Hi Skyline team,
I'm wondering if there is a way to mark a large number of transitions as "Not Quantitative" after filtering according to some criteria. In the screenshot I've attached, I'm looking at the Document Grid and have filtered transitions according to "Shape Correlation" (i.e., shape correlation < 0.9). Now I want to deselect "Quantitative" for every transition left in the Document Grid. There are on the order of 10,000 transitions, so I can't do this by hand. Is there an automated way to do this?

The other approach would be Actions > Delete Transitions, but I don't think this is what I want, as removing transitions will effect peak scoring.

A related question is after marking transitions as non-quantitative, should I perform rescoring with Refine > Reintegrate? I'm primarily concerned with peptide quants, and ultimately want to export a Skyline document with peptide quants for each run. If I want the changes I make to take effect, do I need to do rescoring?

Thanks,
Lincoln

Since version 2.0 DIA-NN no longer generates speclib files for its spectral libraries but instead only parquet files. I used to be able to import the speclib libraries into Skyline but since they are no longer generated by DIA-NN I wonder how I can import the parquet libraries into Skyline. The direct link from DIA-NN to Skyline (Skyline button in DIA-NN) never worked for me but importing the DIA-NN generated spectral libraries worked well until DIA-NN version 2.0 when the speclib libraries are no longer generated. The DIA-NN developers told me that Skyline will soon have parquet import capability - is this already a function that is available in Skyline Daily? If, so where can I find it?
Thanks much,
Dietmar

Hi Skyline team,

Is v25 still support on Sciex raw data reading? Thanks,

Heyi

Hello,

Could you please help me understand why I’m not seeing any masses for the +1 charge state, while I am seeing them for the +2 and +3 charge states?

Hi All,

Is there a *.zip for an unplugged installer for AutoQC? We are unable to use the weblink due to our IT policies. I was able to find the unplugged installer for Skyline and installed that without problems.

Regards,

Art

Hi,
I want to create an in-silico MS/MS library that I would do myself, is there any tutorial on this or a dummy file that can be changed. Also, does skyline allow creation of spectra library from QQQ data ?

Hi,

With recent analytical chemistry publication, there's something interesting to do with multiplexed top down PRM (https://pubs.acs.org/doi/abs/10.1021/acs.analchem.8b02699).

While doing some experiments on my side, I thought this was indeed interesting, but there might be something to add to Skyline to complete data analysis of these experiments. Indeed, in case of high molecular weight (with charge > 1), it's very likely that fragments' most abundant isotope would not be the monoisotopic. However, Skyline seems to integrate only the first one (see capture attached). Maybe this feature is already implemented but I couldn't find it.

By adding all/multiple isotopes to target ions integration, this would increase detected signal intensity which would affect results significantly.

I can send data if needed.
Best regards,
Vivian

The Hoofnagle squad is having trouble with our AutoQC authorization. both Alex (aemmanue@uw.edu) and I have tried unsuccessfully to import our config file to new version of AutoQC.

I attached a screenshot.

Thanks for any help
Bill

Hi Skyline team,

As seen in attached, skyline selected a tiny peak due to its closest rt. Could Skyline select the strongest one within rt window?

Thanks,
Heyi

Hey Skyline team!
I’m working on a small molecule metabolomics project using Skyline with PRM data from an Orbitrap. I’ve added a heavy-labeled internal standard (identical to my target molecule except for the isotopic label) and want to normalize my results with it. I’d love to focus the normalization on a specific fragment ion since its chromatogram is cleaner and more specific, with less noise compared to the precursor, which shows more interference.

I’ve already done the peak integrations for both light (target) and heavy (standard) forms. In the Molecule Settings > Quantification tab, I’ve set:

Regression fit: None (no absolute quantification needed)
Normalization method: Ratio to Heavy
Simple precursor ratios: Unchecked (to focus on a fragment)
MS level: 2 (to target MS2 data for fragment ions)
Units: Default
Figures of merit: All set to None or blank

Could you please confirm if these settings are correct for normalizing the light-to-heavy ratio using a specific fragment for both? If adjustments are needed, I’d really appreciate your advice on how to proceed.

