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,

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

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

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

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

I've successfully implemented using the lists/live reports feature in Skyline Daily - with an exception that I think could be an easy fix (maybe a way around it now as well).

My collaborators had some duplicate Animal ID numbers (think 2 different cohorts of animals #104). They had different chip numbers which were unique 9 digit codes, and then I'm also adding in 'subgroup' and 'sex' into my list. In my runs/raw files, when I had duplicate animal IDs, I used a shortcut of animalID_last 2 digits of the chip (so 104_01 and 104_38 to differentiate two animals without having to include the 9 digit numbers).

The lists feature currently does not currently support that format - it didn't like either '_' or '-' and so I'm currently only getting successful imports for the unique animals. I was thinking one way around would be to replace the dash with 0 - an easy find/replace on my list and update to my runs. But I feel like this might be something that happens to more than one person and if we could incorporate a character into the unique ID line on the lists that would be great.

Please let me know if you think I've missed something or another easy work around already exits. Thanks as always!

Hi, I'm experimenting with small molecule work in Skyline and have a few questions that I have not seen addressed in the documentation or other questions.

1. When building spectral libraries, right now I am converting a zenoTOF wiff2 file into mzml, and selecting scans manually for entry into an ssl file. This works, but has been very tedious. In the documentation, there is a list of supported search engines, but they all seem to be for proteomics. Is there a supported search engine for small molecules to aid in library building/annotation? If not, are there any faster ways that would be useful for me to be aware of?

2. With the above method of creating library entries, I often have high-abundance precursor peaks in the library spectrum. Ideally, I'd like to filter these out from quantitation/selected transitions automatically. The 'Precursor m/z exclusion window' under Transition Settings > Filter seems like it should do this, but in practice it has not worked to filter out these peaks. Can you advise?

Thank you for any help you can provide!

Sincerely,
Morgan Mann

Hi, can anyone suggest any tutorials to characterize peptide sequence coverage? We're working on tryptic digestion of monoclonal antibodies (Trastuzumab biosimilar) and developing a peptide mapping method.

Thanks,
Jinlai

Is there an available version of Skyline for MAC OS?

Hello! There are a few options for calibration models for quantitation (I'm currently working on some small molecule quantitation). Is there any functionality in Skyline or Skyline-daily to evaluate goodness of fit? For example, a way to visualize a residuals plot or perform statistical testing? Thanks!

Hi skyline team,

1. If I have a batch I’ve already analyzed and want to add more metabolites to it, what’s the best way to do so while ensuring the updated batch retains the manual integrations I’ve already adjusted?
2. I seem to have accidentally disabled the feature that colors the peak area in red. Now, I only see the black boundaries for selected peaks. How can I restore the red shading?
3. Lastly, if I want to update the retention time for an existing molecule, what is the correct way to do so?

Thank you so much for your assistance,
Michal

Hello Skyline team, I have generated a prmPASEF data with timsTOF flex. However when I loaded into the the skyline, the precursor for M, M+1 and M+2 are 3 straight lines instead of forming peaks. I wonder why it is like this? and is there a general protocol for how to use skyline to analyze the prmPASEF data?

Hi Skyline Team,

I would like to request a feature that would allow us to remove a specific result file from a Skyline document based on the file name when using the Skyline command line interface. Currently, removing result files by date is possible, but being able to specify the file name directly would be incredibly helpful—especially in cases where the wrong result file was imported by mistake.

Hi Skyline Team,

We’ve encountered an issue in our lab where some team members are unable to open Skyline files directly from their file locations after upgrading to Skyline Daily 24.1. Interestingly, this issue disappears when we revert to Skyline 24.1.

We’re unsure why this happens, but to address it, we were wondering if there’s a way to use the command-line arguments to automate the File -> Share -> Version process. I’ve reviewed the documentation but couldn’t find any information about specifying the version through the command line.

Since we manage a large number of Skyline files, manually updating each file is highly time-consuming. If it’s possible to use Skyline Daily's command-line functionality to streamline this process, it would greatly help our team access files in earlier versions.

Best,
Anirudh

I just noticed that some proteins that contain a certain peptide several times are not filtered by protein uniqueness. This could be problematic with absolute quantification. Maybe an idea to add that option as well in the future?

thank you!

