There are some unimod modifications that aren't in Skyline like Cys->Asn. I'm trying to automate the process of creating .sky files and it looks like adding this modification isn't allowed because the unimod database is incomplete. Is there any way to add custom modifications using SkylineCMD or Skylinerunner?

Hi,I have 2000 proteins that I want to verify using Skyline, but integration is a big problem. I can ensure that the retention time is 0.5 minutes earlier than the ID. Therefore, I want to narrow the integration range through peak boundaries. However, when using PRM Conductor for screening, I encountered an error message saying "Index was outside the bounds of the array". Could you help me solve this problem?

Hello,

I am using Skyline (64-bit) 24.1.0.414 for my analysis. I am trying to compare a few different samples that have heavy labeled peptides to determine the best ratio at which to use our endogenous sample to heavy peptide. I am looking at 2 oxonium ions for 1 heavy and 1 light peptide per sample. I've found the Peak Area - Replicate Comparison bar graph very useful to look at the peak area ratio to heavy, by clicking on "normalized to heavy". This will be very useful as we identify the best ratio and compare normalized samples to our internal standards. I've been using the custom report tool to export each of the heavy and light Total Areas for each sample and peptide. However, which column name do you use in the report tool to project the Peak Area Ratio to Heavy values we see normalized in the graphs? I have attached a file of what my graph looks like, so you can see their approximate values.

I've already tried exporting with Total Area Ratio and Ratio to Standard, but these have completely different values than what my graphs are showing. I've confirmed that the Total Area Ratio is just the Light Total Area / Heavy Total Area, but we would like to know how the Peak Area Ratio to Heavy is calculated and exported, as these are not the calculations we are seeing? The closest I could come up with was the sum of all Heavy and Light Total Area / Heavy Total Area, this gives me a similar curve, but yet the values are still not accurate for all my samples.

For instance: The first sample in the graph has 855524 Light Total Area, 979855 Heavy Total Area, Total Area Ratio of 0.873, however, the Peak Area Ratio to Heavy Graph value is 1.922. The last sample in the graph has 1600330 Light Total Area, 136575 Heavy Total Area, Total Area Ratio of 11.717, and Peak Area Ratio to Heavy Graph value is 25.439.

Thanks for your help!
Erin

Hello,

I'm using a triple quadrupole (Shimadzu 8060) and I'm performing a survey scan (i.e. DDA) with an inclusion list. There are no other functions in the method.
The inclusion list with the precursors m/z is on the transition list of Skyline and I am able to extract the MS1 spectrum. However, it seems that Skyline does not extract the MS2 triggered scan.

I have tried different settings for MS1 and MS/MS but I cannot extract both MS1 and MS2 scans even if they are present in the RAW file.

Can you help me with such issue?

Thanks in advance and big ups for the fantastic work you're doing!

Gioele

Hello,

I would like to implement this GNPS library (https://gnps-external.ucsd.edu/gnpslibrary) but I get the following error on Skyline:

D:\Databases\ALL_GNPS_NO_PROPOGATED.msp (line 1195456): No peaks found for peptide Phakellistatin 13.
Skyline (64-bit) 24.1.0.199 (6a0775ef83)
System.IO.IOException: D:\Databases\ALL_GNPS_NO_PROPOGATED.msp (line 1195456): No peaks found for peptide Phakellistatin 13.
at pwiz.Skyline.Model.Lib.NistLibraryBase.ThrowIOException(Int64 lineNum, String message) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Lib\NistLibSpec.cs:line 1598
at pwiz.Skyline.Model.Lib.NistLibraryBase.CreateCache(ILoadMonitor loader, IProgressStatus status, Int32 percent, String& warning) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Lib\NistLibSpec.cs:line 1060
at pwiz.Skyline.Model.Lib.NistLibraryBase.Load(ILoadMonitor loader, IProgressStatus status, Boolean cached) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Lib\NistLibSpec.cs:line 661

Do you know where this problem could be coming from?

Many thanks and best regards,

Gioele

Recently, our acquisition software was updated from ofofControl 6.2.901 to timsTOF 6.0.8.0. Unfortunately, this seems to result in the following error for data acquired with timsTOF 6.0.8.0: "Maximum expected frame size exceeded." Please find the full error description below.
I encountered this error using Skyline-daily (64-bit) 24.1.1.398 (e8afca524). The error occurs when attempting to import the data file (specifically when acquired in timsTOF 6.0.8.0: 2024GLY00161-GSL-A01_GA2_1_433.d) into Skyline, causing the program to fail during the import process. This specific file acquired with timsTOF 6.0.8.0 does not contain any TIMS data, so I was wondering whether this could be the cause of the error.
I have included screenshots of the acquisition software of the files. Additionally, I have uploaded the Skyline files to Panorama (https://panoramaweb.org/Leiden University Medical Center - Tissue Glycomics/Troubleshooting/project-begin.view: AHE_24_024_GSL_Error.sky.zip).
Thank you in advance for looking into this issue.

Kind regards,
Agnes

At 15:25:
Failed importing results file 'Z:\instruments\TIM\Data\Tao\20250221-GSL-MS2\2024GLY00161-GSL-A01_GA2_1_433.d'.
Maximum expected frame size exceeded.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'Z:\instruments\TIM\Data\Tao\20250221-GSL-MS2\2024GLY00161-GSL-A01_GA2_1_433.d'.
Maximum expected frame size exceeded. ---> System.Exception: Maximum expected frame size exceeded.
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, Boolean getBinaryData)
at pwiz.ProteowizardWrapper.MsDataFileImpl.HasSrmSpectraInList(SpectrumList spectrumList) in C:\proj\pwiz\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1304
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\proj\pwiz\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 624
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasCombinedIonMobilitySpectra() in C:\proj\pwiz\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 698
at pwiz.Skyline.Model.Results.FileBuildInfo..ctor(MsDataFileUri msDataFileUri, MsDataFileImpl file) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1409
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 226
--- End of inner exception stack trace ---

Hello Skyline Team

I received the following warning message when trying to open the file:

Failure opening //mac\Home\Desktop\Griffith University\Milk project\Data Analysis\Experiment 1\N-Glycans\Skyline MAIN LIST revised, without samples.sky.
The file contains an error on line 1111 at column 16

Is it possible to restore the file? I uploaded it.

Thank you in advance.

Beste regards,

Alessandra Tozzi

Dear Skyline team,
I am trying to analyze my msms.txt output file from MaxQuant.
I did a DDA experiment, where localize several PTMs which I defined myself. Specifically, I am looking at ADP-ribosylation.

I imported the mqpar.xml and the modifcations.xml as requested, but i get the following error:

"Skyline (64-bit) 24.1.0.414 (5b5ea5889c)

System.IO.IOException: ERROR: No matching mod for ad in sequence AE(ad)PVEVVAPRGK (line 861). Make sure you have provided the correct modifications[.local].xml file."

My ADP-ribosylated peptides are reported as (ad) by MaxQuant, but it seems that this is incompatible with Skyline. It is not clear to me how Skyline interprets the modifications.xml file, since the shorthand notation found for several PTMs is not found in that file. Similarly, it is also not clear to me if i would be able to define these PTMs myself elsewhere in Skyline to circumvent the modifciations.xml import.

