raw data and try to view the results in Skyline. One thing I do not understand is that I can see all those product ion on the spectrum library, but why most of those product ions were labelled red in the Skyline? For example, if you look at the second peptide “GISNEGQNASIK”, I can see y7, y8 and y10 in the spectrum library, but these product ions were all labelled as red in the list (screenshot attached). The raw data I imported directly into Skyline is the same file I searched with PMI, so the fragment should be exactly the same. I have attached my Skyline file in case you want to take a look at the setting.

Another question is that I set in the transition setting that to monitor y, b, p ion types, why I only can see the precursor ion? I cannot find the way to display the fragment ions.

Hope I have my question expressed clearly, if you have any question or need any further information from me, please let me know.

Thanks,

 Skyline screenshot.png  SST_FA_2.sky.zip 
view request
Skyline stopped integrating in the middle of the peak
(2 responses) dtran25992 2022-09-20

Hi,
I was using Skyline small molecule to quantify this molecule ran on Shimadzu LCMS 9030 QTOF. The data was run using DDA method then raw data was imported to skyline for quantification.
I had a standard curve and a few actual samples that are run in triplicates
However, for some of the replicates, Skyline just stopped extracting/integrating in the middle of the peak (red box).
I went back to the Shimadzu's software and try manual extracted ion chromatogram of that peak and saw it's there in full.
I've never seen anything like this before, hopefully somebody can help,
Thank you very much,
Duc

 Screen Shot 2022-09-20 at 10.29.05 AM.png  Screen Shot 2022-09-20 at 10.37.38 AM.png 
view request
Fail to import Wiff2 files from X500R into Skyline Daily
(4 responses) sarah lennon27839 2022-09-14

Dear Skyline team,

I am struggling to import wiff2 from X500R files in Skyline Daily. Please find attached the error reported by Skyline and the wiff2 file.

The same file converted to mzml using MSConvert imports fine in Skyline.

Would you have any suggestions ?

Thank you very for your help,

Kind regards,

Sarah

 Skyline_error_message.txt  JC20200918-MSMS4methodes-IDA-20-PPT1-Serum-POS.wiff2  JC20200918-MSMS4methodes-IDA-20-PPT1-Serum-POS.wiff.scan  JC20200918-MSMS4methodes-IDA-20-PPT1-Serum-POS.timeseries.data 
view request
Failed importing results- .wiff file from SCIEX triple Quad
(14 responses) sarahmacpherson676 2019-06-19

First, thank you for offering this software, I’m new to Skyline and I am excited to use it.

My data files were collected from another lab using SCIEX triple Quad (5500). I am looking at small molecule data (around 200 metabolites). I was given a .wiff file containing the multiple runs and the .wiff.scan. However I am unable to upload either(file-->import-->results). I’m not sure if this is a file issue, software or something on my side. I have tried using the newest version of Skyline and Skyline Daily.
Attached are the errors that I receive for the .wiff file and .wiff.scan
Also attached is the .wiff and .wiff.scan files
Thank you for your time

 error messages skyline.docx  cell_metabolomics.wiff  cell_metabolomics.wiff.scan 
view request
Easy way to print out list of molecules from "find results" window in the "feature scores" tab
tony considine 2022-09-13

I am trying to find an easy way to print out a list of small molecules that will fail a scoring model on account of having less than 3 isotopes. I can find the molecules by going to edit current scoring model, highlighting the row in the "Available feature scores" grid and then hovering the mouse underneath the "unknown" bar in the graph on the "Feature Scores" tab.

I can push the binoculars button and Skyline will fill in the "Find Results" window with the list of small molecules which are missing that score. However, I cannot find a way to easily print out that list of small molecules. I have tried using the menu item:
File > Export > mProphet Features
and exporting all of the feature scores to a CSV file and then look at it in something like Microsoft Excel.
The "PeptideModifiedSequence" column will contain the name of your molecule, and you can look for "#N/A" in the corresponding score column.

However, the issue is that I am using a dummy dataset to just find out which molecules will be excluded from the scoring model on account of having less than 3 isotopes. It looks like if it is not a hit from the dummy dataset I still get a lot of "#N/A" in the corresponding score column and these might have 3 or more isotopes. What would be ideal is if there was a way to just export the compounds ID’ed in the “find results” window with the list of small molecules which are missing the score from the scoring model. Any help would be greatly appreciated.

view request
Could skyline extract the LC area of isomers which separated by ion mobility
(2 responses) sxu374 2022-09-12

Dear Skyline support,

I've used LC-IM-MS/MS to separate small molecule isomers. The isomers have the same m/z, retention time, and MS/MS spectra, they could be srperated by diffrernt drift time(CCS). I hope to calculate the ratio of these isomers according to their LC peak area, not the IMS spectra intensities or area. Could skyline extract the LC area of isomers which separated by ion mobility?