Also, after applying the normalization, how can I view the normalized values in Skyline to check and export them? Lastly, in the final report I export, will it include both the original values and the normalized values?

Thanks so much for your help!
Michal

Hi team,

I’m currently working with an indexed FASTA file that includes proteins from multiple species, which has led to identifications from non-human sources as well. Is there a way to filter or retain only human proteins and exclude those from other species?

Thank you.
Akhila

Hello Team,
We are currently in the process of submitting data to the PRIDE database, which requires both the raw data files and result files in the mzIdentML format.
Could you please guide us on how to extract the result file in mzIdentML format from Skyline after analyzing our data?
Thank you for your support.
Thanks
VC

Dear Skyline team,

I am using LC-QExactive (Orbitrap) and measured samples with Full Scan/ddMS2 method.
My issue is that I always have an message "do you want to enable Auto-select and use the document settings to manage these new transitions?".
If I do not click "Enable", I cannot see my precursor ions (MS1).

Secondly, I cannot see my MS2. I checked it with XCalibur (Qual browser) which is a Thermo software.
I think the main issue is the mass accuracy (10 ppm) does not work for MS1 and MS2, even though they are set in Transition Settings.

It would be really great if you can help me out.

Best regards,
Yohan Seol

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

Hello,
Is there a way to generate oxonium ion profiles in skyline by extracting specific m/z ranges (such as: 204.08-204.10) across a DDA spectrum? I am working in glyco peptides and need this information to further confirm the glyco-modifications. Oxonium ions are found as fragments of the precursor ions.

Thanks a lot in advance for your help.

Manousos

Hello team!

I’m working on a proteomics project using Skyline with PRM data from an Orbitrap Fusion. I’ve spiked 50 fmol of heavy peptides for all the samples and want to normalize my results with it.

I am attaching the image as a reference and the zip file is shared through https://skyline.ms/files.url PRM_24_1_05072025.sky.zip). Could you please check the file and let me know why I'm not getting fold change values for most of the proteins?

I’d really appreciate your advice on how to proceed.

Thank you
Akhila

Hello, I'm working on the Small Molecule part of Skyline and I'd like to know how to set the integration threshold (not the display threshold). I don't want intensities below 1E4 to be integrated, for example. Thank you and have a nice day. Amandine

Hello,

IQ-X mzML files converted via MSconvert (3.0.25120-02fc3ac) can not be loaded and give the error:
"
At 2:28 PM:
Failed importing results file 'C:\PostDoc\Project_MetallPreliminary\Yasin\Metalls_in_Mushrooms\after_capillary_replacement\mzML\BB.mzML'.
[pwiz::CLI::msdata::ReaderList::read] Invalid cvParam accession "1003411"
"

looking into the file the accession looks like this:

  <referenceableParamGroup id="CommonInstrumentParams">
    <cvParam cvRef="MS" accession="MS:1003411" name="Orbitrap IQ-X" value=""/>
    <cvParam cvRef="MS" accession="MS:1000529" name="instrument serial number" value="FSN50178"/>
  </referenceableParamGroup>

Thank you for maintaining the software!

Hello,

I think I have a settings issue. I'm looking at the transition ions for a specific peptide and skyline is only displaying b10, b11, b12 and b18, b19, b20. When I go to transition settings > filter and change product ions to 6 it only adds b21, b22, b23 to the visible transition ions BUT I am only interested in transition ions below b12 (i.e., b11, b10, b9, b8, b7, .... etc). Do you have any suggestions for how I can visualize these b ions?

p.s. I don't believe the lower end b ions are being filtered out as a result of not being produced by the mass spec because in some cases b10, b11, b12 are not produced by the mass spec, but they are still displayed, so I think it is a settings issue.

Best,
Chelsea

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 ---

when I add the peptide in skyline, and insert files of .lcd from shimadzu 8050.
there is no LC information.