So far i tried to rename modifications, subset the file, use different MQ versions, but all to no avail. If there is a work-around that would allow be to analyze my results in Skyline then that would be great. Thanks a lot for a great piece of software,
Best,
Jonas

Hello,

I am trying to set up Skyline for the analysis of GC-MS data from an Agilent single quadrupole instrument. I followed Pawel Sadowski's protocol for Shimadzu and also tried adapting the guidance provided here: https://skyline.ms/announcements/home/support/thread.view?rowId=43600. However, no matter what changes I make to the transition list, the files do not process correctly. Skyline identifies only the precursor ion and cannot detect the fragments.

That said, when I manually click on the chromatogram, I can see that the MS/MS spectra have been recorded and are readable from the files.

I have attached the Skyline file, the GC-MS data file, and the transition list to make it easier to review the data.

Thank you very much for your help.

Best regards,
Daria

Hi Skyline Team,

We are a public forensic laboratory and use Skyline to interpret LC MRM results. As our samples can come from unknown environments (e.g. a body fluid stain on a piece of clothing) being able to evaluate noise is important.

To get a reasonable measure of noise contributed by potential contaminating substances, we would like to measure all mass spectrometry data for 30 seconds before and after the signal peak. Is it possible for Skyline to access and report average intensity from these two 30 second intervals so we can average them and use them to calculate S/N?

Thanks,
Heyi

Hi Skyline team,
I am trying to set up a Skyline doc using a standard fasta, some mzML files and a custom blib file. During the "Import FASTA" step, I get the following error message:

Importing the FASTA did not create any target proteins

I've uploaded the blib to the file sharing portal, it's called ljh-cust-lib.blib. This blib was generated by applying BlibBuild to a handmade .ssl file. It's entirely possible that something is off with the blib, but I'm not sure what. I don't think the problem is with the fasta or mzML files -- I've used them to create Skyline docs many times.

Thanks for the help,
Lincoln

Hello Skyline,

I hope you are doing well.

I would greatly appreciate assistance regarding the quantification of some compounds I am analyzing, please?

I am trying to quantify multiple analytes which were spiked in my standard with different concentrations.
I prepared the transition list and sorted the compounds with their internals standards using "Molecule List Name" adding "heavy" label type for my internal standards (see file attached).

Following that I adjusted Molecule Quantification Settings for Linear Regression with Ratio to Heavy normalization and regression weight of 1/x.

Then, I assigned the Sample Types and Analyte concentration to my raw data with Concertation Multiplier for my analytes of interest.

However all I am getting is "Error: All of the external standards are missing one or more peaks" when checking the calibration curve.
However I can see that the peaks are detected in my standards and unknown samples.

I would really appreciate some help,

Thank you,
S

Hello! I love the Skyline Software.

I just have one request: I work a lot with the Retention Times > Molecule Comparison Graph.
However, I have a lot of small molecules in a bunch of separate molecule lists (see picture). Is there a way to view that graph for one molecule list only instead of having one huge graph with all molecules from all lists combined? It would really help me work.

Best regards
Helena

Hi!
I am working with a Q exactive in PRM mode. I created a transition list with my internal standard and thiol precursors. All compounds work well except my internal standard. I get a message saying Chromatogram information unavailable. I am sure about my retention time and fragmentation (I got them with a full ms d2). I don't understand that with the same parameters, I get a signal on Chromeleon for my internal standard. It is too bad cause I need my internal standard signal to continue working with Skyline, which is way more user-friendly...
Is there anything I could try
Regards,
PN

Dear Skyline Team,

What is the recommended workflow for processing FAIMS-acquired MS data in Skyline, from mass spec acquisition (CV selection/optimization) to data analysis (ion mobility visualization and library creation)? Additionally, how does Skyline handle multiple CVs per precursor, and what is the best way to extract and utilize FAIMS CV values?

Best,
Sofanias

Hi Skyline team,

Could you please help me understand the line, dot line, shaded area in skyline file (see attached file)? Thanks,

HY

Hi Skyline Team,

I recently conducted an LC-MS/MS experiment analyzing several metabolites, and I would like to use Skyline for data analysis.

I have prepared an Excel table (attached below) as a transition list. Would this format be suitable for data import, or would you recommend modifying it in any way?

Additionally, my dataset includes calibration curve samples. Most of the documentation I found on your website is focused on peptides. Could you kindly provide simple instructions on how to create a calibration curve using a precursor ion and a single fragment ion?

For all samples, I added an internal standard (spiked in). Where can I define it in Skyline if I want to monitor its stability throughout the experiment?

Thank you very much for your support!
Michal

Hello,

I'm wondering if it is possible to report the areas for MS1 peaks using only the monoisotopic peak. In the attached screenshot I have the monoisotopic peak, the M+1 peak, and the M+2 peak. I believe that the area reported in the document grid for 'Total Area' and 'Total Area MS1' are both the combined areas of these three isotopic peaks (in the document for the attached example this value is ~209k which matches the value from the 'peak area replicate comparison' view). I would like to have this value along with a value reporting only the area of the monoisotopic peak (the blue XIC) if possible. I know I can get this if I change the transition settings/MS1 filtering to only look at 1 peak. But then I have to continually go back and forth between 1 and three peaks. Is there an additional field I can add to the report that has the peak areas split out by isotopic peak?

Let me know if I can clarify anything. Thank you!

Simon

Hi Support Team,

I'm using Skyline (v23.1) for a targeted lipidomic assay with UPLC-MS/MS data. Waters developed the method, and I obtained the transition list from a former PhD student. There should not be any issues with the transition list. While I can see PA/LPA chromatograms in MassLynx, they show "Chromatogram Information Unavailable" when imported into Skyline. Since all my other results were processed in Skyline, I also need to extract PA/LPA data there. I have attached my example skyline file with one of my raw data. Please let me know if there is a way to resolve this issue.

Best Wishes,
JS

Dear Support Team,

I hope this message finds you well. I have recently obtained results from LC-QTOF analysis and am seeking to determine the potential proteins and peptides present in my sample. I would like to inquire whether it is possible to perform a bottom-up analysis using Skyline for the identification of these proteins and peptides.

If this is feasible, I would greatly appreciate it if you could recommend any relevant tutorials or instructional videos to guide me through the process.

Thank you for your assistance, and I look forward to your response.

Best regards,
Smith 13

I am doing PRM assay. I spiked my CSF sample with Internal standard heavy peptide at x fmol/ug. So for1ug of CSF (1ug/ul), I spiked my internal standard at 64fmol/ul after getting the LOD from the calibration curve. In the peptide settings-->> quantification i used the Unit as fmol/ug.

When i used SKYLINE for Quantification, it gives the quant of the peptide in similar fmol/ug in the excel sheet when I exported the Peptide Ratio results. Do i have to use peptide molar mass for further calculations or report the quantification data straight from Skyline?

Dear Skyline support,

I am working to create a standard PFAS Skyline method that can be used to quantify a fixed list of 40 PFAS molecules. Ideally, the goal is that the method can be used with different sets of raw data, only with adjustments in RT if needed.

We have 40 unique PFAS analytes, 24 extracted internal standards (EIS), and 9 non-extracted internal standards (NIS). The EIS are added prior to sample extraction. The NIS are added after the extraction is completed, but before injection into the LC/MS system.