Hi Skyline Team,

I tried opening Skyline today, but it's not launching. Similarly, Skyline Runner isn't opening either. Everything was working fine yesterday, and I haven't made any changes since then. Could you suggest any steps I can take to troubleshoot and figure out what's happening?

Thank you for your help!

Best regards,
Anirudh

I am trying to find the best way to get let's say up to 10 unique peptides per protein that match my length and modification criteria. I have a spectral GPF library active and want to select those preferentially. For those that don't match any of my library or are only below ten peptides, I want to fill up with the best ranked.

I feel like there is a workaround here:)

Dear Skyline,

I am trying to generate a calibration curve using heavy and light peptides in a background matrix that contains my endogenous light peptide.

Could you provide guidance on what I need to do.

Conventionally I would spike in a constant heavy amount of peptide with changing light peptide in a constant background matrix conc. However, my matrix contains endogenous light peptide.

What would you suggest? Keep light peptide spike in constant and vary the heavy peptide?

Many thanks,
Mehul

Hi Skyline Team!

I have two calibration curves. One for external standard and one for surrogate standard. Surrogate standard was spiked into an unknown sample at specific concentration let's say 3ppb. From the surrogate standard calibration curve in Skyline I can find out the "recovered" concentration of that surrogate standard in unknown sample and I am now wondering how to apply this value to calculate recovery-collected concentration value of target compound in unknown samples.

Hi Skyline team,
I have a use case where I'm comparing the performance of several spectral libraries for the same set of raw files. It would be great if I could compare different spectral libraries within the same Skyline document without reimporting the raw files. That is, I want to keep the settings in the doc exactly the same and just compare the results of loading spectral library A vs spectral library B.

I know that I can go to Peptide Settings > Library > Edit to import a new library, but I get the feeling that Skyline doesn't actually update the settings according to the new library. The only way that I've been able to successfully perform this task so far is by creating separate Skyline docs for each library, which is cumbersome and slow.

Is there anyway to do this within the same Skyline doc?

Thanks,
Lincoln

HI,
While importing a raw data file from Waters instrument, I got the following error on Skyline. can someone please help ;

At 14:48:
Failed importing results file 'O:\CCNR.group\LCMS1_002_HPMB_BA10_30_01.raw\LCMS1_002_HPMB_BA10_30_01.raw'.
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Generic Error

Inner exceptions:
Exception type: System.Exception
Error message: [pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Generic Error
[pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: Generic Error
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_SpectrumList() in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 507
at pwiz.ProteowizardWrapper.MsDataFileImpl.get_HasSrmSpectra() in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 742
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\Users\nicksh\git\skyline_42_installer\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 194

I am running heavy standards with the corresponding non-labeled ones neat in PRM mode. I have added all precursor adducts in Skyline. If I don't know the transitions or want all the transitions that are optimal in my set-up, is it possible to automatically extract them based on the mass shift between heavy and light?
Or do I need to buy small molecule spectral libraries?

(coming from proteomics, so sorry for my missing knowledge)

Thanks a lot!

I use the version : Version: 3.0.24196-4dd34ff in MS convert
I acquire data from Waters Vion qTOF in MSe mode.
When I try to convert the UEP files to mzML files, I can see both the MS level data. To look at the data i use Msnbase package in R , by loading the data with readMSData function.
When I try to convert the Masslynx. raw files to mzML files with MS convert , then load in R with the function mentioned above. It reads all the spectra as MS1 ..
Kindly please help.. I am hereby attaching a sample file..

Hello,

I have an mzML file of unscheduled PRM data where I was continuously targeting some low abundance peptides. I've been searching the data with Fragpipe and then using the Skyline document generated by Fragpipe to look at the chromatograms for the targets. There are a few of the targets that Fragpipe fails to find, but I'd still like to extract chromatograms for their precursor and product m/z values to try and diagnose what's going on.

However, I can't figure out how to manually specify a peptide and its charge state, and then have Skyline extract chromatograms for the m/z values of its precursors and products. I've only been able to extract chromatograms for peptides that were successfully identified in the Fragpipe search.

I've tried using Skyline's built-in search with both MS Amanda and the built-in version of Fragpipe, but they also don't find the same targets, so I'm still not able to extract the chromatograms.

Is there a way to load an mzML file into Skyline, specify a list of peptides and charges, and then extract chromatograms for those targets from the mzML file, regardless of any associated search results?