Looking forward to hearing from you!

Thanks,
Anne

view request
XL Peptides IMS library saves different entry per file
(4 responses) Juan C. Rojas E. 2022-09-08

Hi Skyline Team,

Today I noticed that when I try creating an IMS library for XL-peptides with "Use Results" and multiple files a different value is stored for each precursor for each file. This does not happen for "normal" linear peptides.

I know that multiple Ion mobility values might be stored to represent multiple conformers but I am not sure that is what is identifying here. So I am wondering if this was intended to allow for multiple conformer annotations or is it a glitch for only XL-peptides?

Instrument: timsTOF Pro
Skyline-daily version: 22.1.9.208

As always, thanks for your time and the great work!
Sincerely,
Juan C.

view request
System.IndexOutOfRangeException when using GetReport() with raw times/intensities
(1 response) thr26 2022-09-09

Hey, Skyline team,

I've encountered a rather odd error while trying to programmatically access a report. When I try to access a report that contains either the Raw Times or Raw Intensities column using GetReport() the program throws System.IndexOutOfRangeException: 'Index was outside the bounds of the array.'

_toolClient = new SkylineToolClient(args[0], "Skyline Tool")
_report = _toolClient.GetReport("Tool Report");

Including either one of the following lines in my tool's report file causes the error. I can programmatically access any report that doesn't contain these two columns.

<column name="Results!*.Value.Chromatogram.RawData.Times" />
<column name="Results!*.Value.Chromatogram.RawData.Intensities" />

Viewing the report in the document grid works as intended, and it's specifically when the tool calls GetReport() that I reach the error. My only guess as to the cause is that the IReport object being created is trying to store too much information, since each of these arrays are over a thousand elements long. Do you know why this is happening?

I'll also attach the report file in case it's helpful.

Thanks,
– Tommy

 ToolReport.skyr 
view request
Limit on number of runs?
(4 responses) phains 2022-09-08

Hi all,

first off, I love Skyline and I'm a long time user. I really appreciate all the hard work for such a great (free!!) product.

This pertains to the latest version of Skyline with Sciex data.

I am currently using Skyline to assess some DIA runs. I use the term "some" lightly. I've loaded a smidge over 700 runs and they all seem to load okay. But when I go to review the data it is quite clear that I cannot view all the files. If I go to Manage Results, all the files are listed. If I remove a good number then I effectively remove the files I can browse freely. I assume I'm hitting some limit here. If this is the case, can you tell me what that number is, and if not, can you suggest what may be happening?

I, and others in our group, have seen similar issues when loading very large numbers of files in Skyline.

thanks,

Peter

view request
Absolute quant on Skyline: plotting the calibration curve using differently heavy labelled peptides
(1 response) abhinav bioc1213 2022-09-09

Hi Skyline team,

I am struggling to perform absolute quant using Linear fit and 1/x*x weighing using Skyline. And the reason for this is that I am using 1 amino acid (1AA) K/R and 2 amino acids (2AA) (K/R + amino acid after K/R) heavy peptides to establish calibration curve. so I have three peptides os same sequence but three different masses (Endo light, 1AA heavy and 2AA heavy).

I am using 1AA heavy peptide with varying conc. and 2AA heavy peptide as internal standard (fixed conc.) to establish the calibration curve. This is due lack of surrogate matrix for our sample types. I am analysing the sample (unknown) by spiking same conc of 2AA IS and taking the ratio of Light/2AA to calculate the peptide conc. in unknown. All these works I am doing is in excel by exporting the sum transition peak area from Skyline separately and plotting the calibration curve.

The problem on Skyline is:
Skyline is not giving option to select 2AA heavy as IS and taking ratio of 1AA/2AA heavy to plot the curve. How can I modify the setting to achieve 1AA/2AA heavy ratio for calibration curve and Light/2AA ratio for Unknown analysis in Skyline?

