Hi,
I am using EncyclopeDIA wizard to create chromatogram library and analyse my wide window datasets, however when I attempt to do it I am getting a Percolator error. Please see the attached log for details. I would appreciate if someone could let me know if there is anything on my side I can do to fix this.

Thank you.

Kind regards,

Michal

Hi All,

I have acquired my PRM data and using Skyline for computing my Fold change using MS stats.

I have 2 peptides corresponding to one protein and would like to know the Fold change at the protein level.
I am getting a foldchange at peptide level. I would like to know how can i get it right at protein level. Do i have to do any offline calculation for protein level?

I followed the Tutorial 20_1, however, my data is displayed in Fold change of each peptide with Adjusted P-Values.

I am ready to share my Skyline document too. Could you please provide me with some ideas how to do so. Than would be very helpful

Thank you.
Varsha

Dear Skyline team,
Thanks for an amazing tool and support.

I'm trying to set up a library for DDA with MS1 filtering, which works perfectly fine for many of my samples. However, one specific sample consistently gives an error (attached) while importing the pepxml with the Skyline wizard. I have both pepxml and .raw files in the same folder. I've also tried to use .msf file instead of pepxml: I can build a library if Score threshold=1, which is rather useless, but not when Score threshold=0.05 (same error message as attached).

I've used Proteome Discoverer 1.4 for my pepxml and .msf files.

Can you please help?

Many thanks
Adnan

Dear Skyline Team,
I would like to use Skyline to analyze immunopeptidomics DDA, DIA and directDIA (DIA-Umpire) data. I'm using the wizard to do it but I'm stuck at the creation and importation of fastA file. We usually use "unspecific enzyme digestion" for normal Discovery approach and I don't know how to do it especially for directDIA (DIA-Umpire).
Your help to overcome this would be great.

Thanks in advance for your help

Best

Hui Song

Hello,

I have acquired Full Scan MS2 data from Orbitrap Exploris 240. I wanted to know if Skyline is able to identify product ions based. I see green dots next to the precursors ion indicating that the precursors are present however all b,y,a ions are red. Is Skyline able to detect data only in MRM format or all MS/MS data?

Dear Nick, Brendan and/or colleagues from Skyline support,

To satisfy a prerequisite of a journal, reviewer and to attempt to comply with FAIR data principles, we are trying to upload MRM3 data to Panorama. I have previously asked for help to upload the data making a method, we have found a work-around and have succesfully uploaded data to Skyline in a method.

Unfortunately, a substantial part of the data-files failed to upload, while others do. Uploading the 'faulty' data provides this error message:

At 10:05:
Failed importing results file 'C:\Users\Hyung\Surfdrive\Projects\2020\P20-0001_COVID-19\Data\20240425_Exp134_RepositoryUpload\220503_Exp111_batch02\renamed_files\220503_019_MRM3_30089A1_10uL.wiff2'.
[WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\Hyung\Surfdrive\Projects\2020\P20-0001_COVID-19\Data\20240425_Exp134_RepositoryUpload\220503_Exp111_batch02\renamed_files\220503_019_MRM3_30089A1_10uL.wiff2'.
[WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object. ---> System.Exception: [WiffFile2Impl::getInstrumentSerialNumber()] Object reference not set to an instance of an object.
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\pwiz\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 200
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 295
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 191
--- End of inner exception stack trace ---

It seems to me that the built-in msconvert module is throwing an error, pertaining the serial number of the MS. I checked the original Sciex data file that fails in Sciex OS (version 2.1.6.59781), and it does not seem to be corrupted, at least it opens normally. The MS data and metadata (serial number in the 'sample info') are also retrievable using Sciex OS (explorer module).

I also tried to convert the data in MS convert (version: 3.0.24094-d2966db), but I am not able to execute it properly. It also returns an error ([WiffFile2Impl::setSample()] Value cannot be null.). I think my colleague has previously reported this. I also tried to look in previous reported Skyline issues if I could find an answer to my question, but I was unable to find a solution for this problem.

Back to the Skyline upload: Is there a solution to upload all of our files?

Best regards,

Hyung Elfrink
Leiden University, Metabolomics and Analytics Centre.

P.S. I have attached my method file and I can share the data files per FTP.
P.P.S This is a link to the data that succeeded to upload, to Skyline and subsequently Panorama: https://panoramaweb.org/Leiden University - MAC/COVID-19 Mass Spectrometric MRM3 repository/targetedms-showPrecursorList.view?id=204275

Hello, I trying insert a transition list with M+H-H2O as adduct, but i receive the error message " calls for removing more O atoms than are found in the molecule" . What can be wrong in my list?
Many thanks

Hi,

I have a question about how Skyline is pulling transition data from Thermo .raw files and I'm going to try and attempt what I mean but I can provide a skyline file to show what I mean. Some background I'm putting in Full scan ddMS2 data from an Exploris 120.

The issue we are seeing is for compounds that we have only a 2 m/z difference between the native and IS. For example, Native mass is 712.9437 and the IS is 714.9540. I've attached a picture for now to show what I mean. We have an injection for a blank and we see now peak for the native mass, but we see fragments appearing. We know the native and IS can produce similar fragments because of the low mass labeling.

So the question I have is Skyline just pulling an ion channel for transition and that means if anything produces that fragment mass at that time then it would be captured and shown? This would explain why we see fragments from the IS when there isn't any of the native present in the blank to produce those ions.