Sorry if this is already answered somewhere--I've been reading through the tutorials and other forum posts and I keep thinking I've found the answer, but it's always not quite what I'm looking for.

Thanks,

Caleb

"G12D_MSMS_BEH_100ng" can be imported into skyline and "20241213 G12D.sky" is the processed data.

While "G12D-CSH_MS2" can't under same procedures.

I'm wondering what causes this.

I use the version : Version: 3.0.24196-4dd34ff in MS convert
I acquire data from Waters Vion qTOF in MSe mode.
When I try to convert the UEP files to mzML files, I can see both the MS level data. To look at the data i use Msnbase package in R , by loading the data with readMSData function.
When I try to convert the Masslynx. raw files to mzML files with MS convert , then load in R with the function mentioned above. It reads all the spectra as MS1 ..
Kindly please help.. I am hereby attaching a sample file..

Hello!

I would like to export a report or transition list containing predicted RTs and the top n transitions from Koina for a given list of peptide targets. Is this currently possible? If so is there any documentation for how to do this?

Thanks for your help,
Vincent

Hi,
Using the neat feature of the export (multiple) methods. Since Skyline is usually installed on personal computers and not on the MS-linked computer, it would be great to generate those methods anyway. Is this a thermo requirement, or is there a workaround to install the necessary software on the user PCs as well?

thank you!

Skyline-daily

An error occurred attempting to export.
ERROR: Registry key (Software\Thermo Instruments\TNG\Stellar) not found. Stellar is not installed on this machine.

Command-line: Method\Thermo\BuildThermoMethod -t -m "W:\User\yx.meth" "..transitions.txt"
Working directory: .....\

OK More Info

Skyline-daily (64-bit) 24.1.1.284 (bc93c2813)

System.IO.IOException: ERROR: Registry key (Software\Thermo Instruments\TNG\Stellar) not found. Stellar is not installed on this machine.

About 8 peptides were tested by waters QTof under MSMS mode. Peptide 1 resulted in "Chromatogram information unavailable" When I used skyline to analyze raw data, while others could work under same settings.

I am confused and want to know how to solve this.

I uploaded two peptieds information and results(peptide 1 and 2) in the file.

Dear Skyline team,

Two years ago a former colleague of mine posted a question titled: "Sciex TripleQuad7500 .Wiff2 support" (roosso 2022-07-05 22:41).
We still encounter this problem, also with the newest Skyline versions.
Some methods only generate ".wiff2" files. We think this happens when we apply experiment scheduling (3.5min of ESI- followed by 3.5 min ESI+)
On import of the .wiff2 file in Skyline we get the error below:

"Failed importing results file ...
The sample .... contains no usable data."

This also happens for methods where both the .wiff and .wiff2 files are generated and we try to import the wiff2 file.
I attached a Skyline file which we use for our 'System Suitability Test', including some raw data we would like to import. The .wiff file succeeds, the wiff2 file fails to import.

The last two years we used work arounds, so that SciexOS would generate .wiff files.
At moment we develop a method which requires experiment scheduling, and so will only generate .wiff2 files.
Could you please advise how to import wiff2 files?

Thanks a lot!

Regards,

Jeroen Kooistra

Hi,

I was wondering if it was possible to have a peptide number per identifies proteins in the customs report and export it ?

Have a nice day,

Tania

Hi Skyline team ,

I am having a bit of trouble adding the background proteome to a skyline document.
I have downloaded a Fast fie from Uniprot , and than in Skyline document I go to Setting >Peptide Setting >Digestion tab and add the background proteome . how ever in my target list the peptides are not assigned to the proteins as they should be . Can you help me fix this ?

P.S. The skyline document is for a Surequant Assay on there samples . So the peptides of interest and the the heavy and light precursors are in the list of targets .

Thank you for your continued support,
Best,
Sogol

Hello Skyline team.
I am working on post-translational modifications and am using skyline to export PRM lists. I would like to generate a list of peptides containing only a specific modification based on the peptide search results, is there a way to do this?
Unfortunately, search results exported from PD cannot be filtered for specific PTMs and .pdResult files imported into Skyline cannot be filtered for specific PTMs.

Yours,
Zimeng

While you provide great tutorials and fantastic support, I was wondering if a readme is available with the full documentation of all features, settings, GUI usage, etc.? That would be fantastic for knowledge-based chatbots. Especially in the beginning, where finding some of the features sometimes can be a a little difficult.