Kind regards,

view request
problem with the recent change in the way rdotp is calculated
(8 responses) Antoine 2022-09-01

Hello Skyline team,

The recent change in the way the rdotp is calculated since Skyline v21.2 Released on 1/4/2022 causes us big troubles with data integrity.
We already published papers that used this value as a filter for the identification of the endogenous peptides. We are currently in a process of a publication and we cannot upload the Skyline files on a public repository because the rdotp values are not the same between the manuscript and the skyline file.

Could you at best re-introduce the original rdotp in skyline, or at least provide a way to convert the new values to the old values? This is really problematic for publication.

Thanks

Antoine

view request
Dotp not show up for all molecules in Skyline team
(1 response) ychiu2 2022-09-08

Hello Skyline team,
Thanks for making this awesome software. This has help me a lot!
I have a question about the spectral library and dotp value (in small molecule mode). I have 4 different targets in one spectral library and then I import this library into my skyline document. The spectral librayr was made using the same skyline document but importing the high concentration standard and then export to "spectral library" and then load it back into the skyline document for data analysis. However, dotp only shows up in one of my target. I would like to see dotp show up for all 4 targets. I am not sure how to address the question better, but hopefully with the attached Skyline zip this can let you understand my question and help me.

Thank you

 Sep2022_4MC.sky.zip  Untitled.png 
view request
EIC cut or not shown for some or samples in DIA
(1 response) dennisjakob 2022-09-07

Hi support team,
I am working on an isotope tracing experiment where we used a DIA method on a Bruker Impact II instrument to record the data.
In general I am very satisfied on how skyline handles the data but I have a problem with some ion traces.
For example sample QC-pool_2: I have two Isomers listed in the Molecule List "MGDG". One traces for the second Isomer is shown, for the first it is not. Also, and this occurs for multiple molecules and samples, the ion trace is shorten on both sides. You can also see this in the example of MGDG and sample QC-pool_2.

I uploaded the skyline document with the name 'MS012_share.sky' to the share folder. Attached you find a screenshot from Bruker DA with the EIC of the MGDG and the raw data file of QC-pool_2 in case you need to reimport this particular sample.

I hope you have an idea what is wrong here.

Best,
Dennis

 QC-pool_neg_2.d.zip  QCpool2_1272.PNG 
view request
Writing to a Skyline data file using an external tool
(9 responses) thr26 2022-08-31

Hey, Skyline team,

As the title suggests, I've been developing an external tool (in C#) and have run into a roadblock with implementing some of its functionality. I'm currently trying to write data generated by the tool to the Skyline file itself, data that can be displayed in a column of a custom report in the document grid. However, I can't quite figure out what I need to access in order to assign the data to its intended column/location in the document grid.

I've made the connection from my tool to Skyline with the SkylineToolClient object, and I can read information from it as well as an IReport object using a custom report. The goal of this tool is to read data, generate a flag based on some criteria, and then write the results back into the Skyline file (which are displayed under a column that the custom report specifies). This last part is what I've been having issue with, as the arrays that the IReport objects expose to the user don't seem to have the functionality to update the underlying data file. The values can be easily set by accessing IReport.Cells, however I have indeed realized that the array is not accessing the data itself, only a copy that's presented to the user.

Am I able to have a tool write data to a Skyline file in this way, with it eventually being displayed in the document grid? And if so, what do I need to access to do it?

Thanks for your help, and if I need to provide any more clarification then please let me know.
– Tommy

view request
Replicate Comparison: Replicate Data not Showing up
(3 responses) noaheearls 2022-08-31
I am trying to analyze a dataset with 9 replicates. The Replicate Comparison for the Retention Times and Peak areas only present values for one replicate, though the chromatography shows peaks for the other replicate. Is there a setting or method to attribute the data in the replicates in a way that can be viewed in the replicate comparison? See Attahched for an example peptide:
 Screenshot 2022-08-31 112950.png 
view request
Losing proteins during dda import from orgs other than human
(2 responses) jdemeter 2022-08-31

I am using the 'Import DDA peptide search' wizard to import results from a dataset generated by msfragger/fragpipe. The dataset contains peptides from human and a couple of viruses, with all the detected proteins included in the fasta library that I used during loading of the dataset into skyline. All viral peptides are in the generated spectral library, but none of them - and none of the proteins - made it into the target list, only the human proteins/peptides are there. I am wondering if I overlooked a setting that may have this effect. Any help would be greatly appreciated.

I am using skyline-daily v22.1.9.208.