Per EPA guidelines, the quantification of PFAS analytes is done with respect to EIS, while the quantification of EIS is done with respect to NIS. Please see attachment "equations-for-eis-nis" for the equations that relate these quantities.

Each analyte is referenced to one EIS, while each EIS is referenced to one NIS. Please see "attachment-2" for the detailed list of these references.

1. Is it possible in Skyline to do quantification this way (i.e., for each analyte, specify that I want the analyte to be calculated based on an EIS, and I want that EIS to be calculated based on an NIS).

2. You will see in attachment 2 that certain EIS may be used as reference for multiple analytes. For example, PFOS-13C8 is an EIS that is used as reference for 5 analytes. Is there a way to enforce these settings directly in the Skyline, or would I have to do this via the transition list?

Please let me know if you require further information. Thank you so much!

Best,

Kyle Nguyen
UNC-Chapel Hill

Hello.
We used a blank sheet in skyline to upload a DDA raw file of a tryptic digest of a protein. We have of course downloaded the corresponding fasta file at the beginning of the procedure. After running skyline, we visualized briefly some peaks, but when the processing was over, no graphical results were displayed and mainly all the fasta peptides (and their PTMs) were preceded with a red cross.
We have also applied and followed the "skyline DDA search for MS1 filtering" tutorial with the same raw file and same fasta file. The searching of spectra ended in one minute with the following message: no spectra found for the new library skyline.
Do you have any explanation for this?
Do we need some kind of library for the processing to be successful?

Thank you

Dear reader,

For a while now, we have been able to process data from the raw .D file format which is used by Agilent Masshunter (Chemstation). I have been using a common fragment ion for our standard instrument performance test, data which we then tunnel to Panorama to plot the outome of each injection. We use pretty standard/basic alkane mixture for this, so far this did not give us any troubles. however, lately we have been thinking about adding an additional layer of confidence, and ease of use with regards to retention shifts whenever a new column is installed. (or during overtime deterioration of column resolution). We have the option to add the molecular [M+]+ peak (obtained through electron impact ionization).

However, here's where I find myself at a "dead-end". The previous method seems to be able to still process data files, but the newer one... you see something is imported from each datafile, but then nothing really happens, nothing is shown or can be send to Panorama. I have added the Skyline method in the .zip. I generated it via the file->share option in Skyline.

The two Excel files are the transition lists I have been trying to import and use.

Skyline version: (64-bit) 20.2.0.343 (a7a9e8c4f)

As there a at least two relevant questions about this in the past I have been trying the suggest approach, which, unfortunately was unsuccesful so far.

Would this still be a conversion issue/skyline unable to read its MS1 data? Which originates from EI? Curious to find out what might be the solution to my issue/question.

Thank-you in advance for your time and effort.

Kind regards,
Jurre

Hi Team,

I was just searching for information about dotp in Skyline, and this thread (below) was really helpful!
https://skyline.ms/announcements/home/support/thread.view?rowId=20003

I have a couple of questions regarding the implementation of spectrum preprocessing and the calculation of dotp (equivalent to the normalized contrast angle).
What would be the easiest way to perform these tasks? I know there are some resources available for the Skyline command line interface and its source code.
I assume there might be a suitable commandlet in these resources.
If you’re available, I would appreciate any advice you could provide on this topic.

Best regards,
TY

Hello everyone
We have the SYNAPT XS instrument and my task is to discover the proteins from cells.
I am trying to repeat the steps from this "Skyline Data Independent Acquisition" tutorial paper.
I am getting error during the export of Isolation schema, there is no option to save at .csv format, only .mrm.
"Import Peptide Search" sections.
Also I have problem with uploading the results, it is showing that it is running/importing the .raw or .mzML file, I can see the process of importing, but at the end I do not have any results imported.

Please see attached files, and if you have any suggestions, please share with me your thoughts.

Thanks beforehand.
Best regards,
Gor.

Greetings,

Firstly, I want to praise your efforts in creating this application, it is powerful and has quite clear documentation.
Still, I have encoutered problem almost immediately. Analysis was performed on Shimadzu LCMS-8030 with ESI ionisation in both negative and positive modes (centroided). Generic output of Shimadzu was converted to .mzML format. After pasting a transition list (I tried different options - attached) and after importing raw results I get an error: At 11:22:
Failed importing results file 'D:\Program Files\skyline\data\mzML-F10_flav_MRM_5-95_004.mzML'.
polarity mismatch (full text is attached).

I tried to change some transition options but without any success (current settings with .sky file will be attached in 'file sharing' with name "flavonoids_refined_table_gao_ren.sky.zip").
In this .zip I also attached one of .raw file, if it would be of any help

My system: Windows 20 (64-bit) Home, version of Skyline: (64-bit) 24.1.0.414 (5b5ea5889c)

With gratitude,
Paul I.

Hi,

I am trying to import RAW data acquired from an Agilent 6410B system and I get the following error:

Failed importing results file 'D:\Skyline\Microcistine - comparazione 6410B e 8060\Agilent febbraio 2025\2025 02 20\25LA00436 Totale.d'.
[MassHunterDataImpl::getSignals()] Specified argument was out of the range of valid values.
Parameter name: Data Not found - deviceNameOrdinalNumber
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'D:\Skyline\Microcistine - comparazione 6410B e 8060\Agilent febbraio 2025\2025 02 20\25LA00436 Totale.d'.
[MassHunterDataImpl::getSignals()] Specified argument was out of the range of valid values.
Parameter name: Data Not found - deviceNameOrdinalNumber ---> System.Exception: [MassHunterDataImpl::getSignals()] Specified argument was out of the range of valid values.
Parameter name: Data Not found - deviceNameOrdinalNumber
at pwiz.CLI.msdata.ChromatogramList.size()
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasChromatogramData() in C:\proj\skyline_24_1\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1265
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 231
--- End of inner exception stack trace ---

Do you know why I am getting this error? I have imported files from such a system before (using a similar LC-MS acquisition method) and it worked before.

Gioele

Hi all,

I was trying to install the Skyline-daily unplugged version (v24.1.1.398) in a computed with limited internet access and I got the following error:
"Application cannot be started. Contact the application vendor"

Attached is a text file with the details of the error.

This is the first time I encounter this error. Any ideas what might be causing it?

Sincerely,
Juan C.

Hi team

I want to use the RMS method for signal to noise ratio calculation using peak heights of individual peptides.
Where can i get the background height data? And should i combine the background height for each transition/precursor to get the SNR for a given peptide? Or is there a better way to do the RMS based SNR calculation?

Thanks
Ramesh

Hi team

I have two peptides light and heavy (8 amino acids long). The heavy peptide has a single 13C on the 3rd amino acid so differs from light peptide by 1 Da only. I want to spike the heavy peptide in the light and monitor their +2 charge precursors (m/z difference 0.5 only). I have generated a skyline document and it gives me details for both but I was wondering how to verify that the peaks are being correctly identified- I am getting same product ions for both. Also, the M+1 and M+2 of light is same (upto 3 decimal places) as M and M+1 of heavy. I am acquiring data on Thermo Q Exactive Plus in PRM mode using 1.6 m/z isolation window and 0.5 m/z offset. I have set skyline ion and method match tolerance to 0.01 m/z under the transition settings.

