support

Welcome to the Skyline support forum. If you have a question about using Skyline, or if you encounter a problem, you can post your questions here.

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

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

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

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

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

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

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

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

If you are including a Skyline document, please use Skyline's File | Share menu item (choose "Complete" if asked), which prepares a single zip file with your document and all the needed supporting files in it. Then upload that .sky.zip file to the Uploads page. If the actual raw data files are needed to illustrate a problem, those will need to be zipped up and uploaded separately.
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Showing: limited to 100 requests
Finding targets based on isotopic pattern
j k o nygren 2024-10-10 07:23

Hello Skyline team,
I'm new to this software and I'm trying to figure out if it can help me with what I currently need to do.
After going through some tutorials, webinars and questions in the forum, I thought it better to ask if this is possible.

I am analyzing polychlorinated alkanes (PCAs) using direct injection and HRMS data.
A common way of doing this with HRMS data is to, for each congener, extract the two (or three) most abundant m/z of the characteristic isotope clusters of highly chlorinated compounds.
For example for C14H20Cl10, that would be [M+4], [M+6] and [M+2], see attached image.
The relative intensity between the most abundant m/z and the second (and third) most abundant m/z are used to identify the congener.
The most abundant m/z is then corrected by the injection standard and used for quantification.

What I hope that Skyline can help me with is to match the theoretic isotopic pattern to the experimental isotopic pattern, and integrate the potential EIC on matches.
So far, I've learned how to get matches based on masses but not on isotopic patterns of chlorine. Is that possible and if yes, are there some functions/tools that I am missing?

For a deeper explanation, see the materials and methods section of Bogdal et al 2015: https://pubs-acs-org.vu-nl.idm.oclc.org/doi/10.1021/ac504444d#_i2

Best regards,
Jonatan Nygren

 example_IsotopicDistributionC14H20Cl10_fromChemCalc.png 
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Why is the "Agilent Automation" feature not available on my skyline operation panel?
(1 response) 2378572477 2024-10-09 01:21

Hello

"I want to optimize CE using 'Automation' as shown in the figure."

But why doesn't my operation panel have 'Automation'?

"Please see the attachment."

Thanks,
Zhaoxuanxuan

 问题.docx 
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How to Combine Skyline and Mass Hunter:
z15839328872 2024-10-09 01:33

Hello, dear Skyline team!
I want to optimize collision energy using Skyline now. I saw a tutorial that said it could be used with Masshunter, but why can't I use it? Here's a screenshot of my problem.
Zhouyanan

 1cf8a69077bad72e332ed054e3e7703.png  0b495051cb27bcf1a12a2aa9490ddb8.png  681a0f3f969f710450b66fd3bacdf5f.jpg  skyline 使用——筛选合适的肽段和离子.pdf 
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How to use Skyline to optimize MRM parameters
(2 responses) z15839328872 2024-10-08 01:48

Dear Skyline Team,
I'm having some issues with Skyline to optimize MRM parameters, and I don't know how to automatically optimize collision energy and declustering voltage. Since my previous protein and peptide identification was done by sending samples, using Thermo Fisher Fisher Scientific's fluid, the raw data was imported into Skyline and the ion pairing information was exported. Later, I used my own Agilent instrument to verify that the peak was too low, and I wanted to optimize the MRM parameters, but I haven't found how to use Skyline optimization, do you have any good suggestions? Or am I using a 2023 version that needs to be updated?
Zhou Yanan

 69efae6c4496f44cc462b4e13cf997f.png 
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Import results from FRAGPIPE to create spectrl libraries
(8 responses) afshari1 2024-10-01 12:47

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

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Export MRM to Waters TQ CE formating should be "eV"
(5 responses) per larsson 2024-09-25 10:51

Version:Skyline-daily (64-bit) 24.1.1.254 (ba32f7f50)
I have had problems to export MRMs to .exp file for waters TQAbsolute. I was able to find the cause. I used a template file with an MRM created in Masslynx.

Skyline writes the "CollisionEnergy(V)_10.000," (for a collision energy of 10eV) this results in an error in Masslynx since collision energy is not regognized.

In masslynx files the collision energy is written as "CollisionEnergy(eV)_1,10.000" the missing e results in that the CEs are not read by masslynx. This can be fixed in word by replacing the wrong term with correct but it would be better if skyline developers fixed this.

Also please make it possible to export retention times (start and stop) and polarity from skyline to masslynx

Regards,
Per

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importing multiple complex crosslinked peptides
(3 responses) sri ramarathinam 2024-10-02 21:49
Hi
Thanks again for making such a fantastic tool as Skyline!
I might be missing something, but I was unable to import crosslinked peptides. I was trying to replicate what was done in following ASMS presentation https://skyline.ms/wiki/home/software/Skyline/page.view?name=Crosslinking#:~:text=Support%20for%20crosslinked%20peptides%20was%20first%20added%20to%20Skyline%20version

In this poster there is a dialogue box that says add crosslinked peptides (screenshot attached)- I am not sure how to get to this screen and specifically how to add multiple peptides using the String based representation. If I try via Edit>insert Peptides it recognises "-" as invalid character so I am assuming this is someplace else.

If I have over 100 crosslinked peptide combinations (2 or 3 possible links) and can generate a text output, would be nice if I could import them into Skyline all at once in format that was demonstrated in the poster:
PKEPTIKDEA-PEKPTIDKEB-PEPKTIDEKC[+57.021464]-[+138.06808@2,3,*][+138.06808@7,*,4]

Many thanks again
Sri
 crosslinked_peptides.png 
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How to develop MRM method for Metabolites
(1 response) Drymoglossum 2024-10-02 05:35

I have untargeted DDA data for metabolite samples and have used public .msp libraries from RIKEN for MS-DIAL data analysis.
I have access to .raw files of the samples & .msp libraries.
I identified over 100 differentially abundant metabolites that I want to further investigate.
How can I develop an MRM method for these metabolites?
Does anyone have a latest tutorial or guidance on how to approach this? I’ve heard that Skyline might be useful, but I'm unsure where to start. Any advice would be appreciated!

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Spectral Library build Issue
(1 response) whdgka75 2024-10-01 18:54

We are trying to build a library using raw files of mass spectrum data contributed in other existing studies.

But as the skyline is updated, some problems arise.

During the initial file upload process, RAW format files cannot be uploaded. (picture 1)

At this stage, only ".dat" file formats are read, and ".mzid" and ".mzxml" files are not readable.

At this stage, may I know if there are any file formats that can be uploaded?

If only a specific file format is possible to uploaded, I would like to know if the library cannot be built if the contributed mass spectrum data does not have that file format.

We built the library by uploading the ".dat" file in the first step and registering the ".raw" file in the next step.

In the next step, enter “search condition”, and under “modification” I want to select carbamidomethyl(M).

However, carbamidomethyl(M) is said to already exist, but cannot be found in the list. (picture 2)

In this case, carbamidomethyl(M) is not in the list because it is a modification that is applied by default, and I would like to know if it is applied automatically or if another error occurred.

 picture1.png  picture2.png 
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Unscheduled PRM data analysis
(1 response) afshari1 2024-10-01 11:18

Hello,

I have generated an unscheduled PRM method using Skyline for 200 precursor ions. Could you please provide a guide on how to analyze the resulting PRM data in Skyline? Moreover, Is it necessary to import spectral libraries for this analysis?

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Lost my points across the peak visual in chromatogram viewer
(3 responses) Lindsay Pino 2024-09-30 19:02

Hi all :)

I seem to have lost my little red scan number demarkations with the small red number that indicated how many scans were across my peak when I was on the Transition Targets level to view my chromatograms. I attached a minimal doc that I think captures the issue, I'm sure I'm just missing some Magical Checkbox in the settings!

If you need another other information or details, please let me know

Thanks,
Lindsay

 nopoints4skyline.sky.zip  skyline screenshot.png 
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PFAS-Specific adducts not uploading in transition list
(4 responses) karamj33405 2024-09-27 12:41
Hello,
My name is Kara Joseph with the Baker Lab at UNC Chapel Hill and we are working on expanding our library for PFAS with some new adducts, however the adducts listed below cause errors when inserting the Transition List. I have included the original adducts we prefer to use below. Do you have any suggestions on how to format these while retaining the structural information for the losses?