Thank you,
Janos

view request
Can I visualize my LCMS data acquired with Shimadzu (.lcd files)?
(1 response) dimitris korovesis 2022-09-01

Hello,

I just download the Skyline beta, based on a thread for reading .lcd files so that I can open my LCMS data. These data concern assessing the purity (e.g. in the UV trace) of samples from organic reactions and checking whether small molecules I synthesised in the lab have the expected mass. Is Skyline beta the right software to use for looking at my data?
After installing and running the software, I chose "molecule interface" and clicked on "Blank Document". Then I tried opening my data, either by dragging it in the Skyline or through "Open" but doesn't open it. Probably I'm doing something wrong so any help on how to easily open my data would be greatly appreciated. For convenience in case someone wants to test it, I have attached the lcd file.

Thanks a lot.

Best,
Dimitris

 Test.lcd 
view request
precursor isotopic envelope
(3 responses) bobxiong 2022-08-31

Hi Skyline Team,

In Skyline Proteomics interface, theoretical isotopic precursors with m/z values lower than the monoisotopic precursor were displayed. My understanding is that the monoisotopic species is of the lowest m/z (I could be wrong though as I am not familiar with the various ways of determining the isotopic envelope). I attached a screenshot here. The Skyline zip file was uploaded to https://skyline.ms/files.url (file name monomer.sky.zip).

I was trying to understand this to help interpret a related Skyline analysis in Molecule interface.

Thanks a lot,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
Email address: bobxiong@joinn-lab.com

 screenshot_20220831_bx.pdf 
view request
Failure opening document - modification name error
(3 responses) joshuasmith 2022-08-30
Hi all,

We are doing untargeted PTM discovery and I have a document with a large number of modifications that I have manually entered and integrated. I saved the file and when I tried to reopen it, I got the message I have pasted below, and it won't open. I have just spent the better part of 16 hours manually QC'ing this document, and so I would be really, really disappointed if my file is now unusable. Is there any way to fix this?

Thanks,
Josh

__
---------------------------
Skyline
---------------------------
Failure opening C:\Users\Josh Smith\iCloudDrive\Documents\Projects\Adductomics\SHIELD\PAA_analysis\SHIELD_Pilot_PAA_072622_analysisinprogress_backup.sky.
The file contains an error on line 5833 at column 9.
No modification named Unknown 712.3934 (C) was found in this document.
---------------------------
OK More Info
---------------------------
System.Reflection.TargetInvocationException: There is an error in XML document (5833, 9). ---> System.InvalidOperationException: There is an error in XML document (5833, 9). ---> System.IO.InvalidDataException: No modification named Unknown 712.3934 (C) was found in this document.
   at pwiz.Skyline.Model.DocSettings.TransitionLosses.FromLossProtos(SrmSettings settings, IEnumerable`1 lossProtos) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\DocSettings\Loss.cs:line 388
   at pwiz.Skyline.Model.TransitionDocNode.FromTransitionProto(AnnotationScrubber scrubber, SrmSettings settings, TransitionGroup group, ExplicitMods mods, IsotopeDistInfo isotopeDist, ExplicitTransitionValues pre422ExplicitTransitionValues, CrosslinkBuilder crosslinkBuilder, Transition transitionProto) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\TransitionDocNode.cs:line 719
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionListXml(XmlReader reader, TransitionGroupDocNode nodeGroup, ExplicitMods mods, ExplicitTransitionValues pre422ExplicitTransitionValues) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1457
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupXml(XmlReader reader, Peptide peptide, ExplicitMods mods) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1332
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupListXml(XmlReader reader, Peptide peptide, ExplicitMods mods) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1258
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideXml(XmlReader reader, PeptideGroup group, Boolean isCustomMolecule) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 905
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideListXml(XmlReader reader, PeptideGroup group) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 840
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupXml(XmlReader reader) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 810
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupListXml(XmlReader reader) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 651
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 616
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Model\SrmDocument.cs:line 2074
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass744_0.<OpenFile>b__0(IProgressMonitor progressMonitor) in C:\proj\skyline_21_2\pwiz_tools\Skyline\SkylineFiles.cs:line 328
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Util\Util.cs:line 1941
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_2\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in C:\proj\skyline_21_2\pwiz_tools\Skyline\SkylineFiles.cs:line 345
---------------------------
 SHIELD_Pilot_PAA_072622_analysisinprogress.sky 
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Discrepancy between Skyline concurrent transitions and Thermo Xcalibur software
(2 responses) Erik 2022-08-25

Hi Skyline team,

When using the Retention Times - Scheduling graph I always get a totally different picture of how many transitions are concurrent in skyline compared to when I export that transition list to my Thermo method and make the same plot in this software.
For instance with a 4 min window I get max 100 concurrent transitions in skyline but in the Thermo method software I get 350...