I have shared the skyline document and raw files on the portal (file name: light and heavy mix_1-13C). It has 3 files, standard- mix of purified light and heavy peptide and biological- two technical replicates of heavy peptide spiked in a tissue peptide extract. The results of all three are confusing.
In the standard, the M of light peptide hasn't been identified but skyline has identified light specific transitions. Can it be correct? Additional replicate give same result.
In biological, precursors and products both are identified for light peptide and only precursor for heavy peptide. If precursor has been identified, why not transitions?
In biological 2, both precursors and product ions are identified for both peptides. Additional replicates give this result. But are the identifications correct?

My aim is to normalize peak intensity/areas of light with heavy. How should I do that?

Thanks

Hello Skyline Team,
We want to use Skyline to analyze prm-PASEF data acquired from timsTOF HT. We first used Spectronaut to generate the spectral library from DDA-PASEF data (we do not have software to directly analyze DDA-PASEF data) and then exported the spectral library tsv file. As previous post suggested, we imported the spectral library tsv file through "Assay Library". However, it requires us to have iRT information to generate the library. Is there a way we can bypass iRT option since our goal is not for absolute quantification and we do not spike in iRT standards? We also tried to choose a protein from our target list as a standard, but in order for it to work, all the peptides from the chosen protein has to be presented in our acquired prm data, but we only want to monitor 2-3 major peptides, so this is not really work for us. Do you know how can we solve this issue? Is there another way to analysis the prm-PASEF data without using iRT information? Thanks!

Hi,

we have trouble importing an external tool that we wrote in the past into a new installation of skyline daily on windows 11. The error message is a bit cryptic: Failed attempting to extract from xyz.zip. A value of NULL is not allowed. Parametername: key

Could you please indicate the source of the problem?

best wishes, David

For a absolute quantification assay of a single protein I ordered in two heavy peptide standards.

Peptide standard 1 quantifies the protein amount as 40 fmol.

Peptide standard 2 quantifies the protein 20 fmol.

Is it common to have a large discrepancy between two peptide standards when they should in theory give near identical values (if everything is perfect)?

Hello,

I'm comparing quantitation results of a small molecule with two transitions, quantitative and confirmatory, between TargetLynx and Skyline. The results are very similar but the areas I'm finding in Skyline are significantly higher than what is shown in TargetLynx. The integrations seem comparable enough that I would not see this level of discrepancy. In the included picture, is this the correct flow path to show the area for just the quantitative transition? I cannot share data files.

Any help would be appreciated!
Thanks

Couple of queries:

1. A colleague spiked 25 fmols of the heavy peptides in the standard curve but spiked in 20 fmols in the sample and I couldn’t find a place to have different values. I only saw one place to put in the concentration of the heavy peptides.

2. A former colleague had 7 heavy and 2 regular synthetic peptides used for the calibration. When I look at the samples, I see the 2 regular synthetic peptides, but I am not finding any of the regular peptides that correspond to the remaining 5 heavy synthetic peptides and I am wondering if it was because they aren’t there or because they weren’t in the samples that I used for the calibration curve.

*We are new to Skyline.

Thank you for the help.

Hi,

I tried to import SIM-Data files, but only a few of the m/z were found (see attached screenshot). The full-scan files, on the contrary, can be perfectly imported and do show peaks. The SIM files are in the uploaded zip-folder "GCMS_Sim". I asked more experienced colleagues and we tried many different settings but the m/z of many molecules and their fragments cannot be found.

What is the problem?
Thank you in advance,
Stefanie

Hi,

I work in an ISO:17025 accredited laboratory and would like to use Skyline for routine small molecule quantitation.
At the moment I use Skyline mainly for method development, but I would like to push for routine use as we have different suppliers and providing a training for LC-MS data handling for each supplier is time consuming and not very effective.

Do you know if Skyline can comply with such guidelines? I don't know if these points are covered:

• Full audit trails that track data changes, including who made the change, when and why.
• Time-stamped records of all actions taken within the system.
• Secure user access management with role-based permissions.
• Generation of customisable reports (this one should be OK).
• Ability to generate comprehensive documentation required for audits and inspections.
• Export options in standardised formats (e.g. PDF with chromatograms) for traceability.

Many thanks for your help and all the best,

Gioele

Hi Skyline team,

How could I show the value in the figure? I tried but still can't do it. Thanks,

Heyi

Hi Skyline Team,

Can Skyline be used to process oligonucleotide LCMS data? I was wanting to use Skyline to detect the oligonucleotide fragments. We know the sequence of the oligonucleotide and we want how an enzyme cuts this when used as a substrate. Or is there another software that can be linked to Skyline to do this?

Kind regards,
Elvis

Hi,

I created a spectral library from PEAKS and when I try to add peptides in my skyline document sometimes I see an error message saying the modications don't match the document settings in spite of all the mods have been enabled in Peptide settings.

I appreciate any help in sorting out what I'm missing here

Kind regards,
Leandro

Hello team

I have acquired MS1 and PRM data on QExactive Plus for small peptides.

1. Is there a way to extract MS1 peak resolution values using skyline?

2. Also, what is the right way to get the number of data points across a peak? I exported the peptide transition results but I am getting very different values for same precursor between technical replicates- analysed with same transient time, peak width etc.

Thank you
Ramesh

Dear Skyline Team,

I have a problem which I am sure will be an easy fix, but I don't know how.
In the attached Skyline file there is a measurement of SRM plasma and the internal standard lipid mix I am using.
Now I would like to do 2 things:
First, normalize my endogenous analytes to my ISTD. For this, as far as I know, i have to set the ISTD as surrogate std, if I want to normalize the other endogenous analytes to it.
Here lies the first challenge for me, because every time, I try to set the ISTD to surrogate standard or add the concentration, the same is done to my corresponding endogenous analyte, which should stay an unknown. I am sure that there is just one setting wrong, but I could not figure out which one.
Secondly, I would like to perform a quick single point calibration, like what was done in the Webinar 13. Probably because of the same reason as the point before, it is currently not working, altough I tried to keep the settings as they were explained in the Webinar. Can you explain to me what went wrong there?

Looking forward to your reply.

All the best
Thomas

Dear Sir,

I insert the non-tryptic peptide: QGARGFPGTPGLPGVKGHRGYP.
I want to add a variable modification (Ox), It does not work for this non-tryptic peptide.
On the other hand, on the same sheet, it works for tryptic peptides.
I use Skyline 24.1 version.

Can we consider variable modifications for non-tryptic peptides?

Best regards

MCM

Dear Skyline team,

I generated a transition list with peptides from from DIA-NN (report and speclib) data to go further into prm-PASEF, i.e. optimization of CE (see Skyline-Targetlist-test.csv). However, the isolation list export for Bruker timsTOF fails due to missing ion mobility values:

Skyline

All targets must have an ion mobility value. These can be set explicitly or contained in an ion mobility library or spectral library. The following ion mobility values are missing:

AEATTLHVAPQGTAMAVSTFR+++
DADPDTFFAK++
DSEITFIKK++
EFNAETFTFHADIC[+57.021464]TLSEKER+++
FKN[+0.984016]GMLHGDK++
FVC[+57.021464]NSGYK++
IPAC[+57.021464]IAGER++
ITQVLHFTK++
LFLEPTRK++
MDVFSQNMFC[+57.021464]AGHPSLK++

OK

But, the values are given in the transition list during the import as Explicit Ion Mobility which seem to be successfull as the corresponding value is given in the spectral library view afterwards. So at least, the value is somewhere, but not accessible for the export...
Note, the box "Use spectral library ion mobility values when present" (Settings > Transition Settings > Ion Mobility) is checked.