M-COOH-HF
M-C2H4-OH
M-CH2-COOH

The only way I can get the Transition List to import properly is as follows:
M-C-2H-2O-F
M-2C-5H-O
M-2C-3H-2O

Thank you!!!
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Koina error
ericw04 2024-09-27 12:01

Hi,

I'm trying to run an EncyclopeDIA search through Skyline with an experiment-specific FASTA file. This has worked for me in the past, however, when I run the search now I get a "GO AWAY" error from Koina before it's finished processing the FASTA and the search fails. The text of error is attached. When I check the Koina configuration in Skyline (Skyline (64-bit) 24.1.0.199 (6a0775ef83)) it says the connection is active.

Any help is much appreciated.
Thanks,
-Eric

 Koina_go away.txt 
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DDA (eclipse orbitrap)
(1 response) zeinab mokhtari31455 2024-09-25 13:09
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No pressure traces in Thermo Fisher Files
(2 responses) sascha 2024-09-24 07:45

Dear Skyline people,

I am trying to extract pressure traces from our Thermo Fisher raw files recorded using a Q Exactive couples to a Dionex Ultimate 3000. However, I am not seeing any pressure traces, although they are stored in as "Pump Pressure" within the file (verifyable using Freestyle). It works for files recorded using a Vanquish HPLC.

Can you help me find out what's going wrong?

I attached you example raw files as well as an example document.

Thank you very much for the help!

 QC.sky.zip  QC_Dionex.raw  QC_Vanquish.raw 
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Skyline-MRM transitions on QqQ with 2 scan segment issues
(2 responses) weigandm 2024-09-23 06:33

Hello,

I collected MRM transitions on an Agilent QqQ. I had 2 scan segments. One segment was from 0-1.1mins, in which solvent was diverted to waste. The second segment was from 1.1mins to the end in which solvent was diverted to MS for data collection. When I import the results into skyline it only shows that first segment from 0-1.1 mins. I want to see the data from 1.1mins to end which is when analytes in the sample will have abundant MRM transitions. How do I get Skyline to be compatible with this data?

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In silico digestion
(1 response) mvm35 2024-09-24 08:19

For in silico digestion using various enzyme.

What do the following mean (X= some amino acid):

[X | P]
[X | - ]
[- | X]

thank you.

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Spectral library generation
(2 responses) mlazear 2024-09-23 12:39

Hi,

Is there a way to construct a spectral library such that Skyline will just "trust" the user that parameters are correct? I am trying to build some empirical (e.g. constructed from DDA) spectral libraries for DIA runs where the peptides have exotic/unlocalized modifications - and I am really struggling with how to get Skyline to accept a spectral library.

Ideally, I can just pass in a list of arbitrary ID, precursor m/z, precursor charge, product m/z, library intensity and have Skyline accept it without any "error" checking (e.g. do not try to match the product m/z to the peptide sequence). Is there a way I can fool Skyline into accepting such a list? I don't want to have to manually add the modifications to Skyline, since I have 1000s of them. I am happy to manually construct a library in whatever input format is needed.

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Top Down Proteomics data
PP 2024-09-23 13:21

Hello,

I wanted to know if Skyline is able to process top down MS data? If so, is there a tutorial for it?

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2024 Skyline Online Course recording
(1 response) skyfall 2024-09-20 08:31

Dear Skyline team,
I won't be able to attend the upcoming online course and was wondering if you will record it? In case yes, would I still need to sign up for the course to watch it?

Thanks a lot
best wishes

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Safety of Proprietary Sequences
(2 responses) seamus kelley 2024-09-19 08:17

Hello, I'm interested in adding Skyline to my group's proteomic workflows. We are working with the proprietary sequences. Does your software protect the proprietary nature of these sequences?

Thank you!
Seamus Kelley

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Help with Small Molecule Library Building, etc.
(13 responses) mwmann 2024-09-10 10:19

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

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Merge two SRM together
(5 responses) nbekhti 2024-09-10 14:07

I have under my skyline file one same metabolite showing in two lines, and I would like to add the second transition into one same SRM
I tried adding transition, copy paste from another skyline file where they show up together, but the second transition is gray with no data when I do this.

How to fix that issue please?
Thanks
Nihel

 SRM dublicates.JPG 
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Replicate comparison - collision energy optimization
(1 response) a b heijnis-2 2024-09-17 04:07

Hey! For our proteomics project we are using the automation tool to connect Skyline with Masshunter from Agilent. Therefore we have two questions.

Recently, we have compared the export methods that we made by going through the steps manually and doing it with the automation tool. Luckily, the results were completely the same!
However, Skyline comes up with the starting point for CE optimization, using step size and step count. We would like to further optimize with the results of the first experiment as an starting point. How could we approach this?

Secondly, when processing our results in Skyline in the replicate comparison, the different CE are always displayed as step (-2, -1, 1, 2 etc.). See figure in the attachments.
We would like to know whether it is possible to have the CE settings shown in de legend as there are many differences between peptides.

Thank you in advance!

Alexander

 Question Skyline.png 
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Trouble with Target Quantification and Integration in Skyline for PRM Data
(5 responses) afshari1 2024-09-13 14:44

Hello,

I am analyzing my PRM results using Skyline and encountering an issue with quantifying certain targets. Specifically, in the attached file, there is a peak at 69.x minutes in the bottom panel, which corresponds to one of my targets. The MS/MS data from Thermo Fisher software confirms this match. However, when I import the same RAW file into Skyline, this target is not being quantified or integrated. Could you please help me understand why this might be happening and how I can resolve this issue?

 IMG_0606.jpeg 
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Wrong XL transition mass?
(4 responses) tjiang 2024-09-12 08:19
Hi, I'm working on generating library and transition list for XL experiments using DSBU as crosslinker. I had some issues with XL peptide mass.

For example, for the XL peptide "AKGILTC[+57.02146]R - EIPLKVLVK -[+196.0848@2,5]", the correct m/z value for the +4 charge state is 538.824, as shown in the attached "peptide_in_datafile.PNG". However, it shows as 485.3038 in the spectra library as in the attached screenshot named "lib.PNG". And in the transition list, it only gives the XL peptide without cysteine alkylation, as shown in the attached "in_transition_list.PNG".

Could you please help? Thank you so much!
 peptide_in_datafile.PNG  lib.png  in_transition_list.PNG 
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Help with SureQuant data analysis
(1 response) darora 2024-09-12 09:03

I´m missing the heavy fragment ions for some of my peptides from a SureQuant run. I can see that these were triggered ( precursor traces)but no fragment ions, unfortunately. These fragment ions were there in the method used for the run. So I´m a bit confused.

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How to update KEGG/CAS/HMDB fields?
(1 response) Will Thompson 2024-09-11 06:48

Hi team,

I have a working document for small molecules (attached) and I'd like to use the document grid to add information into the KEGG/CAS/HMDB fields. However, these fields are grey in the document grid and not editable. Please advise on how to batch-enter values into these fields in a manner such as copy/paste from excel, or importing an annotations file?

Cheers

Will

 P148_ACE_v0p5.sky.zip 
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Error importing MaxQuant msms.txt file
(2 responses) SChen 2024-09-11 08:42

Dear Skyline team,

I encountered an error while trying to import peptide search result (MaxQuant msms.txt file) into Skyline (see attached error message). I have tested this in Skyline Daily 23.1.1.520 & 24.1.1.254 as well as Skyline 24.1.0.199 and got the same errors from all.

I uploaded both mqpar.xml and msms.txt file to https://skyline.ms/files.url. Because the msms.txt file is quite large (50 GB), I uploaded a smaller version (only the top 10000 line in the original msms.txt). Since I encounter the same issue with the smaller version, hopefully you will also be able to reproduce the error on your end. I also had to change the mqpar.xml file name because there is already a file named after that in https://skyline.ms/files.url.

Please let me know if I can provide more information about the issue.

Thank you in advance!
Shimin

 Skyline error.txt 
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Failed to attempt reintegrate peaks_index 6 must be between 0 and 5
(2 responses) vanyabangera 2024-09-10 22:45

Dear Team,

We are failing to train mProphet model in skyline version 22.2.0.527 (841287d47) and 24.1.0.199 (6a0775ef83). But unable to reintegrate. It's showing pop-up "failed attempting to reintegrate peaks. The index 6 must be between 0 and 5". As per the suggestion obtained from previous queries we tried to perform the rescoring but failed to reintegrate. Kindly advice to resolve the issue.