Am I missing something or is this normal?

Greetings,
Erik

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Unable to install Skyline Daily
(2 responses) steven lai 2022-08-25

Hi,

I'm unable to install the latest version of skyline-daily on my computer. I've tried running the setup using "run as administrator" but still get the same error message "Unable to retrieve application files. Files corrupt in deployment". I have also tried re-downloading the setup file, but without success.

This is the information I get in the log:

PLATFORM VERSION INFO
Windows : 10.0.19044.0 (Win32NT)
Common Language Runtime : 4.0.30319.42000
System.Deployment.dll : 4.8.4270.0 built by: NET48REL1LAST_C
clr.dll : 4.8.4515.0 built by: NET48REL1LAST_C
dfdll.dll : 4.8.4270.0 built by: NET48REL1LAST_C
dfshim.dll : 10.0.19041.1 (WinBuild.160101.0800)

SOURCES
Deployment url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application
Server : Apache
Deployment Provider url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application
Application url : https://skyline.gs.washington.edu/software/Skyline-daily13-64/Application Files/Skyline-daily_22_1_9_208/Skyline-daily.exe.manifest
Server : Apache

IDENTITIES
Deployment Identity : Skyline-daily.application, Version=22.1.9.208, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil
Application Identity : Skyline-daily.exe, Version=22.1.9.208, Culture=neutral, PublicKeyToken=e4141a2a22107248, processorArchitecture=msil, type=win32

APPLICATION SUMMARY
* Installable application.

ERROR SUMMARY
Below is a summary of the errors, details of these errors are listed later in the log.
* Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application resulted in exception. Following failure messages were detected:
+ Exception occurred loading manifest from file protobuf-net.dll: the manifest may not be valid or the file could not be opened.
+ File protobuf-net.dll is not a valid Portable Executable (PE) file.
+ PE file does not have enough data.

COMPONENT STORE TRANSACTION FAILURE SUMMARY
No transaction error was detected.

WARNINGS
There were no warnings during this operation.

OPERATION PROGRESS STATUS
* [8/25/2022 3:46:28 PM] : Activation of https://skyline.gs.washington.edu/software/Skyline-daily13-64/Skyline-daily.application has started.
* [8/25/2022 3:46:28 PM] : Processing of deployment manifest has successfully completed.
* [8/25/2022 3:46:28 PM] : Installation of the application has started.
* [8/25/2022 3:46:28 PM] : Processing of application manifest has successfully completed.
* [8/25/2022 3:46:40 PM] : Found compatible runtime version 4.0.30319.
* [8/25/2022 3:46:40 PM] : Request of trust and detection of platform is complete.