Do you have any advice?

Best,

Gianin

Hello,

I generated a peptide mapping dataset using my Agilent 6545 QTOF. The method is an "all-ions" (MSe / MS All) type of experiment, where the 1st scan is a low energy 0V CE, and the next three scans are 15,30,and 45V high energy scans.

I am having issues with:

1. Searching the data
2. Viewing the different high energy scans.

I have been able to make some progress with #2. I used the "Edit Spectrum Filter" option to have Skyline give me peptides for each collision energy. However when select the MS/MS spectrum for my peptides, I always see that the Aquisition information shows the Collision Energy at 15V. This is my first high energy scan. I have to believe that I would get optimal fragmentation information from my 30 and 45V scans as my peptides increase in size. Ideally Skyline should be picking the CE with the most intense fragment ions, but I don't believe that is happening.

For 1, I have no idea where to begin. I thought perhaps Skyline might be able to perform an all-ions based search as it is similar to waters MSe data. But it keeps asking me for an isolation scheme when I chose the type of mass spectrometer. I am unsure how to proceed here. I am also engaging Agilent on this as well (they aren't even sure that I can perform a peptide search on this data at the moment)

You might ask why do this? Well the cycle time is decently fast, like 0.5s per cycle. So we get 10-12 points across our 6s wide UPLC peptide peaks, which is nice. I don't think Agilent's AutoMS/MS can move that fast.

Anyways I digress. Just looking for some help here

Greetings,

For methods with many targets, adding "Collapse All" and "Expand All" entries to the right-click context menu within the Targets pane would be extremely helpful for navigating through compounds.

Happy to move this to the software issue / feature request portion if there is currently no way to do this.

Thank you,
Andrew Psoras

Dear Sir,

I would like to know if the list of peptides generated by ChatGPT (random digestion) + fragment ions (y and b) can be used, in Skyline, as a library in DIA ?

If yes, in what format ? csv?

Best regards

MC Menet

Hi,

I have an issue using this great software: When exporting the tables from the dicument grid despite formatting the numbers as Full precision then its a mix with scientific formatting which hiders further processing in Excel?

Is this a bug?

All the best,
Allan

Dear Skyline Team,

could you please consider implementing support for DIA-NNs .speclib spectral libraries? Its a highly convenient tool for predicted libraries and much faster than Prosit.

https://github.com/vdemichev/DiaNN

With best regards,
tobi

Hi!
I’m trying to streamline my workflow in Skyline Batch by using a previously prepared template (normal skyline document) to process a second batch of samples. The issue I’m facing is that I receive a constant error when attempting to apply the saved template to new chromatograms.

First Batch Processing:

We imported a transition list and raw files into a normal Skyline file.
A colleague manually reviewed chromatograms, identified the best peaks, and applied them to all samples for each analyte.
This Skyline file was saved and intended to be used as a template for future batches.
We also created an iRT calculator from this data.
A report was generated using a pivoted table with peak areas for all samples.

Applying This to a Second Batch:

Now, I want to use Skyline Batch to apply the same retention time borders, peak selections, and chromatogram alignment from the first batch to a second batch.
The issue is that the raw file names are different in the second batch. Could this be the cause of the error?
Instead of manually reviewing and selecting peaks for each new batch, we want this process to be automated so all batches are processed consistently.
My Questions:
Is it possible to use a Skyline file (with manually adjusted peaks and applied retention times) as a template in Skyline Batch for processing a new batch of raw files?
Does the difference in raw file names between batches create an issue?
What is the best approach to automate this process so we don’t have to manually realign peaks for every new batch while using the same transition list?
Is there a better way to set up the Skyline Batch or Skyline workflow to ensure peak alignment and selection are consistently applied across multiple batches?
We are trying to reduce human error and ensure that each batch is processed in the same way. Any advice or best practices would be greatly appreciated!

Hi, team

I am currently analyzing DIA results using the Skyline button on DIA-NN in conjunction with Skyline24.1.
I am processing the identified proteins and peptides in Associate Proteins, and I am wondering if there is any difference in the processing when “Assigned to the protein with the most peptides” is selected in "Shared peptides are:" and when “Remove subset proteins” is additionally selected.
My understanding is that if the former process assigns the peptides to the protein with the most peptides, all subset proteins that can be explained by other proteins will be assigned to other proteins and removed, but as shown in the attached image, after selecting “Remove subset proteins”, Proteins, Peptides, and Shared Peptides were further reduced.
I would appreciate it if you could tell me which Proteins, Peptides, and Shared Peptides are removed by each command (Assigned to the protein with the most peptides, Remove subset proteins).

Sincerely,
Honda

Dear team

I wish to analyse(identify and quantitate) 7 peptides (6-13 amino acid peptides, not proteins) using PRM on Thermo Q Exactive plus. I am completely new to PRM and Skyline and learning them all by myself. Though you already have provided really good tutorials and videos, I still have some queries and  would be really grateful if you can help me in method development and analysis.

1. As per my understanding, centroid data is preferred for PRM on Q Exactive plus. Am I correct? If I collect centroid data on Q Exactive Plus, what do I select under product ion analyzer under MS/MS filtering- centroided or Orbitrap?
2. There are no spectral libraries available for 3 of the 7 peptides I am interested in. I also do not have the synthetic peptide to generate my own library. How can I proceed with their identification in Skyline (without the dotp scores)?
3. If I enable MS1 filtering, the peak area graphs would represent the precursor ion at MS level, while disabling MS1 filtering would mean that peak area graphs represent the unfragmented precursor observed in MS2. Is this correct?
4. I want to calculate fragmentation efficiency of the precursor ions. I have set isotope peak included as none under MS1 filtering and then calculated the ratio of summed peak areas of fragment ions to fragment plus precursor ions (obtained by viewing peak area graphs and selecting split graph). Is this approach correct?
5. I want to check the LOD of my system and have acquired MS data at various sample concentrations. I can see the precursor and fragment ion peaks in XCalibur but I am unavailable to get them in Skyline. I checked and unchecked the 'quantitative' option under transitions but it is not making any difference. How can I distinguish between quantitative and non-quantitative transitions?

Thanks in advance
Ramesh

Dear Skyline Support team ,

I am trying to set up a SureQuant method , I have done a survey run_directed DDA on my internal standards and have imported the raw files into skyline . I was wondering if skyline offers a tool to for peak picking ? or should I import into PD and use CHIMERYS ?

Best,
Sogol

Hi there,

I have some problems with the Skyline software regarding small molecule analysis. The details with screenshots are added as a Word file. In short, I optimized the measuring for optimized retention times with Skyline, but after method optimization one Peak is missing. Why is the peak gone and what can I do to improve my measurements?

Thanks for your help!
VT

Hello! I'm trying to make a calibration curve using your software but an error message appears during the processing : all of the external standards are missing one or more peaks.
I don't have any QC but I've entered the molecule transition on all the chromatograms. The resolved requests didn't really help.
Thanks in advance.