I've checked the same data in Thermo TraceFinder. When we look at the blanks there is no peak for the native and there is not fragments showing up.

Hi,
Is there anyway to export the Candidate Peaks for all the metabolites/peptides. I have tried View -> Other Grids ->Candidate Peaks. But that gives me only a single metabolite. Is there a way to it for all.

Thank you so much!!

Dear Skyline team,

I am having a problem when importing raw files acquired on an Ascend instrument using the SureQuant method (user designed peptides).
I am following the "SureQuant tutorial 2" instructions: https://skyline.ms/wiki/home/software/Skyline/download.view?entityId=14124b80-46d4-1038-b053-e465a393788a&name=Tutorial-2-tuesday-surequant.pdf

My library (build in MQ) works very well and all my heavy peptides are in the library. I can see a very good MS1 signal for them as well as a Library match and the Peptide ID times (see picture attached). Unfortunately NO MS2/product signals, even though I can see them in the Library and listed in the "rank".

I tried importing several times the raw as well as mzML (after conversion with Proteowizard), no success.
I tried several parameters in the MS/MS filtering option (SureQuant, DDA, PRM, centroid, etc...) but NO success, after re-importing the files each time.

I am sharing a very partial version of the Skyline document with just 2 peptides and 3 mzML files, hoping you can help me.
I used "profile" for MS/MS spectra as specified in the Thermo SureQuant template, can this be the issue? should I have acquired the samples in "centroid" mode? this is the only option left that comes in my mind...

I really hope you will be able to help me with this issue since I was really looking forward to set several SureQuant experiments.

Best
Michele

I would like to synchonyse zooming for all the plots in my skyline analysis, but synchronisation works for only a subset of plots, while other chromatogrammes remain unsynchronysed (full RT range is shown). You can see the example in the attached presentation or in the skyline files submitted in a zip archive.

Hello Dr. MacCoss,

I requested skyline-daily yesterday. I was wondering what the criteria are to be eligible to use skyline-daily. I recently do extensive MRM-MS with lipids. Hence, it's crucial for me to to be able to use skyline-daily. I will appreciate if I can have this software.

My email address: 1097064656@qq.com

Thank you.

Best Regards,
Miao Naixin

I would like to synchonyse zooming for all the plots in my skyline analysis, but synchronisation works for only a subset of plots, while other chromatogrammes remain unsynchronysed (full RT range is shown). You can see the example in the attached presentation

Hi All,

I have posted the below message on the Panorama support page, but Josh has got back to me and suggested posting to Skyline support instead, with an example of a raw data file, as he thinks its an issue with importing the pressure trace in Skyline.

As an extra piece of information, the data is acquired from an Acquity UPLC (sample manager, solvent manager and column manager) attached to a Xevo ToF instrument. Acquisition computer was running Windows 7, with Masslynx 4.1.

Thanks,
David

Original post to Panorama support:
Hi all,
I am new to Skyline / Panorama (it was recommended by a collaborator), but ultimately the goal is to use it for monitoring system suitability data on a variety of instruments, mainly Waters QToF instruments, but eventually our triple quad, and maybe UV instruments too if it is possible to get that to work, but it is not high priority at the moment.
So far I have just been using our Xevo ToF instrument to acquire some test SST data (we work with small molecules), and have used Skyline (in molecules configuration) on the instrument computer to get a quick overview of the data, and then Panorama to have a more detailed look once the data is backed up to a server and can be accessed online. In both cases I have been using AutoQC to import the data, to make it as automated as possible.
I have to say I have been quite impressed with what I have seen so far, and think this could be a really useful solution for us to take our SST use to another level, and make it more proactive rather than being used for troubleshooting when things look wrong....
However, I was really hoping that I would be able to also use the column backpressure as one of my SST criteria, and it seems that I should be able to do that from what I have read. Unfortunately, when I follow the instructions how to add a new or custom QC parameter, Panorama says that there are no traces found. If I look in Masslynx I can clearly see the pressure traces, so the UPLC SST method is correctly recording the pressure (and some other parameters) as I asked it to, but this doesn't seem be getting into the Skyline or Panorama files....
Is there some trick or setting somewhere that I am missing so that the pressure traces are also loaded? Or is there a known problem with importing this data from a Waters .raw file?
Any suggestions would be gratefully received... I feel I am probably doing something wrong, but I have no idea what it is....!
Thanks,
David

Has anyone experienced this error before? I saw another post regarding this and someone mentioned to update the visual C++ version. The current version installed on my computer is correct and I still see this error.

Thank you in advance.

Alright, at 5am this morning my runs stopped because my C drive was low on memory. The run that was in process was at min 9 of 11.5, and all my target windows were within that. Obviously it's a bit of a bum file since it got stopped prematurely, but all the data I need are theoretically in it . Anyway to force it into skyline? Maybe if I convert to a non .raw format first?

Or I can just re-run the sample, but would be interested to know! Happy to share said file if it would be helpful.

Thanks!

Hello,

I'm interested in learning the sequence and modifications necessary to achieve the correct m/z for semaglutide ((MWT 4114)? Please let me know

Dear Skyline

I have been experimenting with small molecule data import into skyline daily 23.1.1.520 from a waters HDMSe experiment. I have m/z, charge state, RT and ion mobility information, but not molecule identifications. I would like to have multiple precursor ions imported. Although I appreciate skyline can't calculate isotope dot product with no formula I thought it would be able to import [M+1], [M+2] etc signals. Tweeking the transition settings for filter and Full-scan back and forward is not triggering these precursor inclusions on the list. Is it possible to set these precursor transitions up at all?