ERROR DETAILS
Following errors were detected during this operation.
* [8/25/2022 3:47:31 PM] System.Deployment.Application.InvalidDeploymentException (ManifestLoad)
- Exception occurred loading manifest from file protobuf-net.dll: the manifest may not be valid or the file could not be opened.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.Manifest.AssemblyManifest.ManifestLoadExceptionHelper(Exception exception, String filePath)
at System.Deployment.Application.Manifest.AssemblyManifest.LoadFromInternalManifestFile(String filePath)
at System.Deployment.Application.DownloadManager.ProcessDownloadedFile(Object sender, DownloadEventArgs e)
at System.Deployment.Application.FileDownloader.DownloadModifiedEventHandler.Invoke(Object sender, DownloadEventArgs e)
at System.Deployment.Application.FileDownloader.OnModified()
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
at System.Deployment.Application.SystemNetDownloader.DownloadAllFiles()
at System.Deployment.Application.FileDownloader.Download(SubscriptionState subState, X509Certificate2 clientCertificate)
at System.Deployment.Application.DownloadManager.DownloadDependencies(SubscriptionState subState, AssemblyManifest deployManifest, AssemblyManifest appManifest, Uri sourceUriBase, String targetDirectory, String group, IDownloadNotification notification, DownloadOptions options)
at System.Deployment.Application.ApplicationActivator.DownloadApplication(SubscriptionState subState, ActivationDescription actDesc, Int64 transactionId, TempDirectory& downloadTemp)
at System.Deployment.Application.ApplicationActivator.InstallApplication(SubscriptionState& subState, ActivationDescription actDesc)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivation(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl, Uri& deploymentUri)
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
--- End of stack trace from previous location where exception was thrown ---
at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
at System.Deployment.Application.ApplicationActivator.ActivateDeploymentWorker(Object state)
--- Inner Exception ---
System.IO.IOException
- File protobuf-net.dll is not a valid Portable Executable (PE) file.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.PEStream.ConstructFromFile(String filePath, Boolean partialConstruct)
at System.Deployment.Application.Manifest.AssemblyManifest.LoadFromInternalManifestFile(String filePath)
--- Inner Exception ---
System.IO.IOException
- PE file does not have enough data.
- Source: System.Deployment
- Stack trace:
at System.Deployment.Application.PEStream.PEComponent.ReadData(FileStream file, Int64 position, Type dataType)
at System.Deployment.Application.PEStream.ResourceDirectoryEntry..ctor(FileStream file, Int64 address)
at System.Deployment.Application.PEStream.ResourceDirectory..ctor(ResourceSection resourceSection, FileStream file, Int64 rootResourceAddress, Int64 resourceAddress, Int64 addressDelta, Boolean partialConstruct)
at System.Deployment.Application.PEStream.ResourceSection..ctor(FileStream file, SectionHeader sectionHeader, Boolean partialConstruct)
at System.Deployment.Application.PEStream.ConstructPEImage(FileStream file, Boolean partialConstruct)
at System.Deployment.Application.PEStream.ConstructFromFile(String filePath, Boolean partialConstruct)

COMPONENT STORE TRANSACTION DETAILS
No transaction information is available.

 J851U9WL.log 
view request
Trouble with building a mProphet re-integration model for advanced peak picking
(7 responses) susannah hallal 2022-08-17

Hi,

I hope you're well

I'm having trouble training a mProphet reintegration model that can separate targets from decoys. I have followed the advanced peak picking tutorial, but I am uncertain if there is an error in my Skyline document settings (or my spectral library) that may be contributing to this.

I had set my peptide and transition parameters in Skyline and then imported a spectral library generated by protein pilot (for approx. 8000 human proteins including PepCalMix iRT peptides) and generated decoys peptides (same number as target peptides) using either the shuffle or reverse sequence method. After importing DIA data, I trained a mProphet reintegration model but it does not separate the target peaks from the decoys. Also, I want to integrate peaks with a q-value <0.01, but most of the peaks in the model have large q-values

I noticed that multiple features were greyed out (retention time difference, library intensity dot product, etc) - I deleted the unknown peptides to use these features, but that did not improve the model.

If I integrate peaks with the default method, I get approx 1500-2000 proteins at q-value <0.01. I can identify a similar number of proteins (q-val <0.01) in my samples using the same spectral library with other software. I'm aware that the default method works quite well, however as I have a large target list, I was hoping to use the mProphet model for more accurate peak picking. Any support to understand and rectify this situation would be much appreciated.

Kind regards,
Susannah

view request
"ERROR: No spectra were found for the new library" When making a simple DIA document
(5 responses) ehubb004 2022-08-08

My lab is just starting to collect DIA data on a Lumos and we've run into an issue while testing. After we do a DIA run and attempt to build a Skyline document there is an error (ERROR: No spectra were found for the new library) at the end of the DDA Search step of the import peptide search process. Our assumption is that there is some issue with the instrument's DIA settings, but we can't find an actual issue. I've attached the error message, the instrument settings, FASTA file used, the .raw file (collected on a sample of BSA tryptic digest), and the .mz5 file that Skyline made.

view request
Slow import of Agilent MRM data into skyline
(3 responses) vincentroyrichard 2022-08-19

Hello!

We're having a recent problem where the import of MRM data specifically from our Agilent 6495 system seems to be slower than normal. Specifically data import quickly reaches 99% then hangs for about 15-20 seconds per data file before completing the import. I'm running Skyline Daily 22.1.9.208 on a Windows 10 workstation. Import speeds for equivalent data generated with a Sciex QTrap 6500 is normal. Any assistance would be greatly appreciated.