Thanks in advance.

Kind regards
Vanya K N

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Skyline command line error "report does not exist"
(6 responses) sstoychev23513 2024-09-10 06:00

Hi Skyline team.

We are setting command line based data extraction where a custom report is meant to be exported at the end of the analysis. When trying to export a standard report template everything runs fine but we get an error when we try to export a custom report. I have attached some sreenshots. Please advise further,

Thank you

Stoyan

 image (6).png  image (7).png  image (8).png 
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Failed to attempt reintegrate peaks_index 6 must be between 0 and 5
vanyabangera 2024-09-10 22:48

Dear Team,

We are failing to train mProphet model in skyline version 22.2.0.527 (841287d47) and 24.1.0.199 (6a0775ef83). But unable to reintegrate. It's showing pop-up "failed attempting to reintegrate peaks. The index 6 must be between 0 and 5". As per the suggestion obtained from previous queries we tried to perform the rescoring but failed to reintegrate. Kindly advice to resolve the issue.

Thanks in advance.

Kind regards
Vanya K N

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Failed to attempt reintegrate peaks_index 6 must be between 0 and 5
vanyabangera 2024-09-10 22:36

Dear Team,

We are failing to train mProphet model in skyline version 22.2.0.527 (841287d47) and 24.1.0.199 (6a0775ef83). But unable to reintegrate. It's showing pop-up "failed attempting to reintegrate peaks. The index 6 must be between 0 and 5". As per the suggestion obtained from previous queries we tried to perform the rescoring but failed to reintegrate. Kindly advice to resolve the issue.

Thanks in advance.

Kind regards
Vanya K N

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File format conversion from raw to .sky etc
(1 response) sachin burange 2024-09-10 11:53

Dear Sir,

,sky format is propriety of MacCoss lab ? If yes, how do we convert raw file to this format ? Is .sky format can be used for AI/ML ?

Sachin

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Bug report: Chromatograms randomly switch between RT and iRT
(2 responses) whitney stutts35805 2024-09-10 08:49

When using iRT for the replicate comparison plot, I get different a mixture of RT and iRT plots in the chromatogram window for different samples. I would like both the RT comparison plot and the chromatogram to show the same thing for all samples, in this case iRT instead of RT on the x-axis of the chromatogram.

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Transparent Fragment Ions
(1 response) darora 2024-09-10 07:10

Hi,

I see a signal for my selected fragment ions for peptides but somehow its transparent. I don´t quite understand the reason why and can it be used for atleast saying that this peptide was trigerred with atleast fragment ions

 Screenshot 2024-09-10 160923.png 
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Importing lists of peptides using SkylineRunner.exe / CMD
(1 response) Jonas Becker 2024-09-09 08:25

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

 peptides.txt 
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Standard defining / LOD-LOQ calculation
(1 response) oyku su yildirim 2024-09-09 01:18

Hi,

How can I define my data as standards, blank, and unknowns?

Also, how can I calculate LOD-LOQ using 10 blank data?

Sincerely

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skyline for linear curve
(2 responses) longzhen8866 2024-09-06 01:03

Hello, how to use skyline to obtain linear curve for peptide (PRM data)? is there any SOP? Thanks~

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Small molecule transition list adducts not loading, access to Skyline 23?
(4 responses) trevor adams 2024-09-04 07:17

Hello,

Our group had previously been using a custom transition list for glycomics data analysis that included both protonated and sodiated adducts. However, recently when trying to use the same list it does not seem to properly extract the sodiated XICs, even on old data files where it had worked fine before. Based on the timeline of this problem, it's possible that this began with Skyline 24. Is there a way I can install Skyline 23 to troubleshoot? I can't seem to find any archive of older releases.

I have attached the transition list in .csv format here. I have only tested this on Agilent .d files.

Best,
Trevor

 glyGen_transitionList.csv 
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question about Skyline deal with small molecular SRM
(1 response) longzhen8866 2024-09-05 02:04

There are many transitions listed for one precursor. I just want to keep the highest 3 transitions when I export to be a SRM method. How can I do that?

 1.PNG 
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Could I please get the exact masses and isotopic abundances that skyline uses?
(1 response) pavel shliaha 2024-09-04 03:22

I tried to predict mz for certain compounds using publically available isotope exact masses and my calculations agree with the raw data much worse than skyline calculations (I interpret this as skyline using more accurate exact element massess). Is there a way I could get the element masses out of skyline and also understand where the developers took them from?

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Convert .blib to .tsv`
(4 responses) ronald cutler 2024-08-30 08:09

Hello,

I have built a spectra library in Skyline and now would like to use this in DIA-NN. Since the BiblioSpec output is an SQLite3 file and DIA-NN needs a .tsv as specified here: https://github.com/vdemichev/DiaNN?tab=readme-ov-file#spectral-library-formats.

I have found this program which might be able to do the trick, but wondering if I can have any help with this? https://gist.github.com/icedraco/78d9879f2171ecebcbfb

Thank you very much for your time,
Ronnie

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Spectral Library Build Error
(7 responses) wootena 2024-08-22 07:15

Hello,

I am trying to build a spectral library using timsTOF data that was searched using Peaks Studio. I exported the db.pep.xml file from Peaks.

I am receiving an error message that reads ERROR index 10095 out of range in mzXML file ".\081023 C1 SDS 1 - 45 min_Slot2-1_1_1119.d.mzxml. I know this type of error was posted on the support forum previously and mentioned "this type of error was due to pep.xml file claims to have a spectrum match #, but there aren't that many spectra in the mzXML file."

I'm not quite sure how to fix this issue, but I would appreciate any feedback.

I have uploaded a folder with the error message, raw, mzxml, and pep.xml file to the file sharing folder for skyline support titled "Skyline Issue timsTOF" .

Thanks,
Ashley

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New LOD/ LOQ Calculations
(4 responses) lilian heil 2024-08-28 08:20

Hi Team!

I updated to Skyline daily and when I use the new bilinear turning point LOD calculation, almost everything has LOD of the highest point on the curve. I have heard from a few others that they are seeing the same issue. Do we need to somehow update the Skyline document for these calculations to work?

Thanks,

Lilian

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Error saving user configutation file when running Skyline in Apptainer
mauraisa 2024-08-26 15:08

I am trying to run Skyline from the command line inside of an apptainer container as part of a Nextflow workflow.

There are a couple of steps in the workflow which are failing with the error:

Error: Failed saving to the user configuration file.
Failed to save settings: A configuration file cannot be created for the requested Configuration object.

Specifically, the steps that are causing the error are when I try to run protein parsimony in Skyline and when a new report template is added to a document.

If I set the --verbose-errors flag, this is the stack trace:

Error: Failed saving to the user configuration file.
System.Configuration.ConfigurationErrorsException: Failed to save settings: A configuration file cannot be created for the requested Configuration object. ---> System.Configuration.ConfigurationErrorsException: A configuration file cannot be created for the requested Configuration object.
   at System.Configuration.MgmtConfigurationRecord.SaveAs(String filename, ConfigurationSaveMode saveMode, Boolean forceUpdateAll)
   at System.Configuration.ClientSettingsStore.WriteSettings(String sectionName, Boolean isRoaming, IDictionary newSettings)
   --- End of inner exception stack trace ---
   at System.Configuration.ClientSettingsStore.WriteSettings(String sectionName, Boolean isRoaming, IDictionary newSettings)
   at System.Configuration.LocalFileSettingsProvider.SetPropertyValues(SettingsContext context, SettingsPropertyValueCollection values)
   at System.Configuration.SettingsBase.SaveCore()
   at System.Configuration.SettingsBase.Save()
   at System.Configuration.ApplicationSettingsBase.Save()
   at pwiz.Skyline.CommandLine.<>c__DisplayClass136_0.<HandleExceptions>b__0() in Z:\pwiz\pwiz_tools\Skyline\CommandLine.cs:line 4438
   at pwiz.Skyline.CommandLine.HandleExceptions[T](CommandArgs commandArgs, Func`1 func, Action`1 outputFunc) in Z:\pwiz\pwiz_tools\Skyline\CommandLine.cs:line 4451

It looks like Skyline is trying to save settings to a config file which does not exist or can not be written to because of the user's permissions in apptainer. I understand why saving previously used settings makes sense in the GUI, but I am not sure why this would be useful or desirable when running Skyline from the command line. It is especially pointless in a container where the user settings do not persist.