As I develop targeted assays to share I would like to implement the CPTAC recommendations for assays using the MSinspector external tool - I've downloaded it but am getting an number of errors. I suspect many of the issues are related to it being coded in python 2.7 which is end of life. (Can't get pip installed, can't get pandas installed etc etc). All errors in the attached file. I've been messing around trying to install things in the cmd line and there are just too many issues getting versions for python 2.7.

Do you have any recs or could you give me the contact info for Yin Lu to see if they are planning on updating this tool at all? Thanks!

Hi,
Please help me set up quant where the starting concentrations and dilutions are different for analytes. When I go to document grid, I see where I can enter concentration, but it is the same for all analytes.

Thanks,
Sheher

Hello,
I am currently preparing to submit my Skyline data to PRIDE, which requires files in either mzTab or mzIdentML format. Could you please guide me on how to generate these file types from Skyline or through any alternative method?
Additionally, I would appreciate any guidance you can provide on the overall submission process to PRIDE.
Thank you very much for your assistance.
Thanks
VC

Hi Skyline Team,

I'm new to using Skyline, and I've went through some of the available tutorials to start me off.

I want to create a spectral library using dia-PASEF data, so I can do a peptide search of the data.

I've been able to upload my data using: File > Search > Run Peptide. I've managed to upload my .d folders and select my settings under the DIA workflow. My trouble is at the Conversion settings (where DIA-Umpire creates the pseudo-DDA files for searching), the preset instruments available do not have an option for the timsTOF.

How do I go about continuing with building the spectral library? Would you suggest that I should rather use dda-PASEF data for spectral library building and search my dia-PASEF data against that instead?

Thank you for your time.

From: Rethabile

Hi team!

I've been building Skyline v 24.1 docs from DIA-NN 1.8.1 results using the “Import DIA Peptide Search” walk-through. The good news is that 4 out of my 6 files look great! The sad news is that 2 of the 6 files throw these errors. I'm not sure what could be different about these two files compared to the four that successfully imported. I'm not sure where to troubleshoot! The Skyline file is kinda big but I can get that shared if useful?

More random details if helpful:

• Isolation scheme: Results only
• Same error when I use Skyline-daily

Thanks!
Lindsay

Hello,
I spiked in my heavy peptide into the sample matrix (cell digest) containing my endogenous peptide, but during the analysis on Skyline I noticed that the endogenous peak appeared to have split into multiple peaks. When I was looking through other requests about peak splitting, Skyline staff members previously warned against integrating differing retention times between the heavy and endogenous (light) peptides, which of course makes sense.

So, my question is this: How do I handle this peak integration, if at all? From where I'm sitting, the endogenous peak doesn't appear to be quantitative, but I'm not familiar enough with Skyline to figure out if the problem is a lack of knowledge on my end, a sample degradation problem, a meaningful biological artifact, or a methods/sample preparation problem. For reference, I am running a PRM proteomics experiment on Skyline version 24.1.0.414.

I've attached images of the heavy peak, the endogenous peak that Skyline recommended I integrate, the endogenous peak that I manually wondered if I should integrate, and an overlap of the precursor ions for both the heavy and light peptides (blue is the heavy, red is the endogenous).

I first assumed that I have a PTM on my endogenous peptide, but that would change the overall mass and my PRM scan wouldn't have picked up my peak. I have an amino acid in my sequence that perhaps could have undergone racemization (my cell pellets were >1 yr old), but I wasn't sure if there was a simpler or more likely explanation before I attempt a new experiment. For reference, this peptide comes from a membrane bound glycoprotein. To my knowledge, there are no known glycosylation or phosphorylation sites on my peptide.

I apologize for the novel, but I thank you for your time and look forward to your response.

Sincerely,
Olivia

Hello,

I am hoping to get some more information on the application of the q-value filter when setting up groupwise comparisons on the protein level in Skyline version 24.1. I am preparing to run pathway analysis on proteins that have been identified from DIA proteomics data in Skyline, so I am trying to generate high-quality groupwise comparisons with a p-value and log2 fold change for each protein. Implementing a q-value filter of 0.01 results in many missing abundances in my comparisons.