Thanks very much,
Vincent

view request
Retention Times: Bars from 0 time?
(1 response) alejandro.cohen 2022-08-19

I'm working with the latest Skyline daily on a Targetted PRM on a Fusion Lumos. The Retention Times - Replicate Comparison pane is showing a single data point in the graph instead of the usual start-end time bar information. The Retention Time Peptide Comparison pane (see images attached) shows bars starting form 0 time. This is not as easy to visualize as the start-end of integration boundaries which allows a quick validation of integration process. Was this changed for any reason??

 Screenshot 2022-08-19 110123.png  Screenshot 2022-08-19 105616.png 
view request
CCS Values from Bruker DDA-PASEF data
(1 response) Premy 2022-08-17

Hi there!

I am working with the Bruker timsTOF Pro instrument and have collected DDA-PASEF for a set of lipid standards. After using LipidCreator to create a transition list, replicate data from a dilution series was analyzed.

When adding "Ion Mobility MS1" to the Document Grid, the column shows up as NAN for every row. I'm not sure if I've missed something in the document setup?

Any help would be appreciated and thank you in advance!

 ccs_bruker_ddapasef.PNG 
view request
importing transition only data to scan for PTM diagnostic ions
(2 responses) janelle hancock 2022-08-16

Dear Skyline developers,
Thanks for developing and continuing to update this great tool.

We would like to be able to identify scans in which particular diagnostic ions occur, regardless of the precursor. Our modifications are Lysine linked ubiquitin or Sumo type peptide remnants. The mods are 4-6 amino acids long. We are running an Q exactive HF in standard TopN DDA mode. The modification itself is a peptide, and upon HCD fragmentation produces a predictable pattern on neutral losses (which remain attached to the parent peptide) and released diagnostic b ions. I've attached an example spreadsheet of one such tryptic remnant, from tryptic digest of the human sumo-like protein UFM1.

The spectra for these modified peptides is normally quite complex and we don't get many good matches using PD or maxquant. If possible I would like to use skyline to identify what scans contain these diagnostic b-ions. Once I know that, I can make an inclusion list to re-run the same samples, acquire better quality spectra, enabling us to get and ID and modification site assignment.

Can you please tell me if I can use skyline to analyze transitions only, without considering what precursor they came from?

Thanks!
Janelle

 UFM1_neutral-loss.xlsx 
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MS1 filtering by DDA on FAIMS-Lumos: Rugged Extracted ion chromatograms
(7 responses) alejandro.cohen 2022-08-16

Long story short: We recently got a Lumos with Faims source, and I run our BSA samples we run as QCs using CV of 40, 60 and 80 in DDA mode. I created a skyline document using the Import DDA Peptide Search, using the .raw files and CRAP Fasta file (Contains BSA) and Fragger (I tried Amanda too). I get the expected proteins and peptides, but the XICs appear rugged, which I suspect corresponds to the traces for the different CVs.
I tried the Ion Mobility library correction (as suggested in previous post), but this doesn't seem to fix the issue. Basically, I'd like Skyline to filter the CV with the best signal for each precursor. Could this be done? Attached my skyline file.

Cheers

Alejandro

 BSA_QC_3.sky.zip 
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Chroamtogram information unavailable
(6 responses) ChanniX 2022-08-15

Hi,

I got an error after I imported the MRM results file, "chromatogram information unavailable" for only some of the specific heavy labelled peptides of all replicates, all light peptides and other heavy peptides are fine. I've attached the screenshot, could you please let me what to do to fix it?

Thanks and best wishes,
Channing

 Screenshot 2022-08-15 101957.png  Screenshot 2022-08-15 102019.png 
view request
N-Glycat Library
(1 response) maryam imaninejad 2022-08-15

Hello Skyline Team,

We started using InstantPC to label released mAb N-glycans to study and analyze N-glycans. For this purpose, a Waters QTof system and an MSe method were used. We could analyze our data using Skyline small molecule interface. However, this analysis was based on the precursor ion and its multicharges. As you know, some of the glycans have the same masses and using precursor ions will not let to differentiate these glycans. We are interested in using MSe and MS/MS data to correctly identify N-glycans. It seems that N-glycat library is the one that can help for this purpose. I could not find this library in your website. Not sure if it actually exists. If not, can you help us know how to use MSe data for correct ID? Is there any other library that can use MS1 and MS2 data to assign detected N-glycans? Most of the N-glycans that we are trying to identify is neutral or monosialylated.

Thank you,
Mary

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