Hi, team

I am currently analyzing DIA results using the Skyline button on DIA-NN in conjunction with Skyline24.1.
I am processing the identified proteins and peptides in Associate Proteins, and I am wondering if there is any difference in the processing when “Assigned to the protein with the most peptides” is selected in "Shared peptides are:" and when “Remove subset proteins” is additionally selected.
My understanding is that if the former process assigns the peptides to the protein with the most peptides, all subset proteins that can be explained by other proteins will be assigned to other proteins and removed, but as shown in the attached image, after selecting “Remove subset proteins”, Proteins, Peptides, and Shared Peptides were further reduced.
I would appreciate it if you could tell me which Proteins, Peptides, and Shared Peptides are removed by each command (Assigned to the protein with the most peptides, Remove subset proteins).

Sincerely,
Honda

I have noticed a pattern where when doing a PRM analysis for a peptide in a complex sample, the peptide is not identified by proteome discoverer, but the peptide transition ions are found in skyline with an idotp > 0.9. Is this a reliable identification of the peptide? It is also observed at the correct retention time based on PRM of a positive control sample.

Is there a reason it is found in skyline but there is not a positive hit in proteome discoverer?

Dear Sir,

I would like to digest a protein randomly.

So ultimately obtain all possible peptides from the hydrolysis of a protein sequence.
Peptides must have between 6 and 20 amino acids.

Is it possible to achieve this with Skyline?

Best

M-Claude

Hello,

I have a batch of raw data that I am trying to import into Skyline. Some files import fine, whereas others show the chromatograms importing but then fail at the end, giving the following message:

At 17:44:
Failed importing results file 'C:\Users\sflannery\Documents\Temp_search_folder\TTP0700_MSQ2951_Pierfrancesco_20250207_PatientPilot_01A_1ugCo-digest_S1-E1_1_66011.d'.
Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\sflannery\Documents\Temp_search_folder\TTP0700_MSQ2951_Pierfrancesco_20250207_PatientPilot_01A_1ugCo-digest_S1-E1_1_66011.d'.
Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index ---> System.ArgumentOutOfRangeException: Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index
at System.ThrowHelper.ThrowArgumentOutOfRangeException(ExceptionArgument argument, ExceptionResource resource)
at pwiz.Skyline.Model.Results.ChromDataSet.get_BestChromatogram() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 89
at pwiz.Skyline.Model.Results.ChromDataSet.SetBestPeak(ChromDataPeakList peakSet, PeptideChromDataPeak bestPeptidePeak, Int32 indexSet) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 1207
at pwiz.Skyline.Model.Results.PeptideChromDataSets.PickPeptidePeaks(ICollection1 dataSets) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 601 at pwiz.Skyline.Model.Results.PeptideChromDataSets.PickChromatogramPeaks(ExplicitPeakBoundsFunc explicitPeakBoundsFunc) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 223 at pwiz.Skyline.Model.Results.ChromCacheBuilder.ScoreWriteChromDataSets(PeptideChromDataSets chromDataSets, Int32 threadIndex) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 107 at pwiz.Common.SystemUtil.ProducerConsumerWorker2.Consume(Object threadIndex) in C:\proj\skyline_24_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ProducerConsumerWorker.cs:line 186
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Results.ChromCacheBuilder.PostChromDataSet(PeptideChromDataSets chromDataSet) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1288
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 433
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256

This is timstof PRM data (.d). I have tried converting to another file format but get the same error. The data will open in Bruker DataAnalysis.
Skyline version is 24.1.0.199

If anyone has any insight I would be very grateful!

Many thanks,
SF

Greetings,

This is my first time using Skyline and I have the following issue. I am using a transition list for small molecules based on an acquisition method from a SCIEX instrument. I have noticed that the imported chromatograms have a relatively narrow Retention Time window. The result of this is that when an analyte's retention time shifts by eg. 0.2 mins between days the peak is cut off (see the attached image). This is only the case for Skyline, as the peak is not cut off on Analyst. I was wondering what options I have if I wish to use Skyline over Analyst for this data. I have tried looking for solutions here or asking a more experienced colleague.

Thank you in advance!

Attached Files: support.sky.zip on the File Sharing Folder.
Version: Skyline (64-bit) 24.1.0.199 (6a0775ef83)
OS: Windows 11

Hello,

I have a batch of raw data that I am trying to import into Skyline. Some files import fine, whereas others show the chromatograms importing but then fail at the end, giving the following message:

At 17:44:
Failed importing results file 'C:\Users\sflannery\Documents\Temp_search_folder\TTP0700_MSQ2951_Pierfrancesco_20250207_PatientPilot_01A_1ugCo-digest_S1-E1_1_66011.d'.
Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\sflannery\Documents\Temp_search_folder\TTP0700_MSQ2951_Pierfrancesco_20250207_PatientPilot_01A_1ugCo-digest_S1-E1_1_66011.d'.
Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index ---> System.ArgumentOutOfRangeException: Index was out of range. Must be non-negative and less than the size of the collection.
Parameter name: index
at System.ThrowHelper.ThrowArgumentOutOfRangeException(ExceptionArgument argument, ExceptionResource resource)
at pwiz.Skyline.Model.Results.ChromDataSet.get_BestChromatogram() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 89
at pwiz.Skyline.Model.Results.ChromDataSet.SetBestPeak(ChromDataPeakList peakSet, PeptideChromDataPeak bestPeptidePeak, Int32 indexSet) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 1207
at pwiz.Skyline.Model.Results.PeptideChromDataSets.PickPeptidePeaks(ICollection1 dataSets) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 601 at pwiz.Skyline.Model.Results.PeptideChromDataSets.PickChromatogramPeaks(ExplicitPeakBoundsFunc explicitPeakBoundsFunc) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 223 at pwiz.Skyline.Model.Results.ChromCacheBuilder.ScoreWriteChromDataSets(PeptideChromDataSets chromDataSets, Int32 threadIndex) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 107 at pwiz.Common.SystemUtil.ProducerConsumerWorker2.Consume(Object threadIndex) in C:\proj\skyline_24_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ProducerConsumerWorker.cs:line 186
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Results.ChromCacheBuilder.PostChromDataSet(PeptideChromDataSets chromDataSet) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1288
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 433
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256

This is timstof PRM data (.d). I have tried converting to another file format but get the same error. The data will open in Bruker DataAnalysis.
Skyline version is 24.1.0.199

If anyone has any insight I would be very grateful!

Many thanks,
SF

Hello,

Is there a work flow to quantify cysteinylation using NR peptide map data? Especially, I am interested in IgG 2 cysteinylation.
thanks
Babu

Hi Skyline support,

I'm a new user of skyline and really loving it. Thanks so much for this amazing tool!

However, I have a problem with uploading the full MS data from my raw file (Q-Ex Thermo) as I also have a t-SIM on the method on the 2 first minutes only to measure an internal standard we spike in and that is not in the range of our full MS method. When I upload the raw data on Skyline, I only see the t-SIM in the first 2 minutes. I saw a previous support ticket with a similar problem and they advice to convert in mzML file and then upload again to see the full MS but it did not work for me; still seeing only the t-SIM scan.

Do you by any chance have another solution I could use?

Thanks a lot in advance for your help and answer.