I have attached a minimal set of files to reproduce the error I am getting with the commands:

unzip minimal_example.zip && cd minimal_example
apptainer exec --no-home -B "$(pwd)" 'docker://quay.io/protio/pwiz-skyline-i-agree-to-the-vendor-licenses:3.0.24172-63d00b1' wine SkylineCmd --in=final_minimized_annotated.sky --report-add=replicate_quality.skyr

Is it possible to modify Skyline so the user settings are not saved when running the command line version of Skyline, or at least print a warning instead of raising an error when the file can not be written to?

Thanks,

Aaron

 minimal_example.zip 
view request
External tool accessing multiple reports and not recognizing path to R
(5 responses) lilian heil 2024-08-19 11:45

Hi!

I am trying to write a tool that plots cal curves. So I need a report that has information on intensity for each replicate, as well as cal curve information (slope, LOD, LOQ, etc). I can write this all to one report, but it takes forever. If I remember correctly, it recalculates LOD and LOQ for every line in the table. I really only need that once per peptide, but I may have 12+ rows per peptide. Normally I just write two reports: one that has info on cal curve (one row per peptide), and one that has a row for each point on the cal curve. Is there a way to get an external tool to use these two reports? Or would you suggest another way to do this? It can take a very long time to write the report as is.

Another small thing, I am unable to get Skyline to recognize the path to R.exe. The only reason I can think it's doing this is because there is a space in the path (C:\Program Files\R\R-4.3.1\bin\x64\R.exe). When I run in command line I normally have to add quotation marks around the path name, but Skyline still can't find path when I manually specify pathname, and the macro also has space in name and it gives the same error. Is there a way around this besides moving R installation directory?

Thanks,

Lilian

view request
EncyclopeDIA workflow - koina library generation error
BoW 2024-08-25 11:30

Hi,

I tried to run the EncyclopeDIA workflow through Skyline using the latest Skyline daily version. I got an error in the step of building spectral library using koina. Attached please find a screenshot of the error I got.

Thanks,
Bo

 skyline_encyclopedia_koina_library.png 
view request
Pointed peaks
(3 responses) vmohanty 2024-08-23 08:28

Hi!

I am trying to optimize my PRM method on Exploris 120 coupled to Vanquish Neo.

I am getting pointed transition peaks on skyline for few peptides for some LC-MS methods. I have attached the pics. Could you explain the reason for pointed peaks and can it be considered as good identification?

I have attached 5 pics. However, I know Pic4 right side seems good. But I am unsure what causing pointed peaks in remaining pics.

VM

 1.JPG  2.JPG  3.JPG  4.JPG  5.JPG 
view request
Selecting multiple peptides disregards non-quantitative transitions
Juan C. Rojas E. 2024-08-23 07:25

Hi there,

I am trying to prepare some EIC plots with multiple peptides selected at the same time. I noticed that when this is done, the summed peptide EIC is disregarding if a transition was labelled as non-quantitative and shows the EIC considering all.

Is it possible to exclude the non-quantitative transitions for the peptide EIC or should I delete them for this purpose?

Thanks in advance!
Sincerely,
Juan C.

view request
Normalization DIA TIC
(9 responses) dkueltz 2015-03-16 10:58
Hi Brendan,
Thanks for the 3.1 release! I was wondering whether there is a way to normalize label-free data against the TICs of DIA MSMS runs (I get those using Bruker DataAnalysis). I have entered a separate column in the Skyline replicates table with the relative TIC integrals for each replicate. But I cannot select that column for normalization under group comparisons. It only shows up as an option for control group annotation and identity. It would be nice to have the option to select this column of DIA TIC values for normalization. I have been using the DIA TICs to normalize each transition in a sample/ replicate-specific fashion to account for small differences in loading or sample ionization after exporting the data to Excel. But with the new group comparison that includes statistics it would be great to be able to do it within skyline to save time.
Thanks for any suggestions,
Dietmar
view request
Precursor exclusion m/z window
(1 response) vanyabangera 2024-08-21 22:11

Dear Skyline team,

We have found a response regarding query on Precursor exclusion m/z window. However, we were not able to understand importance of "Precursor exclusion m/z window" instead the option to use "DIA precursor exclusion window".

Thanks in advance

Vanya

 Precursor_exclusion_m_z_window.PNG 
view request
More significant figures possible in peak boundaries report?
(1 response) sstewart 2024-08-21 17:08

Hi team,
Thanks so much for all these new command line additions!

Is there a way to set number of sig figs in exported reports? My goal is to get higher precision peak boundaries via the command line. I can right click on the column in Document Grid and set to Full Precision, but this isn't sticky. Precision resets to 2 decimal places after I close Document Grid.

Right now, peak boundaries are truncated after two decimal places and this is enough (.06 seconds) to be a noticeable wiggle.

Judging from line 3358 in CommandLine.cs, it looks like number of sig figures might be persisted in viewInfo? I'm happy to submit a patch if you can give me a rough outline of what to do, or maybe I'm just missing something obvious in the GUI.

Thanks as always,
Sam

view request
TMT quantification tutorials
(3 responses) TK_1234 2024-04-09 07:42

Hi Skyline users, a new user here.

I was able to follow the tutorial on "Absolute Quantification.' Now, I'm interested in learning about TMT-based quantitative proteomics. My questions are the following: 1) Is there a skyline tutorial on TMT quantification? 2) If the TMT-labeling is applied only to the samples but not the heavy standard target peptide (used to generate calibration curve), what is the correct way to quantify the labeled samples?

Thank you so much!

view request
Problem with Koina
(2 responses) ericw04 2024-08-19 12:39

Hi Skyline team,

I am trying to run an EncyclopeDIA search through Skyline (v.Skyline (64-bit) 24.1.0.199 (6a0775ef83)) and it fails almost immediately when trying to submit my FASTA file to Koina to generate a Prosit library. A picture of the Skyline error is attached as a PNG. Any advice you have is much appreciated!

-Eric

 Skyline_Koina error_20240819.png 
view request
MALDI support
(6 responses) alesur 2024-08-14 02:11

Dear Skyline Team,

I recently tested the script MALDISkyLink from C. Ashwood for MALDI support and found that it worked quite well. It essentially duplicates the spectrum and brackets it with background values to add a "fake" time value, allowing Skyline to calculate areas. Could you consider implementing something similar directly into Skyline for the import of MALDI data?

The limitation of this script (in my hands) is that it can only convert MALDI spectra one by one, whereas MALDI users usually have thousands of spots to analyze.

It would be a great addition, as Skyline is a fantastic tool for processing and curating large sample batches. MALDI-MS still has numerous applications in proteomics, glycomics, and the world of small molecules.

Thanks

Best regards

view request
Sciex OS (6500+) CE optimization method export error
(4 responses) AL24851 2024-08-12 23:07

Hello Team Skyline,

I am currently trying to export CE optimizing methods for 2x Sciex Qtrap systems (6500+). Both systems got Skyline installed on their own individual PCs. When I try to export CE optimizing method using Skyline, I am getting two different errors from each Skyline (please find attached). I have fed in the same transition list, both have the same transition settings, and I selected the same export method settings. Any idea on how to fix this? FYI, I do not have any problem exporting them as transition lists.

Thanks a lot for your time.

Kind regards,
Alex

 Sciex 1.png  Sciex 2.png  Skyline errors.txt 
view request
Low quality MS/MS spectra in building a spectral library for targeted peptidomics
(1 response) julian matytchak 2024-08-16 03:08

Hi there,

We are fairly new to Skyline and targeted MS methods in general. Our goal is to target certain peptides in a complex sample using a Brukers TimsTOF mass spectrometer.

We have compiled a bunch of DDA data which should be used for the creation of the spectral library. Whenever we look through the data in Skyline, the MS1 spectra of the identified peptides look fine. The corresponding MS2 spectra however, usually have very weird, angled shapes.

In the attached picture SADPNFLRF_MS2, you can see how the spectra for the fragments are triangular. Compared to this one, many other spectra are very messy, some of them making little sense at all (the chromatogram being just a straight horizontal line...)

Does anyone know what could be causing this and/or could share some tips with this kind of method development?