• From my understanding, the q-value essentially descibes the FDR for picked peaks. If it applies to individual peaks (ie, peptides), how is it calculated on the protein level?

• If I've already filtered my data by removing duplicate epptides, removing missing peaks, decoy scoring, etc. earlier in the analysis process, is it necessary to apply the q-value filter? Why is it applied in the groupwise comparison step and not earlier in the analysis process?

• Would you recommend applying the filter for this application?

Thank you for your help!

Hi!

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

Thanks in advance!

Cheers,
Sean

When using an Agilent LCMS 6495D model instrument, with the large molecule iFunnel mode specified in the template method, the exported methods use standard mode.

Hi,

I want to create a peptide library in Skyline using a result file created by Biopharma Finder with the extension .resultsbpf. Has anyone tried this and could help with the process? Thanks!

I am currently trying to export isolation list for PRMPASEF method for which I need spectral and ion mobility library. I wanted to create that with the PEPXML file ontained from PEAKSONLINE database search. However, I am constantly getting the attached error. Therefore, it will be helpful if you guide me what to do further.

I've now successfully used a list with associated datasets in an application - but now I'm wondering if I can connect information from the file names to this list.

I know that we can use a regular expression to define a result file rule pulling some information from the file name to a specific target (although could you remind me of which tutorial described this? I don't have my regular expression worked out quite yet and I know you JUST talked about how to do this recently). For reference file name formats are all: date_source_animal_ID#_timepoint (example 040825_IUSM_animal_26_M2 ) and I want to pull ID# into the document, and then use that ID number to pull in information from the associated list.

Right now, I cannot seem to define that 'ID' as the target for the replacement information, it is only listed in the 'source' section for the result file rules. Attached a screenshot for reference of what I'm seeing.

Dear Skyline Team,

Thank you for your continued development of this excellent software package. The longevity of the project is a testament to its utility.

I am currently developing a scheduled PASEF-PRM method on a timsTOF Pro instrument and I am running into some issues when importing results. I've tried importing a reference DDA run of isotope-labeled peptides and a scheduled PASEF-PRM run of the same sample while adjusting the Transition Settings - Full-Scan - MS/MS Filtering parameters as follows, with varying outcomes:

Import as PRM – no chromatograms
Import as DDA – Few product ion traces (even in DDA run), despite spectra in library
Import as DIA – Product ions observed in DDA run, few products in PRM run

I've attached some screenshots of my observations and a zipped file of the Skyline document. Any suggestions you could provide would be appreciated. Let me know if you need the timsTOF acquisitions uploaded also. Thanks.

Robert

I am interested in attending the Quantitative Proteomics with Skyline course scheduled for April 28, 9:00 AM - April 30, 12:30 PM, 2025. Could you please advise me on how to register for this course?

Hello,

This is my first go through using Skyline/EncyclopeDIA to look at my samples vs. GPF, so there might be something I'm setting up incorrectly, but I keep hitting a GC overload error. There are a ton of entries in the file, and I'll try parsing it to see if that fits, but I wanted to check to make sure it wasn't something in my setup. I had 6 GPF pools, and 15 samples. The blib is created, but the error comes in when the elib is supposed to be created.

1. Is there a way to get around this in Skyline? I'm happy to send over whatever files you need.

Thank you,

Shay

Hello,

I am using the small molecule side of skyline and frequently use the normalization method in the live reports to assign surrogate standards to all the metabolites I am looking at. However, I like to use different surrogate standards for different molecules, so is there a way to assign a normalization method to the molecules in your transition list so that I don't have to go in and manually change it in the live reports for every experiment?

Thanks for the help!
Jaedon

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

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

Hello there, I found an answer that described what I am trying to do, set one surrogate peptide as global peptide for several light peptide from a previous thread but it is not working for me. It mentioned to set the Standard Type of each of these heavy peptides to "Surrogate Standard" and then set the "Normalization Method" for all the other peptides to the appropriate "Ratio to Xxxx". However, I can't see the options to Ratio to Light or Ratio to Heavy after setting the whole peptide as Surrogate Peptide, do I have to change something under the "peptide setting" "modifications" tab? There I have the internal standard type set as "heavy". Do I set the peptides with no heavy counterpart as Standard Type "none"?