Tanks you,
best
André

Getting error message when saving Skyline .sky files to our file server.

Error states "Failed writing to ... the process cannot access the file because it is being used by another process".

Please advise

Hello,

I was in the lipidomics webinar today and was curious if there's a way to do isotope corrections and isotope abundance calculations directly in Skyline?

Thanks!

Hi, I would like to use Skyline for untargeted lipidomics analysis. I have an Exploris240, and LipidSearch for lipids identification. I am doing AcquireX for high-throuput lipidomics. I am also using more than one internal standards for lipids class normalization.

I have never used Skyline, and I would like to know if you have a Tutorial for that.

Thank you very much
Nidia

Good evening,
lately I am unable to import raw files to the Skyline (I just uploaded the newest version). Everytime I try to upload my result files by the usual way, it starts importing and and after few seconds each of the files gives the "failed" which imideately turns into the "canceled" yellow saying. Sometimes first few injections upload, but the rest is always blocked from importing. Everytime the data are not open anywhere else. I tried rebuild the .skyd cache, reexporting the files in case there would be problem in them, saved the Skyline for those data in different folder in case there would be the clash there, checked the read/write permissions. But here I am done with what I had in my mind that could possibly be the problem. Could you help me solve this issue please? I am attaching the error statement.

So I´m facing some method development issues for Surequant with heavy labelled peptides. I followed the tutotrial for Surequant but when analysing my files, I saw there was no signal for my endogenous peptides which were spikedin at 250 ng. Could there be a specific reason for this?

I'm very new to Skyline and am trying to set up my project for some DIA data collected on a Bruker timsTOF HT. In doing so I have a few questions...

1. When configuring the full-scan settings, is it best to use the resolving power that is stated by Bruker for this instrument (60,000)? I ask as I read in another post that the resolution setting in Skyline is used to define the width of the XIC extraction, and if in MS1 it is set a little lower than the instruments stated resolution, it can account a bit better for potential mass accuracy deviation. If going a little lower is useful, do you have any suggestions on what this should be?
2. How do I determine what the ion mobility filtering resolving power should be? Is this something I should be able to ascertain from my data?
   Thank you for your help!

Hello,
I am wondering if we can import results from FRAGPIPE to create spectral libraries on Skyline. If yes, what formats are supported by Skyline.

Thanks

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

I am trying to create the library with msms.txt file from MaxQuant but I keep receiving a error. What do I need to do in order to proceed?
Thank you!

Similar to the existing routine for optimizing CE on a triple quad, is it possible to optimise % HCD and/or % CID on a Thermo Stellar MS?

Dear Skyline team,

I am currently working on generating calibration curves for several peptides. Despite observing good peak areas, I am unable to obtain a calibration curve for some peptides. I have attached a screenshot for your reference.

Additionally, I would appreciate guidance on selecting the appropriate quantification settings, as there are multiple options available (e.g., linear, bilinear). Could you please advise on which method is recommended and how to approach LOD calculation? Is it possible to choose different quantification options for individual peptides?

Thank you.
Akhila

Hello,

I'm running a DDA skyline file to look at certain peptides and it's giving me cut-off peaks near the middle. I'm using an orbitrap mass analyzer to run my samples. The peak and transition settings are attached below. The same samples were ran with PRM and the peaks looked completely fine.

Hi Skyline Team,

I am having an issue in my document regarding the "Enforce Peptide Uniqueness by: Proteins" function.

My target list contains a list of peptides that I thought were unique, but when I copied the peptides and pasted them back in the document, some were flagged as also being present in the background proteome. The proteins I am building a targeted method with are recombinantly expressed and are from another organism entirely.

It was my understanding that when you enforce peptide uniqueness by protein, any peptides in the background proteome should be removed from the target list of peptides, but this does not seem to be happening in my case.

When I select Edit>Unique Peptides, I can see that some of the peptides are shared with background proteins. So I am unsure as to why they are not being removed when I enforce peptide uniqueness.

Any help on this would be greatly appreciated.

-Peter

Hello Skyline team,

I am trying to build a chromatogram library using GPF runs by setting the parameter "run peptide search." When I finish, I get the "No matched passed score filter in." How do I solve this issue?