Best,
Lucille

Hi team,

I have PRM data for more than 100 samples. However, I have not acquired the samples in triplicates as I have biological replicates. Can I still use this data?
Hoping for a response.

I appreciate any help you can provide.

Hi team,

I would like to know why I should select “Remove subset proteins” after selecting “Assigned to the protein with the most peptides” for “Shared peptides are:” in the protein association.
My understanding is that the peptides of subset proteins with only shared peptides are assigned to the protein with the most peptides in the first command and eliminated, However, when I select “Remove subset proteins”, both peptides and proteins are reduced. Is there any exception to this?

Sincerely,
Honda

Hi,

Good day and hope you are well. I wanted to make an enquiry on constructing a calibration curve in skyline. Currently I have multiple replicates per each standard in my calibration however i only know to construct the curve in skyline using one replicate per standard. is it possible to do this with multiple replicates please?

Kind regards,
Elliot.

Hi Team,

I am performing small molecule quant on an exploris120, and have an MS1 survey scan along with several targeted narrow SIM scans (0.4 Da width). Unfortunately, my MS1 survey scan has a scan window of less than 500 Da (m/z 150-400), which means that Skyline assigns this as a SIM scan on import.

Is it possible to adjust this value somewhere in Skyline? I have had a look around and cannot see it. In future I will modify my MS method to have a survey scan greater than 500 Da, however I only just discovered this issue today after running some relatively precious samples.

Thanks for all your hardwork in keeping an amazing program running.

Cheers
Nick

Hi, Skyline Team.

I'm running into issues installing the Avant Garde tool in Skyline. I've tried to install it through both the Tool Store and as an External Tool, but I keep running into issues with specific R packages not installing. The two packages that won't install are stringr and ggplot2. This is happening on several different computers running the most current version of Skyline Daily.

Do you have any suggestions about how to remedy this? I tried installing the specified versions of ggplot2 (3.2.1) and stringr (1.3.0) outside of Skyline through R (v 4.0.3) but there were issues with the tool when I did that. I attached the error message I got related to the tool stoor failed installation of ggplot2 and stringr.

Scott

Hei, I have one Internal Standard (Isotope) per Analyte but I don't get how to assign it as Internal Standard (Small Molecule Interface). Everything I have tried so far didn't work. So if I recalculate with a slightly different integrated IS area it doesn't change the conc. for my analyte. Can you help? I didn't found anything in the tutorials, or I've overseen it.
Thanks in advance :)

Hi Nick,
I am conducting a metabolomics experiment where I analyze a library of metabolites from mice over multiple time points. Every few days, I collect samples and run them on the instrument, but due to normal instrument variations (such as injections from other projects conducted in our lab, cleaning, and calibration), ionization efficiency fluctuates over time.

I have two main questions regarding handling batch effects in Skyline:

1. Batch Grouping in Analysis & Visualization
   I would like to assign my samples into different batch groups within Skyline so that I can compare a specific metabolite’s integration across different batches within a single analysis.
   o What is the recommended approach to organize and visualize batch-specific comparisons?
   o Is there a way to display bar plots for each batch, with individual data points representing the distribution within the batch? Alternatively, are there other built-in visualization tools to observe batch differences without exporting the data to external tools?
2. Normalization Across Batches
   I include internal standards in my samples by adding them to the extraction solvents. Given this, is there a way in Skyline to normalize signal intensities across batches to correct for ionization efficiency fluctuations? Would Skyline support an automated correction approach, or should I calculate and apply normalization factors externally?

I would appreciate any recommendations or best practices for addressing these issues.
Thanks,
Michal

Hi team,
I have raw data which contain full-scan only (without MS2 (PRM/DDA/DIA) data) and woud like to draw MS1 XIC using Skyline.
There are instructions which explain how filter MS1 (and MS2) from DDA/DIA/PRM data and I follow them.
However MS1 XIC did not show in the chromatogram window (instead I could view TIC).
Could you please if I can do what I want to do? If yes, please teach me how I can do.
Best,
TY

Hi Team,

I noticed that when I graph volcano plot in Skyline, some proteins/peptides were missed in the volcano plot. These peptides had good peak and were integrated in the protein peak area report.
I also noticed that different numbers of proteins were included in the volcano plot if using different summary methods (Turkey's median polish or sum of transition area).
Any idea what could be the reason?

Thanks
Qingling

Hello,

I am trying to optimise my MRM method and wanted to use Skyline. I am looking at 3 synthetic peptides, each heavy labeled. In Skyline, I tried to add the fasta with my 3 peptides but it is giving me this chat: This operation has added 3 new proteins with no peptides meeting your current filter criteria. I have also tried to run a peptide seach with no success. I have 3 mzml files corresponding to my PRM runs.

Could I have some info as to how to proceed so I can look at my data?

In case that helps, my fasta with my 3 peptides looks like this:

CRP| DYSLFSYATK^
DYSLFSYATK

Is it actually possible to add information on the fasta to show it is heavily labeled on the C-terminus?

Any help would be greatly appreciated.

Best wishes,
Cecile

Hello,
I tried to build a spectral library for a list of phosphopeptides using Koina on Skyline, but it says it is not supported. I was wondering how I could solve this problem. I have attached the error I encountered.

Hello skyline team, I have done a search for a timsTOF data with Peaks. Now I tried to create a library with the exported pepxml file. but it shows reading spectra from the raw file and take forever. then it shows no spectra in the library. could you please help with this issue?

Dear Skyline Team,
is there way to do PRM CE optimization with Thermo Stellar? I noticed in the method export feature the CE option is greyed out for Stellar.
Is it possible to import a manually set-up CE stepping run (couldn't get this working properly yet)?

Maybe related or not needed in this case: how do I set the regression parameters for normalized (%) CE on Thermo (Stellar) under 'Transition Settings' -'Prediction' - 'Collision energy'

Thanks a lot!

Hello!

I wanted to ask about how to determine signal to noise of a specific feature/ion on my spectra. Is there a way to calculate it on the spectra level?

Thanks!
Justin E :)

Hi Skyline tram,

I have a few technical questions about Skyline:

1. Where do I define mass accuracy for metabolite signal extraction? At what stage should this be set, and can it be applied after the analysis is already complete?

2. After modifying a metabolite (e.g., updating its formula, retention time, etc.), how can I reprocess the batch to reflect the changes?

3. How can I reprocess the batch after adding a completely new analyte to the list?

4. Can the software identify a compound based on its entire isotopic pattern, rather than just the primary peak?

Thank you for your help!
Michal

Is there a database to search for absolute quantification protein data?

Hi, this might be more of a feature request (if there is no other way to do this):
The full scan m/z graph -when doing CE optimization- can be shown by clicking the retention time profile trace for a certain step-energy only if a single transition is selected under targets (plus using the zooming option in the full-scan window). Selecting traces is not possible if the precursor itself is selected under targets (and therefore the summed intensities for all transitions are shown). Selecting the CE-steps from the summed intensities would help find transitions in PRM mode that have not been annotated yet and might be useful to add for quantification.
Or is there another way to show the full scan spectra for the CE-steps?

Thank you!

Hi

I collected some negative PRM mode data on a Q Exactive. Skyline reads it as positive mode; the error message follows:

"Failed importing results file.
This document contains only negative ion mode transitions, and the imported file contains only positive ion mode data so nothing can be loaded."