Thank you very much,

Julian

 SADPNFLRF_MS2.png 
view request
Native Method Export for TSQ Quantis?
Will Thompson 2024-08-16 05:20

Hi Skyline Team,

We recently moved a TSQ Quantis to our lab from another lab in the company, and so I'm starting to work with it and noticed there are no native method export settings that seem to work for this instrument. We are able to work around it by using TSQ Ultra CE settings and using the File/Export Transition List for TSQ Altis, then importing the transition lists into the method editor. Obviously the native method export is an additional convenience. Are their settings I am missing or is there no plan to support this instrument in native method export?

Cheers

Will

view request
Pin the target panel and other view's option
(1 response) ftayyari 2024-08-15 11:21

Dear Skyline Team,

Could you please inform me on how to re-pin the target panel to the left side of my main skyline window? I accidentally unpinned it, causing it to merge with the other tabs displaying the peaks. Additionally, I would appreciate guidance on pinning other viewing options such as RT replicate comparison. Typically, I have them displayed on tabs or separate windows. Thank you!

 Pin_target.PNG 
view request
Request: Consistency between note importing and exporting
(5 responses) Chris Ashwood 2024-06-06 23:14

Dear Skyline Team,

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

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

Cheers,
Chris

view request
Error in DDA Search Library Build
(2 responses) mcrawford 2024-08-14 12:19

I am attempting to perform label-free quantitation on two proteins in human macrophage cells. I prepared the samples using the EasyPep™ MS Sample Prep Kits from Thermofisher (Pierce), from the required amount of cells and passed over the cleanup columns.
I then injected the sample onto an Agilent 6545XT QToF running DDA MSMS over a 50 minute reversed-phase gradient on a peptide mapping column (fairly standard proteomic methods). The TIC itself looks quite dense, so there should not be a lack of data for the algorithm to mine.
I am attempting to build a spectral library for the samples, but keep encountering the following error: ERROR: No spectra were found for the new library.
I was first trying to build the library using the FASTA sequences for only my proteins of interest, but just to double check I also ran the search against the human proteome and encountered the same issue. I tried lowering score cut-offs and loosening mass tolerances, but nothing has prevented this error from showing up.
I have attached the error message, are there other pieces of information I should upload to help figure out the problem?

 Skyline_Error.txt 
view request
MPP Report doesn't work after upgrade to 22.2
(10 responses) denina simmons 2023-07-08 17:55

Hi there,

I am processing my first set of data since I upgraded to 22.2.

I use the MPPreport external tool to export my DDA results, and the tool no longer works. I have attached a screenshot of the error string from the "immediate window".

Can this be repaired? I am trying to find a different way to export my results, and I tried to export report from the file menu, and it sort of works, only my protein groups are not rolled up together, and I am not sure if there is a way to fix that? Or just to fix the MPP report external tool?

Thanks.

Denina

 MPPReport tool error.png 
view request
EncyclopeDIA search - (StatusCode=Unavailable, Detail="GOAWAY received")
Matt Padula 2024-08-14 23:08
Hey all. Trying to run an EncyclopeDIA search and at the end of requesting predictions I am getting this error. I did need to get out IT people to open the firewall to access the Koina server but everything seems connected.

---------------------------
Skyline
---------------------------
Status(StatusCode=Unavailable, Detail="GOAWAY received")
---------------------------
OK More Info
---------------------------
Skyline (64-bit) 24.1.0.199 (6a0775ef83)

System.Reflection.TargetInvocationException: Status(StatusCode=Unavailable, Detail="GOAWAY received") ---> pwiz.Skyline.Model.Koina.KoinaException: Status(StatusCode=Unavailable, Detail="GOAWAY received") ---> Grpc.Core.RpcException: Status(StatusCode=Unavailable, Detail="GOAWAY received")
   at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   at System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   at Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   at Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   at Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Inference.GRPCInferenceService.GRPCInferenceServiceClient.ModelInfer(ModelInferRequest request, CallOptions options) in C:\proj\skyline_24_1\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\GrpcServiceGrpc.cs:line 900
   at Inference.GRPCInferenceService.GRPCInferenceServiceClient.ModelInfer(ModelInferRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) in C:\proj\skyline_24_1\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\GrpcServiceGrpc.cs:line 886
   at pwiz.Skyline.Model.Koina.Models.KoinaModel`6.Predict(GRPCInferenceServiceClient predictionClient, TKoinaIn inputData, CancellationToken token) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Koina\Models\KoinaModel.cs:line 276
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Koina.Models.KoinaModel`6.Predict(GRPCInferenceServiceClient predictionClient, TKoinaIn inputData, CancellationToken token) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Koina\Models\KoinaModel.cs:line 280
   at pwiz.Skyline.Model.Koina.Models.KoinaModel`6.<>c__DisplayClass22_0.<PredictBatches>b__2(Int32 batchIndex) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Koina\Models\KoinaModel.cs:line 422
   at pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in C:\proj\skyline_24_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ProducerConsumerWorker.cs:line 186
   --- End of inner exception stack trace ---
   at pwiz.Common.SystemUtil.ParallelEx.LoopWithExceptionHandling(Action loop, Action`1 catchClause) in C:\proj\skyline_24_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ParallelEx.cs:line 149
   at pwiz.Common.SystemUtil.ParallelEx.For(Int32 fromInclusive, Int32 toExclusive, Action`1 body, Action`1 catchClause, Nullable`1 maxThreads) in C:\proj\skyline_24_1\pwiz_tools\Shared\CommonUtil\SystemUtil\ParallelEx.cs:line 77
   at pwiz.Skyline.Model.Koina.Models.KoinaModel`6.PredictBatches(GRPCInferenceServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, List`1 inputs, CancellationToken token) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Koina\Models\KoinaModel.cs:line 435
   at pwiz.Skyline.Model.Koina.Models.KoinaHelpers.PredictBatchesFromKoinaCsv(String koinaCsvFilePath, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, CancellationToken token) in C:\proj\skyline_24_1\pwiz_tools\Skyline\Model\Koina\Models\KoinaModel.cs:line 479
   at pwiz.Skyline.FileUI.PeptideSearch.EncyclopeDiaSearchControl.Search(EncyclopeDiaSettings settings, CancellationTokenSource token, IProgressStatus status) in C:\proj\skyline_24_1\pwiz_tools\Skyline\FileUI\PeptideSearch\EncyclopeDiaSearchDlg.cs:line 695
---------------------------
view request
Skyline for MAC OS
(2 responses) ana normando 2020-03-17 10:50

Is there an available version of Skyline for MAC OS?

view request
MAC OS Version
(4 responses) tranjoh1 2015-11-09 12:18
Hello,

I am very new to this software and I am wondering if there is a version of Skyline for MAC OS, and if so, where may I find the link?

Thanks.
view request
Chromatograms are not visible in the Skyline when imported from SCIEX 7500
(1 response) deepak ahire 2024-08-14 13:07

Hi,
I am trying to visualize my proteomics samples after running on Sciex 7500. The samples do get imported in the Skyline but I don't see any chromatogram of corresponding peptide transitions.

view request
request for help with msconvert
tm92 2024-08-13 18:42

I reached out 08/13/2024 about difficulty filtering in msconvert to filter thermo ascend data by scan event. I beleive the issue is related to syntax, as the GUI MSconvert operates when scan filter is not used.

view request
quantification analysis - manually input normalization factor
(3 responses) lorrain 2024-08-13 13:19

Hi,
is there a tab that we can add to the results grid so that we can manually assign a normalization factor for each replicate?
i.e. we know for each replicate/data file, different amount of total peptides were loaded, and we hope to assign a normalization factor for each of the data file such that the final quantification results will be normalized to total peptides.

Thanks!

Lorrain

view request
Looking for Peak Picking Algorithm Adjustment Documentation
(3 responses) jeff 2024-08-05 07:56

I'm trying to use Skyline to replace Tracefinder in my workflow. I'm collecting HRMS on a Thermo Orbitrap in MS1 full scan mode. I've got a list of targeted analytes, and while Skyline in general works well, all the documentation I can find for small molecules recommends making manual adjustments to peak integrations, which is not feasible for the number of samples I need to process per day.

I haven't exhaustively read every document, so I'm hoping there is a resource I've just missed!

view request
Ratio to standard N/A after enabling FAIMS compensation voltage filter
(3 responses) qing yu2 2024-08-08 17:43

Hi,

I have a list of peptides that I am trying to quantify. I spiked in heavy standard and collected the data with FAIMS. if I don't use compensation voltage filter, everything looks fine. But if I select compensation voltage filter, the peak looks nice but in the report all ratios become N/A. Any idea what might have caused it?