I am trying to setup system suitability monitoring as per https://panoramaweb.org/home/wiki-page.view?name=autoqc_setup_tutorial.
However, when I import search result to the skyline, it only shows precursor chromatogram but no product ion. The experiment was run through Orbitrap Exploris 480 with Full MS + target MS2 method. I am not sure if I done the setting wrong on Skyline side or method setup side. Please see my skyline document and method attached below.

Regards,
Kittinun

Hi guys,

I am trying to export all the chromatograms of the species detected with a contaminants template published by Rardin (https://link.springer.com/article/10.1007/s13361-018-1940-z) in a RAW sample acquired in full scan.
However, when I "Right Click --> Copy Data" on the chromatogram and I have only 100 intensities while the documenport the group name (i.e., protein.name) TIC for each contaminants - would it be possible? I ts contains more than 700 of "MS1 filtering". I would like to click on "Export --> Chromatogram --> Precursor" but I do not see "Retention Time" and "Intensity" column.

Many thanks and best regards,

Nesgio

Dear support team,

We are trying to build a spectral library from Bruker TimsTOF data, analyzed and exported as mzXML in PEAKS studio 11.

When adding data to a new spectral library in Skyline, the same error happens every time the data is being read from the mzXML file.
I attached a compressed folder where you can find the pep file, the exported mzXML file as well as a text document with the entire error code log.

Note that the data is from run 142 and the error code is from loading data from run 141, though the data is practically identical and the same issue occurs with both.

We are running Skyline (V23.1.1.520) as well as PEAKS Studio 11 on windows10.

Thank you in advance,

Julian Matytchak
Lab technician at the Temmerman lab
KU Leuven
Belgium

Dear Skyline support,

I have managed to do this before, but now I am again running into issues when importing MyriMatch search results into Skyline. I am trying to import these peptide search results to build a library in the "peptide settings -> library -> build" menu. However, Skyline does not recognize the files (MyriMatch results parsed through Peptide Prophet - this has, however, worked before). It would be great if you could please have a look at the attached example file here and let me know what is wrong with it or why Skyline can't load it.

In general, we have previously managed to use .mzXML and files like the attached one to build libraries with the DDA with MS1 filtering workflow.
It is my understanding that I load the raw files (.mzXML) using "Import Peptide Search, build spectral library, start from DDA raw (search and build library)" and the peptide search results (pep.XML) under "peptide settings -> library -> build"? Is this correct or am I completely off?

Skyline recognizes both the .mzXML files as well as thermo .raw files when I use the "Import Peptide Search, build spectral library, start from DDA raw (search and build library)" but after the percolator it always says "Search failed". Do you know why that is? I attach the corrsponding mzXML file, too.

I would really appreciate your help on this!

Many thanks,
Claudia

Hello,

I wonder how to simply export the chromatograms with the Skyline CLI using the sky and skyd files without raw files.

I could successfully export the chromatograms only using the skyline files without raw files in the Skyline GUI version. In the directory, there are sky, skyd, skyl, and sky.view files. But no raw files.

I tried the following command but it didn't work. I might need to specify the replicate file names to export the chromatograms from though.

SkylineRunner.exe --in=test.sky --chromatogram-file=test_xic.tsv --chromatogram-precursors -chromatogram-products --chromatogram-base-peaks --chromatogram-tics

Thank you in advance.

Hi Skylien team,

In our lab (Markus Aebi Lab, ETH Zürich) we have two issues with Skyline files as they are based on raw data that apparently have to be in the same storage location (path) as the skyline file...
Issue/question 1) If I change the storage path of my raw data from a location to another after having loaded the skyline files, I cannot open the skyline files again anymore, as the raw data are not found. Is there a way to get around it? like changing manually in the skyline file the "path" of the raw data before opening them again?
Issue/question 2) As we are running into storage problems (because of the huge amount of raw data), we wondered whether skyline read zipped raw data?

Thank you very much for your help, hope I could formulate the questions clear enough...
All the best,
Ilaria Affolter

Dear Skyline Team,

This is Regine from the Paulovich lab with another question about CE optimization. As part of our CE optimization process, we do a preliminary CE optimization run using a 3V step size. We then want to do a refinement run with a 1V step size. In the Transition Settings, we checked the “Use optimization values when present” box and we have “Optimize by: Transitions” chosen. However, we noticed that the csv file that we export for the refinement run does not have the center CE value corresponding to the best CE value that was found in the 3V data for each transition. Instead, the resulting csv has the same center value defined for each transition, despite the fact that the center value is quite different for some of the transitions. How can we force it to use the previous, 3V data to choose a best CE value as the center CE value for the 1V step size optimization? (We've been manually adjusting the csv files so far before running our refinement mass spec run.)

I'm attaching a couple of files that show this problem.

Thanks very much in advance for your help!

~Regine

Hi Skyline Team,

When building a library from a .pdResult file, I have noticed a discrepancy between the number of “high confidence” peptides found in PD and the number of peptides imported in. When using a Percolator q-value of 0.01 or 0.05, the number of peptides imported into library is often a fraction of the “high confidence” peptides found by CHIMERYS. I am using Skyline daily 23.1.1.520 and Proteome Discoverer 3.1 with CHIMERYS searches primarily. I have attached a few examples. I would be happy to provide additional info or upload the .pdResult files. Do you mind taking a look at this?