Thank you very much,

HHB!

Dear Skyline Team,

I found this tutorial of AlphaPeptDeep predicted library build-up in the Skyline

https://skyline.ms/wiki/home/software/Skyline/page.view?name=Build AlphapeptDeep Library

However, the Skyline (version 24.1.0.414) that I use does not have this option in the Build Library window. Could you please indicate how could I get this function to build up a predicted library with AlphaPeptDeep in Skyline?

Additionally, I found that AlphaPeptDeep has an online version, and I built a library with the online tool. While the library generated was in .tsv and .hdf format, which I tried and found cannot be recognized by Skyline. If the built-in function is not available for the moment, is there a way to upload this predicted library?

Best,
Ziliang

I will likely have to do this manually but just wondering of there is a quick way within skyline to calculate 95% CI for LOD using SD of blanks and slope of calibration curve

Hello everyone,

I would like to add the following 15N-labeled product to Skyline:

L-Tryptophan-15N₂, with the molecular formula C₁₁H₁₂¹⁵N₂O₂.

Thank you very much for your assistance.

Best regards

Is there a way to calculate monoisotopic mass of a peptide without digestion?

Hello, dear Skyline support!

Is it possible to arrange quantification of concentration, using only the concentration of IS that is structurally relative to the target molecule? I need the concentration that is calculated according to the formula: C(tm)=Int(tm)*C(IS)/Int(IS), where tm -- target molecule, IS -- internal standart, Int -- intensity of the signal.

I have already tried labeling IS as heavy (it really is an isotoped molecule), but I can only calculate the ratio and the normalized area.

Maybe it is not quite accurate to ignore the external standart calibration curve, but to I need the calculation, I mentioned.

I would be very grateful for your help!

Best regards,
Ismailova Diana

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.

Hello, Skyline team!

is there any way to extract all the visualisations of chromatograms, peak areas etc in one report? I could copy and past the pictures step by step and create my own report in Word, for example, but it seems to be too long, cause I hve plenty of small molecules in the analysis... Using R or such tools is ok, but I already like the pics from Skyline. What should I do? I attach my file here for the example.

Thanks!
-- Diana

I will be sharing two skyline documents, both with data imported from DIA-NN 2.0.2 (report-lib.parquet.skyline.speclib) and 3 Astral raw files

"100SPD_2_4_8_Th.sky" here I added a dabase containing UniProt human fasta plus some entries of contaminants annotated as CON_xxx
"100SPD_2_4_8_Th_woc.sky" using same database but without contaminants.

Hello, dear SKyline team!

We have a problem uploading mass NISt Library (msp format) for small molecules. Do you have any recommendations?

Best regards,
Diana

Hello
We have results generated from a timsTOF bruker under the format of a " .d folder". This .d folder contains a file named .d (0 KB), and other files such as .tdf .
When we try to load these results on skyline, the folder .d is not visible. However the file .d is visible but unfortunately skyline cannot be run with this file because it is empty.
How can we open our tims TOF results on skyline?
Thank you for your help.

Hi Skyline Support Team,

I'm a new user learning targeted proteomics data analysis with Skyline and have some questions about normalization approaches for my experiment.

My current setup includes:

• Around 2-3 peptides per protein
• Around 2-3 transitions per peptide
• One transition for each labeled peptide (for chromatography and instrument variation normalization)
• BSA protein as a global standard for sample processing normalization

I would appreciate your guidance on the following:

1. Is it possible to normalize multiple unlabeled transitions (e.g., Transition A & Transition B) using a single Heavy Labeled Transition (Transition C) in Skyline? As an example, can we normalize transition A over C and B over C for the proteins relative abundacnes analysis in skyline?

2. For proteins where we don't have labeled peptides, can we use labeled peptides from other proteins as normalization references? If yes, then could you please advice how to process that in skyline?

3. We're using BSA as a global standard for sample processing normalization. Is there a way to implement a two-step normalization process where we first normalize all samples using their respective BSA peak areas/ratios, and then apply a second normalization using the heavy labeled peptides?