Thanks,
Qing

 Capture.JPG 
view request
Panorama upload
(1 response) adriana.paesleme 2024-08-08 05:29

Dear,

We are having some issues in submiting the skyline file to Panorama. We are receiving the error below. Could you please help us to troubleshoot this issue? Thank you very much. Best, Daniela

08 Aug 2024 05:17:48,369 INFO : Starting to run task 'org.labkey.targetedms.pipeline.TargetedMSImportTask' at location 'webserver-high-priority'
08 Aug 2024 05:17:48,370 INFO : Starting to import Skyline document from Corridas_PRM_Amostras_Injeçao1e2_184pepsquantificados_2024-08-08_09-14-59.sky.zip
08 Aug 2024 05:17:48,370 INFO : Expanding Peps_Mix_Saliva_PRM_Unscheduled_364peps_17fev23.blib
08 Aug 2024 05:17:50,589 INFO : Expanding PRM_Final_21_07_23.blib
08 Aug 2024 05:17:55,067 INFO : Expanding PRM_Final_21_07_23.redundant.blib
08 Aug 2024 05:18:16,718 ERROR: Import failed
java.lang.IllegalArgumentException: malformed input off : 26, length : 1
at java.base/java.lang.String.throwMalformed(String.java:1242)
at java.base/java.lang.String.decodeUTF8_UTF16(String.java:1198)
at java.base/java.lang.String.newStringUTF8NoRepl(String.java:732)
at java.base/java.lang.System$2.newStringUTF8NoRepl(System.java:2398)
at java.base/java.util.zip.ZipCoder$UTF8ZipCoder.toString(ZipCoder.java:199)
at java.base/java.util.zip.ZipCoder.toString(ZipCoder.java:66)
at java.base/java.util.zip.ZipInputStream.readLOC(ZipInputStream.java:302)
at java.base/java.util.zip.ZipInputStream.getNextEntry(ZipInputStream.java:124)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:106)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:82)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:69)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:64)
at org.labkey.targetedms.SkylineDocImporter.extractIfZip(SkylineDocImporter.java:1005)
at org.labkey.targetedms.SkylineDocImporter.importSkylineDoc(SkylineDocImporter.java:317)
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:249)
at org.labkey.targetedms.pipeline.TargetedMSImportTask.run(TargetedMSImportTask.java:63)
at org.labkey.api.pipeline.PipelineJob.runActiveTask(PipelineJob.java:833)
at org.labkey.api.pipeline.PipelineJob.run(PipelineJob.java:1075)
at org.labkey.pipeline.mule.PipelineJobRunner.run(PipelineJobRunner.java:40)
at jdk.internal.reflect.GeneratedMethodAccessor1156.invoke(Unknown Source)
at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
at java.base/java.lang.reflect.Method.invoke(Method.java:568)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invokeMethod(DynamicEntryPoint.java:312)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invoke(DynamicEntryPoint.java:259)
at org.mule.impl.DefaultLifecycleAdapter.intercept(DefaultLifecycleAdapter.java:193)
at org.mule.impl.InterceptorsInvoker.execute(InterceptorsInvoker.java:47)
at org.mule.impl.model.DefaultMuleProxy.run(DefaultMuleProxy.java:470)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$CallerRunsPolicy.rejectedExecution(ThreadPoolExecutor.java:1486)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.reject(ThreadPoolExecutor.java:391)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.execute(ThreadPoolExecutor.java:865)
at org.mule.impl.work.ScheduleWorkExecutor.doExecute(ScheduleWorkExecutor.java:39)
at org.mule.impl.work.MuleWorkManager.executeWork(MuleWorkManager.java:277)
at org.mule.impl.work.MuleWorkManager.scheduleWork(MuleWorkManager.java:244)
at org.mule.impl.model.seda.SedaComponent.run(SedaComponent.java:483)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:650)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:675)
at java.base/java.lang.Thread.run(Thread.java:833)
Caused by: java.nio.charset.MalformedInputException: Input length = 1
... 39 more
08 Aug 2024 05:18:16,745 INFO : Failed to complete task 'org.labkey.targetedms.pipeline.TargetedMSImportTask'
08 Aug 2024 05:18:16,747 ERROR: malformed input off : 26, length : 1
org.labkey.api.pipeline.PipelineJobException: malformed input off : 26, length : 1
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:276)
at org.labkey.targetedms.pipeline.TargetedMSImportTask.run(TargetedMSImportTask.java:63)
at org.labkey.api.pipeline.PipelineJob.runActiveTask(PipelineJob.java:833)
at org.labkey.api.pipeline.PipelineJob.run(PipelineJob.java:1075)
at org.labkey.pipeline.mule.PipelineJobRunner.run(PipelineJobRunner.java:40)
at jdk.internal.reflect.GeneratedMethodAccessor1156.invoke(Unknown Source)
at java.base/jdk.internal.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
at java.base/java.lang.reflect.Method.invoke(Method.java:568)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invokeMethod(DynamicEntryPoint.java:312)
at org.mule.impl.model.resolvers.DynamicEntryPoint.invoke(DynamicEntryPoint.java:259)
at org.mule.impl.DefaultLifecycleAdapter.intercept(DefaultLifecycleAdapter.java:193)
at org.mule.impl.InterceptorsInvoker.execute(InterceptorsInvoker.java:47)
at org.mule.impl.model.DefaultMuleProxy.run(DefaultMuleProxy.java:470)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$CallerRunsPolicy.rejectedExecution(ThreadPoolExecutor.java:1486)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.reject(ThreadPoolExecutor.java:391)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor.execute(ThreadPoolExecutor.java:865)
at org.mule.impl.work.ScheduleWorkExecutor.doExecute(ScheduleWorkExecutor.java:39)
at org.mule.impl.work.MuleWorkManager.executeWork(MuleWorkManager.java:277)
at org.mule.impl.work.MuleWorkManager.scheduleWork(MuleWorkManager.java:244)
at org.mule.impl.model.seda.SedaComponent.run(SedaComponent.java:483)
at org.mule.impl.work.WorkerContext.run(WorkerContext.java:310)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:650)
at edu.emory.mathcs.backport.java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:675)
at java.base/java.lang.Thread.run(Thread.java:833)
Caused by: java.lang.IllegalArgumentException: malformed input off : 26, length : 1
at java.base/java.lang.String.throwMalformed(String.java:1242)
at java.base/java.lang.String.decodeUTF8_UTF16(String.java:1198)
at java.base/java.lang.String.newStringUTF8NoRepl(String.java:732)
at java.base/java.lang.System$2.newStringUTF8NoRepl(System.java:2398)
at java.base/java.util.zip.ZipCoder$UTF8ZipCoder.toString(ZipCoder.java:199)
at java.base/java.util.zip.ZipCoder.toString(ZipCoder.java:66)
at java.base/java.util.zip.ZipInputStream.readLOC(ZipInputStream.java:302)
at java.base/java.util.zip.ZipInputStream.getNextEntry(ZipInputStream.java:124)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:106)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:82)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:69)
at org.labkey.api.writer.ZipUtil.unzipToDirectory(ZipUtil.java:64)
at org.labkey.targetedms.SkylineDocImporter.extractIfZip(SkylineDocImporter.java:1005)
at org.labkey.targetedms.SkylineDocImporter.importSkylineDoc(SkylineDocImporter.java:317)
at org.labkey.targetedms.SkylineDocImporter.importRun(SkylineDocImporter.java:249)
... 24 more
Caused by: java.nio.charset.MalformedInputException: Input length = 1
... 39 more

view request
Unable to select mapped network drive as source of results to import
(1 response) madsT 2024-08-08 05:16

Hi,

I was wondering if anyone has encountered a similar issue. I'm trying to access results files to import from a mapped network drive. However, I can only see the local drive (C:). I've tried making a shortcut of the network drive to "This PC" and restarted as well but still can't see any other option other than C:.

Thanks!