Thank you,

Wes

Test runs using Thermo 240 were able to successfully import into Skyline and peaks were observed.
When importing new data using same instrument with minor modified data starting from 14th June Skyline can import the data but won't show any results.
I cannot confirm if it's a method issue from Mass analysis or else.

Hello,

I need to provide information to my institutions IT services in order to download Skyline.

Please may you provide me with the following information:

1. Software terms and conditions URL
2. Software privacy policy URL
3. Software accessibility information URL
4. Cyber Security information - for example Cyber Essentials or ISO27001/ISO27002 series and if these do not exist then a copy of the application development history,

Thank you,
Nicole

Hi,

Could anyone tell us how to export dot p value after compare library std spectrum vs midas data. We can find this value which present in spectra match (see attached). But can't find a way to export these values.

Thanks,
Heyi

When the measurement file (.lcd) obtained from the Lab solution was imported into skyline, peaks were detected only for some transitions (with green marks), but not for others.
However, on the lab solution, all transitions showed peaks.
Any solutions would be appreciated.

The version of the software : 64-bit Skyline 23.1
Measurement and analysis method : proteomics data

Below is a screenshot of the skyline screen.

Hello,

I'm trying to build a new library in Skyline using a DIA-NN predicted library output. I get the error, saying Bibliospec supports up to version 3, but my library file is version 8. How do I correct this issue? Thank you.

Good morning,

this is Regine in the Paulovich lab. We have been optimizing CEs for numerous peptides using a 6500 Qtrap and have seen that not all peak area measurements for the different voltage steps are being shown for some transitions on the Skyline Replicate plots, when viewed using the Single Transitions setting. We're attaching a file that shows this issue for the GTELWER peptide, for the light y5+ transition. Only 4 measurements are shown, whereas for the other transitions, all the voltage step measurements are shown. We've checked the raw data using the Qtrap's Analyst software, and the data are there, they are just not showing up in Skyline.
Would you please look into this issue so that all measurements show up in the Replicate plots? Thank you very much in advance!

Best,
Regine

I have all amino acid labelled in peptide VINKQTPNRQIW with 13C and 15N. When I used Peptide Settings, modifications, N and P have the same mass, and can’t distinguish one to another in skyline and skyline doesn’t pick up the signals.

I have identified several K-acetylated peptide sequences in the Fragpipe analysis of my data. I would like to see the peak groups and spectra of these peptide sequences in Skyline.

The peptide segment acetylated on the K-terminal is shown below：
ALAAGGYDVEK[42.0106]NNSR
GK[42.0106]YYAVNYPLR
IEGEGSVLQAK[42.0106]LK
ISIC[57.0215]SSDK[42.0106]R
NGLSLAALKK[42.0106]
NGLSLAALK[42.0106]K
n[42.0106]SETAPAETATPAPVEK[42.0106]SPAK

I insert the peptides as below:
ALAAGGYDVEKNNSR
GKYYAVNYPLR
IEGEGSVLQAKLK
ISICSSDKR
NGLSLAALKK
SETAPAETATPAPVEKSPAK

And I also set modifications in peptide setting as attached figure 1. However, the peptide in the document failed to display the acetylation marks, as illustrated in attached figure 2.
I don't know where the operation went wrong.
I look forward to your reply.
Best wishes.

Dear Skyline Team,

I hope you've had a great ASMS. I've been working on my reports, and noticed that when I import a transition list, only one form of note is available, "Note". However, when I try to export the same note from the document, there are four different note types (transition, precursor, molecule, molecule list). Skyline seems to default that "Note" in the transition list import is a "Transition Note", and this cannot be changed in the transition list import stage. This becomes problematic when exporting MS1 data with isotopes because only the monoisotopic line in the export features the imported note.

Could the transition list import feature please be updated to include all existing note forms? I would like to say that a note is a precursor note, ensuring the note is specified across different isotopes coming from the same precursor.

Cheers,
Chris

Dear Skyline team,

When having two modifications in the same Skyline document, and we choose to see modifications as mass difference, the mass difference will be the same for modifications of masses when rounded to first decimal digit will identical.

Example 1:

Formyl (K) [+27.994915] and Dimethyl (K) [+28.0313] will both appear as [+28] when choosing: View > Modifications > Mass Difference.

Example 2:
The same applies to trimethyl (K) [ +42.04695] and acetyl (K) [+42.010565] .

If I want to export 'Peptides' report from the Document Grid in the 'Peptide Modified Sequence' column, both can be seen as the same peptide if the 3-letter format was not chosen.

Could you please keep the rounding to the 2nd decimal digit?

Thank you,
Hassan

Good afternoon,

I am writing you because I cannot view the chromatograms after importing the files.

The message that i see is: Chromatogram information unavailable.

I tried to change in settings -> Transition list -> instrument -> Method match tolerance m/z ... but it didn' t work.

I also tried in edit -> modify molecule -> Explicit values -> RT window ... but it didn't work

I am using Skyline (64-bit) 23.1.0.268 (e59d0dc56)

Waiting for your answer,

Thank you in advance,

Luz Alonso Dasques.

Hi,
We have Skyline oven mod.: ECOE61T2D0.
A few times coming error code 203-CPUA: ACS IC broken on ACU.

Could you pls give advice, what is problem?
The unit is working only 0ne year.