4. When reviewing protein abundance reports ( Simply Control vs Treatment normalized Peak area or area ratio), how can I determine the calculation method used (e.g., whether Skyline is averaging or summing the responses from each peptide's transitions)? Is there a way to customize these calculations?

I'm happy to share my Skyline documents if that would help in addressing these questions.

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

Best regards,

Hi Skyline Team,
I was trying to import my Spectronaut report files into Skyline, but there is no chromatogram been showed.
In the report file, it contained "Modified Peptide Sequence" "Relative Intensity" "iRT" "Precursor m/z" and "Product m/z" as Skyline indicated in the error message. After I modified my .tsv file, the file is successfully imported, but chromatogram place is empty. And under the "View" tab, the chromatogram button is grey that I cannot select. Could you help me with it? Thanks!

Hi,

I'm doing lipidomics with truple quad and have transition list for the compounds.
I can't see the chromatogram in some compounds out of ~600 compound list. I tried like wider retention time window and selecting nclude all matching scans, but still It show chromatogram information unavailable (also did re-import).
I can see the peak in masshunter if I extract the transition that does not show in skyline.
Could you please help me with this?
Thank you so much

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

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

Dear Skyline support,
I run lipidomics using waters xevo tqxs. Managing the many MRMs in the Masslynx software is to time consuming (adjusting RTs manually) so I am trying to learn skyline. My main objective is to be able to write all MRM experiments into a *.csv file that can be used as a compound database. I can then use skyline to build the *.exp method for each application and update the RTs in excel.

I manage to insert transition list with the parameters needed in a way that seems correct into Skyline but I have problems to get all needed settings exported to the *.exp file.

Problems:

1. The problem is that when I export method I am not able to get the right polarity set for each MRM in skyline into the *.exp file, they will al be ES+ even if listed as ES- in my Skyline file.
2. I have the retention time and retention time windows in Skyline but am unable to get export this to the *.exp file the start and stop time will be at the same value.

If I can find a solution to these problems I would be able to write and modify all my methods from templates in excel and export them very quickly as needed to Masslynx. Changing RT times and windows would be a very simple task in excel and making selections for MRMs for a new application would be very simple as well. To make this work I need to find the solution to export the scheduled RTs and the polarity for all MRMs from skyline to masslynx.

I am using Masslynx 4.2 SCN 1045 and Skyline 23.1.0.380 and run this on the workstation used for controlling the instruments.

In case any one can help me I attach my csv file that I use to import into skyline, pasting into "insert transition list"
I attach my skyline file
I attach my .exp template for the export
I attach a raw file with results from the lipids in the template

Would be extremely nice to find a solution and I am think there should be lots of other users that would have use of this solution.
Excel->Skyline-> Masslynx for method setup would be great

Regards,

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

Dear Skyline support.

we have 7 potential protein standards for IBAQ MS experiment.

Before I order in the protein standards I would like to check if the complete (no missed cleavages) in silico tryptic digest of the protein standards give over lapping peptide sequences to my in silico digest proteome.

Can Skyline be used to search this ?

Many thanks,
Mehul

Hi Skyline team,
I've encountered an issue where Skyline sometimes does not recognize the correct peptide peak/the peak I want to export data for. When I say it doesn't recognize the peak, I mean that it is not selected and that it is not selectable - I can't click on it to change the peak selection. The only way to select it is to manually drag the boundaries around it.

We are trying to standardize analysis as much as possible and so would like to avoid needing the user to arbitrarily move peak boundaries. Is there a way to force Skyline to select the peak?

You can see in the attached screenshot an example where in one injection the peak is not recognized (left) but in a duplicate injection of the same sample Skyline selects the correct peak (right).

Thanks for your help,
Erin

Dear Skyline-Team,

I hope you can help me with a question regarding a Skyline tool.