 Capture.PNG 
view request
Optimum transition full scan and other tips for filtering targeted MS2 collected in Astral
mlane 2024-08-08 07:54

Hi,
We're evaluating some Astral data with Skyline doc previously optimized for PRM on Lumos Fusion (MS2 filtering matched Fusion Orbi res 60K, At 400 m/z and selected PRM and Orbitrap as instrument in the MS2 filtering). I would like to know if you've observed any issues or have experience with Astral run in targeted mode. We use the latest Skyline Daily build Skyline-daily (64-bit) 24.1.1.202 (c511d22bf). We noticed the Astral data seems very noisy with very choppy peak shape so curious if alternate instrument selection (under full scan filter settings), resolution, etc. would improve this (we already have Savitzky-Golay smoothing active). The Astral experiment acquires targeted MS2 scans for ~60 peptide precursors. The Astral method has Experiment 1 and 2 in parallel: Exp 1 set as Orbi for full scan MS and resolution of 240K, while Exp 2 Astral MS2 scans were set for 1.6Da isolation and 20ms injection. Should Skyline MS2 filter be PRM & Orbitrap or should it be TOF (PRM?)? What would be optimum resolution for Astral MS2...80K? Are there any other settings we should be concerned with, especially since this instrument is scanning so much faster than our older instrument?
Thanks!
Monica

view request
No product ion chromatograms found error - crosslinked data - 24.1version
(4 responses) David26 2024-08-07 07:50

Hello,

I have the following error message : "No product ion chromatograms found" and indeed I don't see any fragments for any ion in any of the proteins monitored. Sometimes the error message don't appear but the signal associated to the fragments is extremely low (a line on the 0)

What I tested :
• crosslinked peptides / non-crosslinked peptides
• changing transition settings / peptide settings (and re-importing the results each time that I was doing significant changes) for the transition/peptide settings modified I tried to stick with what you said in this similar request : https://skyline.ms/announcements/home/support/thread.view?rowId=43123
• using different raw files with different protein concentrations to verify if it was a sensitivity problem (I can send the data if needed)

Thank you in advance for your help,

Best regards,

David

 Test_XL_07-08-24.sky 
view request
Error when importing peak boundaries
(4 responses) Yasin 2024-08-04 16:34
Hi,

I am trying to import peak boundaries for small molecules. This works most of the time but sometimes I run into errors like the one below. I realize that I can change the names of my molecules (which are in my case just numbers) but this is sub-ideal when I want to retain ids to compare to other tools. Any chance this could be fixed? I have attached the file in case it helps. Also I am on a Windows 11 system.


---------------------------
Skyline
---------------------------
Failed reading the file E:\dt_skyline_boundaries.csv.
A protein sequence may not contain the character '5' at 0.
---------------------------
OK More Info
---------------------------
System.IO.InvalidDataException: A protein sequence may not contain the character '5' at 0. ---> System.IO.InvalidDataException: A protein sequence may not contain the character '5' at 0.
   at pwiz.Skyline.Model.FastaSequence.ValidateSequence(String seq) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\PeptideGroup.cs:line 293
   at pwiz.Skyline.Model.Peptide.Validate() in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Peptide.cs:line 388
   at pwiz.Skyline.Model.Peptide..ctor(FastaSequence fastaSequence, String sequence, Nullable`1 begin, Nullable`1 end, Int32 missedCleavages, Boolean isDecoy) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Peptide.cs:line 57
   at pwiz.Skyline.Model.ModificationMatcher.GetModifiedNode(String seq, FastaSequence fastaSequence) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\ModificationMatcher.cs:line 435
   at pwiz.Skyline.Model.PeakBoundaryImporter.Import(TextReader reader, IProgressMonitor progressMonitor, Int64 lineCount, Boolean isMinutes, Boolean removeMissing, Boolean changePeaks) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\ImportPeakBoundaries.cs:line 359
   at pwiz.Skyline.Model.PeakBoundaryImporter.Import(String inputFile, IProgressMonitor progressMonitor, Int64 lineCount, Boolean removeMissing, Boolean changePeaks) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\ImportPeakBoundaries.cs:line 236
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass793_0.<ImportPeakBoundaries>b__2(IProgressMonitor longWaitBroker) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 1543
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Util\Util.cs:line 1920
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.SkylineWindow.ImportPeakBoundaries(String fileName, Int64 lineCount, String description) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 1546
   at pwiz.Skyline.SkylineWindow.ImportPeakBoundariesFile(String peakBoundariesFile) in C:\proj\skyline_23_1\pwiz_tools\Skyline\SkylineFiles.cs:line 1517
---------------------------
 dt_skyline_boundaries.csv 
view request
report_skyline* CANNOT be created
(2 responses) ksasaki 2024-08-04 23:22

Dear Skyline Team,

I'm working with DIA-NN 1.9.1 and Skyline (64 bit Administrator Install), but DIA-NN doesn't generate "report_skyline.sky/skyd".
Could you tell me how I could get over it?
I'm using the Administrator Install Skyline, because DIA-NN 1.9.1 hasn't recognized the conventional one.
Thank you very much.

Kazuki Sasaki

view request
Kiona server unavailable
(1 response) kylinchang32 2024-08-02 17:17

Hi

When I was trying to set up Koina to generate a spectral library in Skyline, it says server unavailable. Do you know what could be causing this?

Thanks,
Qingling

 Koina.png 
view request
DIA-NN .speclib support
(46 responses) Tobi 2020-05-27 03:03

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

view request
Changing the default isotope envelope for multiple precursors?
(2 responses) ho-tak lau 2024-08-02 09:51

Hello Skyline Team,

Is there a way to change the isotope envelope of multiple peptide precursors?
I am working on some long glycopeptides with many precursors (see attached .png). So I wonder if the M+3, M+4 can be included by editing the .sky using a text editor.

Thanks in advance.
Ho-Tak

 Glycopeptide_envelope.PNG 
view request
problems with RT predictor
(1 response) christiee 2024-08-01 15:33

Dear Skyline Team,

I am hoping you can help me with a problem that is causing me great frustration.

Recently when loading LysC digested PRM data into skyline, there is no longer a predicted retention time associated with the data. This seems to be a relatively recent (we started noticing it around June) problem for us. I've attached two files to this message. The first is a file entitled HA_May2024. This data was acquired in may of this year and as you can see when you open the data, there is a predicted RT time associated with the data. The second file is entitled HA_V2d. This data was acquired on July 27th and as you will see, there is no predicted RT time associated with this data. Interestingly, if you remove the data from the May2024 file and import the data from the V2d file into the May2024 file, there is again no predicted RT time associated with the data. Perhaps even more perplexing is that if I take the data acquired in May and import it into the recently made V2d skyline file, the data has predicted RT times! To me this is suggesting that the HA_V2d skyline document (and associated blibs and iRT databases) are not the problem but perhaps there is a problem with the way the data is being read??? One thing to note is that we are only seeing this with LysC data, our Trypsin digested data does not have this problem.

If possible, could someone on your team take a look at these files and see if they can identify why this is happening.

Thank you for your time,
Christie

NOTE: the data for this request has been uploaded to your file sharing folder. The title of the file is problems_w_RT_predictor_20240801

view request
Issue with opening Skyline files after upgrading to 24.1
khoil 2024-08-01 17:15

Hello,

I was able to successfully install Skyline 24.1 after previously uninstalling the older version. However, my current issue is that now I am unable to open previously saved skyline files by clicking on them and the icon has also gone missing. I can still view them by opening the Skyline software from the start menu and manually loading the file, however this is a huge inconvenience. Attached is a screen clip of the skyline file.

Thank you!

 Screenshot 2024-08-01 171337.png 
view request
Mass tolerance
(4 responses) c3053055 2024-08-01 07:26

Hi Skyline team,

I have found that when I was working on a Full scan spectrum from Sciex QTOF 6600 system, I was not able to extract the correct peaks after entering the molecule's formulas. For example, I input the molecular formula of Molecule 1 as C16H20N6O3, and after adding an H, the software calculates the monoisotopic m/z to be 345.166965, but I didn't get the peak. Only after I manually changed the monoisotopic m/z to 345.1193 which was read from the instrument, I could get the correct peak.

I tried to change the "Method match tolerance m/z" in the Transition settings to 0.6 m/z, but it didn't help. So I wonder what I can do to increase the tolerance so that I don't have to change the m/z manually every time. Thank you!

Best wishes,
Mengchun

view request
MRM method for Peptide having Unusual Amino Acid
(1 response) v kumar1 2024-08-01 05:33

Hi,

I would like to set up DDA method for linear peptide, which got 1unusual Amino acid.