I’m running a PRM of only one long peptide (~34 residues) with thermo orbitrap 480. Although pairs of ion fragment under charge+6 precusor, it's too difficult to get a high quality of PRM result as following attachment. Because there is almost no any signals about +2/+3/+4 precusors, I only choose the highest signals of +6 precursor which also have only some y ions detected. How could I improve my PRM method about this long peptide?

Hi everyone,
I have just started using Skyline after finishing the fantastic online introductory and small molecules course (2 days each).
Working in the forensic toxicology area dealing with small molecule quantification, I gained experience with SCIEX Analyst and Agilent Masshunter.

So here is my question: Is it possible to assign the regression weighting for the calibration curve for each analyte individually (e.g. amphetamine 1/x, methamphetamine 1/(x*x), ketamine none)? This is a common thing in forensic toxicology and would be crucial for a switch e.g. from SCIEX Analyst to your amazing software.

Thank you for your help in advance
Hannes

Hallo Skyline Team,
I recently started using Skyline to investigate my MIDAS runs (MRM triggers MSMS) . I started with simple old experiments and want to change over to complex naturally crosslinked proteins. However already in the simple runs I find discrepancies between Skyline and my Analyst software. I guess some of my parameters are not set appropriately. However after 4h of tutorial reading and testing I am still stuck with the missing MRMs. I attached a document with the screen shots of Analyst and Skyline and I also attached the Skyline experiment.

Thanks in advance for your time and help.
Kind regards

Edda von Roepenack-Lahaye

Hello there,

I am a research assistant who is attempting to find a way to streamline our data analysis and peak integration process. I came across skyline recently, after having used MassHunter for a while, and was intrigued by the Deep MRM tool. However, when I attempt to use it, I am met with a server error (specifically, an internal server error: code 500). I was wondering if anyone might have any insight on how to fix this. Thank you in advance!

Dear Skyline team,

Hope you're doing well. I'm excited by the new feature finding feature but have some hopefully helpful feedback regarding its use. Specifically, I work with negative mode small molecule data, so this may just be an unintended use case.

Skyline assumes the feature finding will return protonated molecules, so it returns the chemical formulae with M+H adducts, despite Hardklor feature finding being performed on a negative mode only raw file. This then causes an issue when it tries to import the raw file as only positive mode transitions are present in the transition list. When I manually modify the adducts and chemical formula to reflect negative mode, the correct molecules are observed and extracted. So Hardklor/Bullseye might be working fine, and only changes to the transition list processing may be needed.

If small molecule use is unintended, it might be helpful to exclude it from the small molecule mode view.

Happy to provide all the associated files if desired.

Cheers,
Chris

Dear Skyline support,

I am running a targeted, scheduled lipidomics (900 total transitions between + and - mode) MRM on a SCIEX 6500+ Qtrap, each batch consisting of ~80 total runs. A handful of those runs are standards; I am using a splashmix which is commercially available and contains ~14 deuterated lipids, 1 per class. On 2 separate batches, I have encountered this error only when importing standard runs - my sample runs have all imported with 100% success rate. My previous batch, I ran 3 cal curves consisting of 7 points each, and 1 of those runs failed to import (1/21 = 5% failure rate). On my latest batch, I ran my highest point 4 times throughout, and 1 of those runs resulted in that error (25% failure). When I use Analyst to view their TICs, the expected peaks are present at the same RTs and intensities of those which successfully imported, so I know data was collected, but it's just not importing. So it seems to be intermittent and it's unclear why.

Any idea what's going on and how I could resolve this?

Thanks,
George

Hi, I'm unable to add any modifications to existing peptides in the target list. When I right-click the peptide and "Modify...", there is no change to the peptide or m/z values below. When I create a copy with a new modification, it just adds the exact same peptide below. Also when I right-click and select "Pick children", I get the "(Not responding)" error and the software crashes.

This used to work perfectly fine. I've been using Skyline-daily 23.1.1.459, and it was working fine last week. I updated to 23.1.1.503 and it still doesn't work. I also uninstalled and reinstalled, but that didn't work either.

I've been using Skyline for a long time, and have never encountered any issues like this.

Thank you for your help!

Hi,
Recently updated to the latest daily version of Skyline (23.1.1.353).
I tried exporting a scheduled transition list from the attached document using SCIEX settings.
The values for retention time were not exported (i.e., zero in all rows).
It works if the document is shared using a previous version (23.1).

Maybe I missed something? Many thanks for your help tracking this down.
Best,
Jeff

Good afternoon, my name is Luz Alonso

I am writing to you because I have the following problem with Skyline (64-bit) version 23.1.0.268 (e59d0dc56).

The baseline of my chromatogram, instead of being fixed at zero, starts at another point on the y-axis.

Therefore this influences my peak integration process due to it not only integrates the peak but also from the y-axis point to zero.

I attach a picture of my problem.

Looking forward to your early response

Best,

Luz.

Dear Skyline,

I currently would like to analysis the disulfide bond pattern for one single protein.
I originally though if i added disulfide bond as crosslinker as indicate in (https://skyline.ms/wiki/home/software/Skyline/download.view?entityId=ac4c6cc4-97c6-1038-8aae-e465a393ee52&name=NicholasShulman2020ASMSPoster.pdf) then all possible linkage of disulfide bond within the same protein will be listed. It turned out I have to add the linked peptide one by one. Since it have only 2 Cys in the protein, the first segment contain 3 variable modification which generate a total of 8 possible peptide, where second segment contain no modification. Considering these two cysteine containing peptide, it will have 88 (1st segment disulfide link to 1st segment) plus 18 (1st segment disulfide link to 2nd segment) plus 1 (2nd segment disulfide link to 2nd segment), 73 possible disulfide peptide is generated.(assume no misscleavage). 73 is still reasonable for me to add it one by one.
I have few question.