I would love to use a tool like Avant-garde to determine the integration boundaries of a large label-free PRM data set. I can apply the tool to DIA or PRM test data containing heavy peptides. Therefore, I'm wondering if it's generally possible to evaluate label-free PRM data with Avant-garde? I have tried different settings of the AvG_Params file, but none has worked so far. Is there something I should consider?

I would be super thankful to receive any tips.

Best wishes,
Terese

Hi Skyline Team,

For each time segment, I have a specific set of transitions that I want the QQQ (Agilent 6495D) to monitor. I’ve written batch scripts using the Skyline Command line to handle this automatically for standard dMRM and MRM workflows, but I'm unsure how to implement this with my current time-based segmentation strategy.

Is there a way to automate this kind of time-segmented transition scheduling for the QQQ, possibly through Skyline.

Thank you
Anirudh

Hi,

I would like to use skyline to quantify my compound discoverer search results. Unfortunately, the ".cdresult" file cannot be imported into skyline just like the ".pdresult" from proteome discoverer. I know that there is a way to do so, at least to import the MS2 spectra into skyline with the included transition list. Could you please help me out?

Ps: I tried to import a file to skyline as annotation file, but skyline tells me that I am missing a column called "elementlocator".

Thanks a lot,
Chengkang

Dear Skyline team
I am trying to get a quantitative method for tryptophan and 10 of its metabolites. all of them have different concentration ranges, but when I type them in, Skyline uses only one set of concentrations. Any idea how I can modify the grid to have different calibrators in different molecules lists?
Thanks
Gio

Hi Skyline Team,

In general I use calibration curve for quantification but needed to use single point internal calibration using a heavy standard spiked into each sample at a constant amount. In the document grid, I have a ratio to standard tab with value and my internal standard concentration tab with the given value. However, the value in the quantification tab for the endogenous analyte value is different from a simple multiple of the ratio to standard with internal standard concentration.

I thought they should match? Is there a regression fit enabled in internal calibration, is that why they don't match? Or did I make a mistake somewhere.

Dear Skyline Team,

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

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

Best,

Gianin

I have an LC-UV-MS data file. We are looking for targets in the extracted sample that are all UV active but also want mass spec confirmation. I was able to successfully import my target list and data file. I have reviewed the EICs for my targets but wanted to confirm UV detection at the EIC retention times as well. I'm unsure how to view the UV trace, or if the UV trace can even be viewed in Skyline?

Hi there,

I want to use Skyline software for Sciex 7500. I have downloaded the latest version of Skyline (24.1). However, I don't see the list of instruments in the Instrument Tab under Transition Settings to select Sciex 7500 (attached the screenshot). How can I make it visible to export transitions list for Sciex 7500 OS software? Am I missing something here? Your support will be greatly appreciated.

Best,
Vipin

Hi Skyline team,

A quick question about dot p value. If there are more than one MS2, dot p value of MRM (4 transitions) is from which MS2 comparison? Thanks,

Heyi

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

Hi,
I am having trouble setting up quant with internal standard. I have the same name for the light and heavy under the molecule tree. Cal curves work with external standard. When I try to do with internal standard it is giving the message .....truncated transitions. Please help.

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,

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

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,
we have a strange behaviour of Skyline when saving files. On some of our computers on which we use Skyline saving the files using the "Save" button does not save the file neither does Skyline save the file after prompting when closing the file. We have to save using "Save as..." with a different name. Interestingly, this is not the case on all computers although they use the same Windows 10-built and have the same hardware. Are we missing something?
Many thanks in advance!
Joerg

Dear Skyline support,

I have done targeted MRM assay with peptides. Now I want to do the same targeted MRM with small molecule named Lactobionic acid.

My queries (total 3) related to:

1. How can I add this molecule to Skyline file?
2. I want to export the isolation list and use it as acquisition MRM method. After acquiring the MRM results, I want to check the presence of lactobionic acid in different samples.

I have followed the attached document which helps me to have an idea.
But I am unable to know the precursor and product charge of Lactobionic acid.

3. Please help me how I can proceed with Lactobionic acid. If you can share a skyline file with all the settings, it would be a great help.

Instrument: Xevo G2 XS qTOF

Thank you
Arpita

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 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

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?