Thank you
Vanya

If data has been acquired using an acquisition method that uses both HCD (beam type fragmentation in a collision cell) and CID (Resonant fragmentation in an Ion Trap) on the same precursor, can those filters be separated in Skyline? Currently, when I try to process with Skyline, it treats the MS2 fragments the same, so the you get a noisy combined XIC.

Subject: Assistance with mProphet Peak Picking in Skyline

Dear Skyline Team,

I hope this message finds you well.

I have encountered some instances where Skyline appears to overlook clear candidate peaks, which results in the mProphet peak picking model selecting incorrect peaks. In the attached screenshot, you can see an example of a very straightforward peak that Skyline does not even recognize as a candidate.

Could you please advise on possible reasons for this behavior and suggest any steps I could take to resolve it?

Thank you for your assistance!

Best regards,
Anirudh

Hi, I am new to Skiline and beleive I must be missing something when importing my raw file. I can open the file in MassLynx with a problem, however, the file looks like it is importing up until the very end and then gives the error message.

At 6:07 PM:
Failed importing results file 'C:\Users\RobertRegister\Dropbox\Skyline\Tocilizumab 61 min scan.raw'.
error reading spectrum function=1 process=0 scan=24757
Under More Info it says

At 6:07 PM:
Failed importing results file 'C:\Users\RobertRegister\Dropbox\Skyline\Tocilizumab 61 min scan.raw'.
error reading spectrum function=1 process=0 scan=24757
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\RobertRegister\Dropbox\Skyline\Tocilizumab 61 min scan.raw'.
error reading spectrum function=1 process=0 scan=24757 ---> System.Reflection.TargetInvocationException: error reading spectrum function=1 process=0 scan=24757 ---> System.Exception: error reading spectrum function=1 process=0 scan=24757 ---> System.Exception: [pwiz::CLI::msdata::SpectrumList::spectrum] Unhandled exception: EOF Scan File Read Error
at pwiz.CLI.msdata.SpectrumList.spectrum(Int32 index, DetailLevel detailLevel)
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetCachedSpectrum(Int32 scanIndex, DetailLevel detailLevel) in C:\proj\skyline_24_1\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 1384
at pwiz.ProteowizardWrapper.MsDataFileImpl.GetSpectrum(Int32 spectrumIndex) in C:\proj\skyline_24_1\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 995
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1109
--- End of inner exception stack trace ---
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.ReadSpectrum(Int32& i) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1160
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.Read() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 987
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.<RunAsync>b__33_0() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 845
--- End of inner exception stack trace ---
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Util\Util.cs:line 1926
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.NextSpectrum() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 950
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatogramsLocked() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 273
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.ExtractChromatograms() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 253
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.SetRequestOrder(IList`1 chromatogramRequestOrder) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 617
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 388
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256
--- End of inner exception stack trace ---
Any idea what is going on? Also, it says Chomatogram information unavailable.

Any help would be appreciated !! Bruce

Hi,

I'm currently quantifying small molecules using Skyline. For some analytes, I have labeled internal standards, but for others, I don't. So, I'm considering using surrogate internal standards from different molecules. I'd like to assign these individually but haven't found instructions on how to do this.

Could you please guide me on where to find this information?

Thank you.

Dear Skyline,

Is there a way to set digestion enzyme as non-specific for the Background proteome? In the context of peptidome analysis having a non-specific background would be quite useful.

Thanks in advance,
Li

Dear Skyline,

I am now encounting an issue when I am importing the spectral library into the Skyline.

I used the .speclib format file and report.tsv files that are generated from the DIANN and renamed them as "NANE.speclib" and "NAME_report.tsv". I imported the spectral libraries through File-Import-Peptide Search-Add files...Then it came an error information which asked me if that report.tsv is the correct one for the .speclib file. Do you know what might be the potential cause for this issue and how I can slove it?

FYI I am using DIA-NN 1.8.2 beta 34 and Skyline daily versions. The error information is also attached.

Thanks in advance!

Best wishes,
Yuxing

Hi Nick,

I am doing ToF MRM for peptide quantitation in my sample with heavy-isotope labeled peptides.