1. Did i miss some feature of skyline that do the above automatically instead of adding one by one?
2. If I do not miss feature of skyline, would this feature be added? As if No. of cysteine and no. of modification of cysteine containing peptide
   increase, then the manual addition of crosslink peptide seem to be impossible.
   Really appreciate and looking forward for your response.

Best regards,
Sam

Is there a way to filter peptide IDs using a Koina dotp filter (comparing the Koina spectrum prediction to the spectral library spectrum as a 'refinement')?

Hi, Skyline teams!
My LC-MS data is glycome from mice liver tissue. After I import my transition list (29 glycan precursors in MS1, and 21 fragment under each precursors in MS2) and raw data, there's nothing showing up in the window. There's nothing in chromatography, peak area graph and retention time graph. Please see the screenshot in the word file I attached.
I was guessing this issue might be due to too much glycans in transition list, so I deleted 4 glycan precursors in MS1, 25 glycan precursors left, and start with a new skyline file with the same setting and importing steps. Then, it worked, and everything showed up.
However, I think I still need to figure out the solution, and want to know how can it work by using my originally complete transition list.

We would like to draw a calibrarion curve for Adenin, and this is where we were stuck.
We aligned the peaks for the molecule and the peak area shown below seems appropriate for the calibration curve but it's not shown.
It's also same for other molecules that we are targeting.
We'd appreciate if we can get some guide for this problem.
Thank you so much.

Greeting

When trying to import data from my last experiment, I get this error for all my samples (not blanks or Hela QC) of this experiment:

Failed importing results file 'C:\Users\PPC-P\Documents\1M_REF_F3_1.5µl_S4-C1_1_12079.d'.
[ReaderFail] [Reader_Bruker::read()] Bruker API does not support Unicode in filepaths ('C:\Users\PPC-P\Documents\1M_REF_F3_1.5µl_S4-C1_1_12079.d')

My data is DDA with ion mobility (timsTOF HT). My skyline file and sample data are saved in the same folder.
Does anyone have experience with this error? Could it be that my data is corrupted?

Best regards,
Jana Kindermans

So this will probably go to Nick,

So disulfide homodimeric peptides load beautifully through the modify peptide interface. And crosslinks between different peptides can also take other chemical modifications in the insert peptide interface. On a mAb there is this sequence:

THTCPPCPAPELLGGPSVFLFPPKPK

And in evaluating it's disulfide integrity, this peptide

THTCPPC[+57]PAPELLGGPSVFLFPPKPK-THTCPPC[+57]PAPELLGGPSVFLFPPKPK-[DSS@4,4]

would be useful but I get the error that the peptide is not recognized by the software. I tried adding another copy of the sequence but there is no field for specifying different proteins in the same peptide entry on the insert peptide field. Also the insert peptide field does not seem to support multiple crosslinks between two peptides which does happen with disulfides.

Is there a way to do this that I'm missing? If it's too specific an issue, that's fine. But I thought that since there are a fair number of disulfide oligomers, it might affect others too.

Best,
Lance

Hi,

I am currently working on a project that involves analyzing data. I am interested in the area under the curve for a product of interest. Could you please guide where to find the area under the curve for a particular precursor and one product in Skyline. I am using Skyline 21.01.0.278 for reference.
In this particular project, I am using the Chromatogram Precursor M/Z Chromatogram Product M/Z respectively, 730 and 660.25
I am sending you one skyline file so that you can see what kind of analysis we are doing.

Thank you for your attention
-Nitha

Hi Skyline team,

I often have this issue but not sure where it's coming from as I download the same SRM list to Skyline.
It shows "No chromatogram available" for some of the compounds, showing as example "aconitic acid" and noticed that on the Document Grid, the "Average Measured RT" is #N/A for this compounds and was wondering what's the issue and how to fix it. (Images attached)
Thanks a lot

Nihel

Peak intensity (height) appears in the document grid as a different value from the actual obtained data. I would like to know the reason for this.

Hi,

In few files peak peaking is not working. We manually look at the TIC and check the data file in mass hunter qualitative analysis, it looks ok. But in skyline processing those file does not show any peaks. In the attached skyline document , the file name "Pos_CC6" where peak peaking is not working.

Hi,

In few files peak peaking is not working. We manually look at the TIC and check the data file in mass hunter qualitative analysis, it looks ok. But in skyline processing those file does not show any peaks. In the attached skyline document , the file name "Pos_CC6" where peak peaking is not working.

Hey Skyline team,

I would like to add to my list of metabolites an internal ID (not an HMDB, kegg.. IDs) and would like to be able to export it with the data at the end of the processing.
I tried to use HMDB ID column but as it is not in the same format it shows an error message. I also tried to use Molecule or Molecule Note, but when exporting the data it does not export that info on the molecule.

Is there a way to enter internal IDs in addition to the molecule name and be able to export them at the end of the processing?
Thank you,

Nihel Bekhti.

Hello,

I am having an issue with connecting to Prosit server through Skyline (version 23.1.0.455) - see the attached snip for details. I have used the test-netconnection command in Windows PowerShell as recommended by Brendan in one of the previous posts and it looks like my computer is able to connect to the server using port 8500. Any suggestions on how this could be fixed are appreciated.