The amino acid is 2 aminoisobutyric acid.

I tried to include in modification but option is either N or C terminal. This modification is at C next to N terminal. Do you have any suggestion, how i can incorporate that.

view request
add functions for mRNA analysis?
(1 response) zhijcao 2024-07-31 06:19

Hi Skyline team,
Thank you for your contributions to the proteomics community.
There is a growing interest using Mass Spectrometry for mRNA analysis in academic and pharm industry. I wonder if functions for mRNA analysis could be added to Skyline. Similar to protein analysis, mRNA sequence is digested into oligonucleotides with enzyme (such as T1), and the oligonucleotides of interest are quantified with MRM.

Thanks,
Zhijun

view request
MSstatsShiny - Connection Problem
oyku su yildirim 2024-07-31 05:20

Hello,

I cannot connect to the website of MSstatsShiny (link below). There is no problem with my internet and I tried to connect in different browsers. Is there a general problem?

https://msstatsshiny.com/app/MSstatsShiny

Thank you
Oyku

view request
diaPASEF Calibration curve
marinedmstr 2024-07-31 02:00

Hello everyone,

First of all, I'd like to thank you for all your hard work. Skyline is a very powerful tool (even if I haven't mastered it perfectly yet) and it allows us to do so many things.

I have a challenge that I think is quite unique. I'm working in diaPASEF on a timsTOF Pro and I'm looking to do absolute quantization in label free mode.
I'd like to know if it's possible to make a calibration/spreading curve on diaPASEF acquisitions from a DIA-NN library?
If so, can you tell me how?

Thanks in advance!

view request
Version confusion
(2 responses) Matt Padula 2024-07-30 16:11

I got my IT people to try and install 24.1 using the setup.exe from here, which is labelled as version 24.1.0.199. But after installation, the About says that it is 23.1.0.268.

In addition, I am confused by the labelling of the Unplugged and Administrator versions as they are both labelled as 23.1. Is 24.1 not available as these versions?

view request
Detection Z score and Q Value.
(5 responses) anirudhkashyap511 2024-07-30 12:53

Hi Skyline Team,
I want to run a Quality check on peaks that have been identified by my mprophet model trained on second best peaks. Could you guide me on what Z score and Q value I need to keep? Thanks!

view request
Unable to install V24
(2 responses) lu yu28579 2024-07-18 05:56

I had the v22 installed on my PC (Win10, 64-bit, 32GB memory), but failed to installv23, now failed again on V24. The message said "Application validation did not succeed. Unable to continue." I have attached the log file.
Could you please advise? Thank you very much.

 Skyline Installation error Log_20240718.txt 
view request
MSstats in Skyline
(1 response) oyku su yildirim 2024-07-30 05:57

Hi,

I am working on MSstats in Skyline, and was following the steps of the attached tutorial. However, the version of skyline is old in the tutorial. Is there any updated tutorial about MSstats?

Best regards
Oyku

 MSstats-SkylineExternalTool-InstallationAndUserGuide-v2.1.6.pdf 
view request
Left-handed switching between ‘tabs’ for many measurements
(1 response) niklas gombert 2024-07-29 05:05

Dear Skyline team,

We really enjoy using your software to analyse our PRM measurements. However, there is one thing which caused us some trouble when dealing with larger data-sets. You are probably already familiar with the phenomenon - you create a method based on an initial measurement, validate it and start a big sample set.

However, in our case (and I can imagine that we won't be the only ones affected), we sometimes have slight shifts in our chromatograms when measuring several hundreds of samples. No problem - the PRM windows are large enough. Unfortunately, however, it can happen that the integration of the peaks does not always work automatically in these cases. Therefore, in the past we have often had to reintegrate ‘by hand’...it would therefore be an absolute quality of life improvement for us if we could ‘jump’ between the ‘tabs’ (i.e. between the individual measurements) in Skyline with our left hand, while we could integrate or reintegrate with the right hand (using the mouse). We know the "ALT + arrow key"-way, but this can only be done left-handed by real piano players ;)

Thank you very much in advance!

view request
Unable to install skyline
(1 response) lijm83 2024-07-27 23:44

Hi Team,
I had tried many times to install the skyline 24.1 - 64 bit and the skyline daily(beta), but I failed.
Sometimes, it says I'm running out of disk space. In fact, the disk space is enough. In other cases, it suggests that it is a network issue. The attachment is one of the log files.
I really need your help. Thank you very much!

 JA1QSBNT.log 
view request
Can I export heavy peptide areas in Report?
(1 response) martinnm 2024-07-27 18:27

Hi Skyline Team,

I want to export the normalized peak areas of heavy and light versions of my target peptides in my Report. I know I can customize my report and include "RatioLightToHeavy" but I was wondering if there is a selection for the normalized area specifically for the heavy peptide?

Thanks!

view request
Exporting Transaction List
(4 responses) oliveirarv1 2024-07-26 07:29

Hello Skyline team,
I upgraded Skyline to version 24.1.0.199 (6a0775ef83) this week. After that, I am not able to export transition list using multiple methods anymore. Normally I select 50 max transitions per sample injection.
In this new version, when I enter with 50 (max transition) it doesn't display the number of methods anymore.
But, If I try to export, I get an error message " The precursor XXX requires 600 transitions to optimize, which exceeds the current maximum 50".

Instrument Sciex 7500 Qtrap, proteomic interface, optimizing methods for quantification of surrogate peptides
Could you please help/advise?

thanks!

view request
Copy & Pasting into Rule Set Editor
(5 responses) mcminn m 2024-07-25 10:49

Hello,

I am trying to copy and paste rules into the rule set editor in the document settings for result files. I have a number of different conditions, and right now I cannot paste in or import a table to set up my result file name rules. Happy to discuss more if needed, thank you so much!

view request
Product ion chromatograms look jaggy
(11 responses) skimura 2024-07-25 09:18

Hi,

I have MRM data obtained by SCIEX ZenoTOF7600.
Product ion chromatograms look jaggy.
What kind of parameter do I need to change or use?

Best regards,
Satoshi

 Jaggy product ion chromatogram (2).pdf 
view request
How to use Peptide spectrum match count?
(1 response) Juan C. Rojas E. 2024-07-25 07:58

Hi there,

I am wondering if there is any default filter being applied for Peptide Spectrum Match Count in the document grid? I see that for some peptides this has 0 despite having a library match. Example in the attached figure.

If data is required I can provide some through Panorama, but this is more of a general inquiry on how this is counted in Skyline and what would happen if there are multiple spectral libraries active in a Skyline document?

I had not seen this PSM counting before, but looks very interesting for generating UpSet plots from a Skyline report.

Sincerely,
Juan C.

 PSM_counting.PNG 
view request
Issue with Import DDA Search in 24.1?
(2 responses) Will Thompson 2024-07-24 17:25

Dear Brendan and Team

Just tried to use the Import DDA Search wizard for the first time since updating to 24.1. It appears that the wizard in the current version has deleted the ability to start from raw files, and instead goes straight to the screen where you point to the search results. To reproduce:

File New/Start
Select Import DDA peptide search

Result: There is no option to choose start from raw files
Desired Result: There previously was an option to start from raw files, search results, etc.

Has this functionality changed in the current version, or is this a bug?

Cheers

Will

view request
Setting match
(4 responses) luzjpaulo25041 2024-07-24 13:30

Dear Skyline Support Team,

I hope this message finds you well. I am writing to seek assistance regarding an issue I am experiencing with the Skyline program. Specifically, I am encountering difficulties importing a peptide with a PTM (Post-Translational Modification) due to a configuration mismatch with my current settings.

Here’s a brief overview of the problem: Skyline indicates that it cannot import a specific peptide, even though there is a similar peptide in the library with the same modification mass that Skyline does recognize. This discrepancy has left me unsure about which settings I need to adjust in order for Skyline to correctly recognize and import the peptides from my library.

Could you please provide guidance on what configuration settings I should review or modify to ensure that Skyline recognizes the peptides with PTMs from my library? Any advice or steps you can provide would be greatly appreciated.

Thank you very much for your assistance. I look forward to your prompt response.

Best regards,

 Ostra_20240724.sky  Ostra_20240724.sky.view  Ostra_20240724.skyl  Ostra_seach.blib  Ostra_seach.slc 
view request