1. Can you please help to calculate LOD and LOQ in skyline?
2. CV calculation?

Please share any tutorials having all these

Thanks
AD

Dear Skyline Team and Community,

I'm generating spectral libraries from ssl files via the command line tool with the following (simplified) code:

"D:\Software\Skyline\SkylineRunner.exe" ^
--in="tool_comparison.sky" ^
--import-search-file="results_tool1.ssl" ^
--out="tool_comparison.sky

How can I set the name of the generated spectral library? Currently, it is automatically named "tool_comparison.blib". Is there any option to assign a name or have it named after the ssl file (i.e. "results_tool1.blib")?

Additionally I receive the following warning: "Warning: Unable to locate results file 'results_tool1.mgf'". This does not seem to cause any major error for my analysis, nevertheless it would be nice to understand why this is happening. The mgf file is in the same folder as the ssl file and naming in the ssl file is correct.

Thanks in advance! Cheers
Jonas

Dear Sykline-Team and Community,

I'm looking for help in streamlining some skyline analysis using the SkylineRunner.exe / command line version.

I have a list of peptides in txt format (peptides.txt file attached). Is there any possibility to add these peptides to the Skyline document using SkylineRunner.exe? When analyzing the data using the GUI, I just copy them into the target list which creates a "peptides1" entry with all of the peptides - something similar would be needed.

I do have a spectral library for all the peptides in "peptides.txt" which I add using --add-library-path, peak boundaries from a search engine which I add using --import-peak-boundaries and a custom report which I can also create using --report-add. The only thing missing to have this completely in CMD is adding the peptides to the document.

Additionally, I add the corresponding raw files using --import-file and --import-replicate-name, i.e.

"SkylineRunner.exe" ^
--in="test.sky" ^
--import-file="condition_untreated1_20220522.raw" ^
--import-replicate-name="untreated" ^
--import-file="condition_treated1_20220522.raw" ^
--import-replicate-name="+treatment_1" ^
--out="id_all.sky"

However when doing this, the two rawfile end up both in the replicate "+treatment_1". How to have them in separate replicates as defined in the command? When I run the imports after each other, it works fine.

Thanks in advance for you help!
Jonas

Hello,

I am working on a project with tmt labeled small molecules. I would like to assign colors for the MS1 peaks I have assigned for each of the small molecules and their tag. I noticed there is a tutorial for this for peptides, but I can not seem to make it work for my small molecules.

Dear Skyline support,

I am looking for peptide quantitation in my sample using heavy standards of my peptides. After making the calibration curve based on serial dilutions of heavy standards, if selected normalization method as heavy in the quantification tab, I am not getting R2 of 0.9. While keeping quantification method as none, I am getting R2=0.9.

1. Am I doing something wrong in making calibration curve?
2. I am not able to see the peptide quantitation in (nmol) normalized to heavy.
3. In the calibration curve, peak area is there on y axis but not light to heavy peak area ratio.

Please guide me, which settings I need to follow to get my results.

Thank you
Arpita

Since diann 1.9 version, diann results could be directly export to skyline. Thanks to this very useful option.
To the slice-PASEF or Diagonal-PASEF acquisition, skyline is able to extract MS1 scan of the peptide but fail to extract MS2 ions.
Something was wrong in my parameters or skyline is not compatible to these acquisition methods?

Thanks
Jerome

Dear Skyline team,

We were trying to build a spectral library using DIA raw files in Skyline v24.1 through import->peptide search. In the build option, while we were trying to add the raw files, it seems the file format was not supported. In the previous versions of the Skyline, we have found an option to "start from -> DIA raw files". Kindly guide us to generate a spectral library using the DIA raw files in the recent version.

SCIEX TOF

I have a set of precursor masses and/or formulas (say n different one), and a set of collision energies (CE) (say m different one). I would like to create n x m individual method files, all with different precursors and CEs. The name should somehow represent the formula/precursor mass and CE, say sample_precursormass_CE.dam

I see that maybe it might be done in a batch using
BuildAnalystFullScanMethod.exe
but I cannot figure out in what format I need to provide the precursor/CE list, and what parameters I have to use with the EXE. I know that I need a file with the precursor and CE values, and a template method file. I was able to do one inside the software, but even if the CSV file has multiple lines, it just created new method for the first line. If you have a tutorial on that or a video, I would appreciate that, too, I could not find it.

If you could send me an example command line , that would be appreciated.

Thank you very much in advance, Vilmos

PS: Bonus: if I could also do the same, PRM methods with given inclusion lists for a Thermo QExactive, that would be phenomenal.