Kind regards,

Michal

I am aware of this page

https://skyline.ms/wiki/home/software/Skyline/page.view?name=adduct_descriptions

and it is helpful.

I have this question, if I know the structure of my fragment ion and it is doubly positively charged with a carbocation at one site and a proton at the other,
what is the notation?

do I use [M+2]
and add a Hydrogen to the fragment molecular formula?

Dear Skyline Team,

I would like to import a spectral library generated from DIANN into Skyline. The library features some customized non-unimod modifications as well as UniMod:4 (carbamidomethylation on cysteine) as a variable modification. Whenever I try to import via "Import DIA Peptide Search" I receive an error Message (attachement). I assume Skyline does not recognize the modification "Unimod:4", probably due to misspelling, and, hence, will not recognize my customized non-unimod modifications anyways. Is there a way to add such modifications to Skyline prior to import?

I also tried to import the speclib without any variable, customized and non-unimod modifications (and UniMod:4 spelled correctly) which was successful.

Best,

Gianin

Hi Skyline team, I used to be a Skyline protein mode user but now switched to GC small molecule omics analysis. Current software I am using can only give me a laundry list of RT with precursor mass without any compound name information. I can import transition list and results file to Skyline but cannot annotate these peaks from RT. So I was trying to figure out how can I actually annotate this peaks using Skyline then output a list of compound peak area list.

So I was thinking to use NIST GC-MS library but clearly I have difficult time find the right one (my compounds are all volatile compounds with <350 m/z, but all library I can find is peptide size). So I was wondering can Skyline annotate those undefined Peaks from GC-MS (Agilent)? If yes, how can we actually do it using Skyline? Thank you very much!

Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan
Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan
Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan
Hello
I downloadsed Honeybee from the Website (PeptideAtlas - Bulk Downloads)(Database Tables:XML) for building BiblioSpec library last time.This is not the correct download path,is it?
When I want to download spectral libraries from Peptide Atlas(Website:PeptideAtlas - Spectrum Libraries),I don not find Honeybee(honey). I can not download spectral libraries about honeybee,is it?
Now, I wonder the way how to bulid the BiblioSpec library.
Thanks,
Zhaoxuanxuan

Hi!

We use the [M-1] to qualify potential interferences and correct species identification for peptides and was wondering if anyone is able to help me enable the precursor [M-1] chromatogram trace for all precursors in a document by default. I've tried manually enabling the option for each precursor, but this is impractical to do for large analyses.

Thanks in advance!

Cheers,
Sean

I imported 32 interact.pep files from FragPipe closed search output, 32 mzML files, and 1% FDR filtered FASTA generated by FragPipe with 2 missed cleavages into Skyline-daily. After MS1 filtering is completed, Skyline does not respond as soon as I click anything. Checking my PC memory and CPU performance, it does not look like they are the reason. Do you have any suggestions to fix this?

I am just learning to use it and when I wanted to learn about MS1 filtering, I found 2 ways to get the tutorial.

1. I went to https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ms1_filtering_zh, the download link opened a new tab called MS1Filtering-20_1_zh-CHS, the download file was actually MS1Filtering-22_2_zh-CHS.pdf.
2. In the Skyline, I went to the homepage > tutorials, after clicking on MS1 filtering, I noticed that the automatically created folder is MS1Filtering-22_2, but the actual downloaded is MS1Filtering-21_1_zh-CHS.pdf.

I'm not sure if this is isolated or if you guys forgot to update something after going through the upgrade.

I have two static (structural modifications):

1. The N-terminus of the peptide is modified by phenylisocyanite.
2. The NH2 group of Lys is modified by propionyl group.

it so happens that I have a peptide that has K on the N-terminus:

KQLATKAAR

meaning the first Lys should have both phenylisocyanite and propionyl

how ever when loading the peptide in skyline only adds propionyl, ignoring phenylisocyanite

if I however add Ala to the peptide N terminus

AKQLATKAAR

I do get A modified with phenylisocyanite and Lys with propionyl.

How do I force skyline to add both structural modifications to K in KQLATKAAR?

Hi Skyline Team,

I have a biomarker assay where I'm using two differently labelled heavy peptides - one is used as an internal standard, the other as the surrogate analyte used for calibration (because my blank matrix has the light peptide endogenously present). I can't figure out how to get Skyline use the calibration curve of the surrogate analyte for calculating the concentrations of my unknown samples where I have signals coming only from the light peptide and the internal standard. Any ideas how to get Skyline to do this?

Thanks!

At the moment, we are currently attempting to create a spectral library using Skyline for a PRM experiment; using DDA data of the full proteome and of heavily labelled peptides, acquired using the Astral equipment.

It is possible to import the raw files, although a message saying that there is " no matching instrument configuration for the analyser type" appears.
Also when setting the MS/MS filtering acquisition method, as it is not possible to find the Astral mass analyser as an option, what would be recommended to use in this particular case?

I appreciate all the attention given,
Best regards,
Nádia

Dear Skyline team,

I am a recent user of the platform so please forgive my ignorance. I have been trying to use the platform to identify 13C labelled metabolites from a metabolomics ms/ms run. I created a target list where each compound was assigned a list name and the isotopologues of the compound where different molecules. I have also the peak integration in all my samples. The step I am stuck on is trying to normalize the intensities/peak area to total ion current and then exporting the report. I noticed that the normalized areas of the isotopologues are assigned "#NA" and I only get values for the M+0 (the unlabelled fraction). I would appreciate whatever help I can get with this issue.