The same issue occurred with all the data files.

When I used the same transition list and raw data on another computer, everything worked without any issues. I can't figure out what's causing the problem.

I'll wait for your reply.

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

Hi Skyline community,

I am using Skyline for small molecules quantification.
For the quantification of some metabolites, I don't have standard but I have two internal standards in the samples and relative response factors of my interest metabolites.
Is it possible to implement these data in Skyline workflow for the quantification of large-scale metabolites?
I have not found the documents in Skyline and please advise me.

Thanks and regards,
Nay Min

Hi team,

I am facing an issue with visualizing all the raw files together. I have 160 raw files along with 11 standards in triplicates, and I am unable to check them all at once. Is there a way to view them collectively?

Thank you in advance.

as skyline only support several type library file. I want to use spectronaut library(.txt) as skyline library for PRM test. How to set?

I would like to perform data analysis using Skyline for a while. I am trying to visualize specific glycopeptide precursors and the transitions corresponding to specific sugar masses. I can add glycopeptides along with their sugar compositions and visualize their transitions. However, after adding the glycopeptides, I cannot view only the Q3 sugar mass transition in MRM mode. For example, the precursor (Q1) mass of the glycopeptide is 1129.59 m/z, and the transition (Q3) mass is 366.14 m/z. I had entered these specific masses in the MRM method, but I am currently unable to view this data in Skyline. I hope there is a solution for this issue in Skyline, and I appreciate your response in advance.

Hi,

I have run two samples on our Exploris 480, one sample in DDA and the second using DIA (selecting the Thermo DIA template from their Method Editor). I am now having an issue when trying to set up the DIA Isolation Scheme in Skyline.

Previously for diaPASEF files I had been able to set up the DIA isolation scheme in Skyline by selecting the "Prespecified isolation windows" button and using the "Import" link to locate and extract the window information directly from the file. However when I try this with the Thermo DIA file I get the following warning (depending on whether or not "Specify Margin" has been selected).

If I select the "Specify margin" option I get "There are gaps in a single cycle of your extraction windows. Are you sure you want to continue?" The Thermo method does have a Window Overlap of 1 m/z and the margin in Skyline is showing up as 0.5002.

If I de-select the option I get "There are overlaps in a single cycle of your extraction window. Are you sure you want to continue?"

I would appreciate if you could help figure out what I am doing wrong. By the way I have gone ahead and used both versions of the Isolation schemes, with and without margins to extract the targets and targets are extracted in both cases.

Thanks in advance
Caitriona

I am running into an error whilst trying to install onto our windows server. I was wondering if it possble to run Skyline on such an operating system?

Many thanks

rob

Edition Windows Server 2025 Datacenter
Version 24H2
Installed on ‎1/‎10/‎2025
OS build 26100.2605
Experience Windows Feature Experience Pack 1000.26100.36.0

Hello,

I have created a spectral library in Skyline and exported it in .blib format. How can I convert this into a .tsv format that is compatible with DIA-NN?

Thank you,
Sofani

Hello,

Thank you in advance for helping with this issue, which seems to be a common problem. I have received the following error using the following:

Skyline (64-bit) 24.1.0.199 (6a0775ef83)
My computer: Windows 10 Home ver. 22H2
Acquisition computer: Windows 10 enterprise 2016 LTSB ver. 1607

Failure opening C:\Users\Thermo\Desktop\250109D_EO_Select5\250109D_EO_Select5.sky.
The file contains an error on line 1799 at column 16.

I can use the program to export data but as soon as I close and reopen on either my system or our instrument acquisition computer I will get this error message. I have attached my files.

I am trying to use skyline to quantify multiple transitions for unmodified and modified peptides of the same sequence using PRM.

Again, thank you!

Hi Skyline Team,

I have written a script to automate the import of transition list CSV files into Skyline. My code is structured as follows; it is looping across all csv files.

%SKYLINE_CMD% --in=%INPUT_SKY_FILE% --import-transition-list=!TRANSITION_LIST! --save --out=!OUTPUT_FILE!

Here:

INPUT_SKY_FILE is my template Skyline file, saved in the molecule interface.
Each TRANSITION_LIST file contains the following headers:

Molecule Name Precursor Mz Fragment Ion Precursor Charge Collision Energy Product Mz Product Charge Explicit Retention Time Explicit Retention Time Window

This script worked correctly when I used the peptide interface and had PeptideModifiedSequence in the headers. However, now that I am working with the molecule interface and using Molecule Name, the transition list fails to import properly.

Could you please provide guidance on how to modify the script or troubleshoot this issue? Are there any specific changes required for importing transition lists into the molecule interface?

Thank you for your assistance.

Best regards,
Anirudh

Hello!

I am trying to build calibration curves for several small molecules using light:heavy normalization (simple precursors), and use Skyline-daily (64-bit) 24.1.1.339 to determine instrument LOD. I am using PRM an Orbitrap Exploris 240. I am using linear regression and 1/x^2 weighting. My "Standard"s are all spiked with the same concentration of internal standard. My "Blank" contains no spiked analytes and the same spiked concentration of internal standard.

By visual inspection and peak area totals, there are some instances where I can't differentiate the lowest points of my curve from one another, or the blanks, which I am expecting. However, some calculated LODs fall within this visually-indifferentiable range. For example, visually I'd expect the LOD for 1,3-Diphenylguanidine to be closer to 0.5 ng/mL, but the calculated value is 0.097 ng/mL. Some analytes (Amantadine and Gabapentin) have negative values. Can you help me to understand why this is? I'm wondering if this has something to do with the presence of my analyte in my blanks due to carryover, or how I've got it integrated, or something different entirely (possibly my understanding/interpretation of how to make my cal curves and calculate LOD).

I'm fairly new to Skyline so any tips or advice you have is greatly appreciated!

Thanks!
Kat

Hi Skyline team,
I have a discovery DIA dataset, searched through Spectronaut, and I'm currently trying to compare the identifications of a protein of interest between two sample groups, which were run alongside a recombinant expression of this protein.
I was hoping to use Skyline to look more into the data, and to provide some additional filtering.
Some of what I wanted to achieve has been possible through Refine>Advanced and setting thresholds here but I was also hoping to filter based on retention time. Is there a way I can set a peptides retention time using a sample I am confident in and then use this as a basis for filtering in other samples?
Thanks for your help!

Dear Skyline Team,

thanks for all the work you have put so far into your programm and community!
I am a young researcher and just starting in the field of Lipidomics.
Currently I have the challenge, that most of the Ion Mobility values which I generated from a standard lipid mix are not displayed in Skyline.
The measurements are done on a Bruker TIMS-TOF, this problem is found in both polarity modes.
For example, in my measurement the CCS value for [HexCer18:1/16:0+HCOO-] is displayed, while the Value for the same compound [M-H] is not. For further explanation a screenshot from Skyline is attached. So I looked this Ion up in the DataAnalysis software from Bruker and found the same ion mobility for the [M-H]-peak. Both Ion Mobilograms can be found in the second screenshot.
To work around this problem, I tried different resolving powers while importing under the tab Settings>Transition Settings>Ion Mobility.
So far, nothing worked, thats why I put my hope in you.
If needed, I will upload the skyline file as well.

Hope you have a nice day.

All the best,
Thomas