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

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,

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!

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

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.

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 ---

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

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

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

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

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?

Dear Skyline support,

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

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

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

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

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

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

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

Best,

Kyle Nguyen
UNC-Chapel Hill

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

Thank you

Dear reader,

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

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

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

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

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

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

Thank-you in advance for your time and effort.

Kind regards,
Jurre

Hi Team,

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

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

Best regards,
TY

Greetings,

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

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

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

With gratitude,
Paul I.

Hi all,

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

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

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

Sincerely,
Juan C.

Hi team

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

Thanks
Ramesh

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

Hi,

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

Could you please indicate the source of the problem?

best wishes, David

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

Peptide standard 1 quantifies the protein amount as 40 fmol.

Peptide standard 2 quantifies the protein 20 fmol.

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

Hello,

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

Any help would be appreciated!
Thanks

Couple of queries:

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

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

*We are new to Skyline.

Thank you for the help.

Hi,

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

What is the problem?
Thank you in advance,
Stefanie

Hi,

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

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

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

Many thanks for your help and all the best,

Gioele

Hi Skyline team,

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

Heyi

Hi Skyline Team,

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

Kind regards,
Elvis

Hi,

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

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

Kind regards,
Leandro

Hello team

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

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

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

Thank you
Ramesh

Dear Skyline Team,

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

Looking forward to your reply.

All the best
Thomas

Dear Sir,

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

Can we consider variable modifications for non-tryptic peptides?

Best regards

MCM

Dear Skyline team,

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

Skyline

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

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

OK

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

Do you have any advice?

Best,

Gianin

Hello,

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

I am having issues with:

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

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

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

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

Anyways I digress. Just looking for some help here