Best wishes,

Mikky

Hello,

I have been trying to upload the transition list attached onto molecular interface of Skyline.
It only gives me an option to do Ctrl-V for loading the transition list.
Whenever I try to upload it that way, it gives me the following error message.
"Data columns not found in first line"

For some reason, the data columns are not recognized by the software.

Could you please let me know how I can go about resolving this issue?

Thank you.

hello sir,
i am trying to statistical analysis using a MSstats shiny but i think there is no availability of tutorial on MSstatsshiny so I am trouble in the uploding the skyline annotaton file please guide me to make a annotaion file for group comparison study.

I am trying to quantify a protein and I have loaded its sequence in skyline to in silico digest it and filter peptides. I have then created a library using prosit and loaded the raw data and picked the peptides that have good signal, peak shape, good dotp with the prosit library (I ignored the RT component of prosit prediction).

Now that I have a list of high-quality peptides from real data, I would like to use the data to create a new library using the experimentally observed RT and fragmentation spectra. However, I can't find a way to do this.

Hence, my question is: how do I take the experimentally observed data loaded into the skyline and curated there to create a library?

Hello.

I am trying to build the spectra library using the PD searching results. After I added the msf. or pdresult. file, an error popped up saying no Mass Spectrum Items. Can you please let me know how to fix this? Or what is the other way to import the PD searching results?

Thanks,
Yanyan

Hello Skyline Team,

First of all, thanks for such a great tool, I've used Skyline for almost a decade now to design and analyze targeted proteomics dataset.

I have recently been exporting my reports for my datasets as multiple individual .csv documents, as I found it cumbersome to export all the information I wanted (fragment ion peak area, MS1 IM, MS1 peak area, total area, fragment ion dotp score, etc.) in a single report and then going through and separating the information into separate tabs. Currently, I take all the multiple individual .csv reports and combine them into multiple tabs into a single Excel Workbook, as this is sometimes easier to manipulate for downstream analysis and is a somewhat more logical document when provided to collaborators. Would it be possible to modify the Report Export procedure so that I can tell Skyline to output into a single Excel Workbook a document with multiple tabs that contain information from different report templates? For example, a new Merged Report Output option that combines the report template from Report Template 1, Report Template 2, and Report Template 5 as three separate excel tabs in a single document? Or, did I just not adequately search the forum for this topic? I currently have a tool to combine the separate single reports into a single merged document, but I still have to tell Skyline to export 5 different reports, and it would be convenient to have a method within Skyline to export a single merged document.

Thanks in advance!
Josiah

Hello, sir. I am facing to a problem downloading R packages . after multiple trials, some R packages cannot be downloaded in MSstats downloding.

Dear Skyline,

Since we are using skyline we always had a bit problems with our reports. Mostly after each update we lost them and had to reimport them.
After Brendan's mail on April 1st, I decided to update again all computer with skyline, after almost 2 years.
Unfortunately a colleague using skyline and a specific template lost her template. gain
She was not able to import it. I tested it on a new Instrument acquiring PC we have since 5 month, where I installed AutoQC for Panorama.
This PC never saw any inhouse template we saved. I tried to import the specific report of my colleague.
As shown in the word file, I could import something, but during import the report name changed ("Report_Template_QQQ_Skyline.skyr" to "Report_JC.skyr") and the imported report did not corresponded anymore of what she paste normally in excel. she could not use this imported report.
I did one other test on my computer. I was able to import one of my templates, but unfortunately stays light-grey, and I am not able to use it.

How could we fix these issues?
Thank you in advance for your support and wish a nice day.

Kind regards
Jean-Christophe

Hello Skyline Team,
I have been unsuccessful in importing raw files from our Orbitrap Astral instrument.
The instrument method is DDA acquisition using FAIMS with three CV settings.
I think I need guidance with Full Scan scan settings. What is recommended with Astral data using FAIMS?
Thank you very much!

Hi Skyline Team,

I have a question, I processed data from a sampleset, and need to add few new runs to this skyline file and would like skyline to consider the same integrations done on the previous samples for the new added ones.
Is there a way to automate this process? or should I go through all the compounds one by one and manually reintegrate each peak? thanks a lot!

Nihel

Hi, in Skyline-daily (64-bit) 23.1.1.459 (f9dd888b9), I tried to build a spectral library from FragPipe pep.xml output of a DDA-PASEF search. The error message indicates that Skyline is looking for a data file (mzml/mgf), which is actually present in the same folder. Any idea why Skyline doesn't recognize it? Noteworthy, the error message appears when clicking next on the spectral library tab (i.e. before selecting the raw data).

Thank you, David

I tried to quantify a peptide containing an artificial, variable modification adding 'C13 H8 O2 Cl2' to lysine. I have DDA data and performed a MaxQuant-search including the artificial modification. When I tried to build the library using the msms.txt file I got the error message "No matching mod for.... Make sure that you provided the correct modification[local].xml file."

I checked the support and found very similar questions two times in 2020 and 2021. Both times the answer was, that 'BiblioSpec is unable to handle ... and Chlorine modifications in MaxQuant search results', but from the next Skyline realease on the problem would be solved. I am working with Skyline (64-bit) 23.1.0.455 (599e74ad2), but obviously I have the same problem. Could you please help me? You will find the mqpar.xml file attached to this mail.