We are planning to develop a mass spectrometry based quantitative assay for the monitoring of certain proteins/ genes. As part of this, we are working on selecting the best proteotypic peptide for each of our target proteins using Skyline.

There are certain proteins which do not have any unique proteotypic peptide. But when we try to apply uniqueness at the gene level, we could see certain peptides considered as unique to a gene. So, I would like to know whether we can consider such peptides (razor peptides) for our quantitative proteomics?

For example, we are looking for a proteotypic peptide of p40 (deltaNP63alpha). However, the peptide sequence for p40 is present in its multiple isoforms. Hence we are not able to select specific proteotypic peptides for p40 isoform (i.e alpha).

--
Jyoti

Hello Skyline Team,

I'm still trying to learn the basics of the software, in particular for the targeted analysis of peptides by PRM. I managed to perform all the steps well described by your tutorials, but I have an issue regarding the number of scans that are imported into Skyline from the raw data. To show you the issue I attached you 3 slides in a pdf file.

In the slide 1 you can see the peak from the raw file (two different visualizations of the same peak are showed). As you can see the peak contains over 40 scans of the same precursor (the instrument used was the Orbitrap Exploris 240 from Thermo).

In the second slide you can see how the same peak is imported into Skyline. As you can see, only few scans (less than 10) are used and the resulting peak has a lower resolution, that means also a lower accuracy when the peak is integrated.

In the third and last slide you can see the only parameter that I've found that should affect the number of scans that are imported, but the option "include all matching scans" was already selected. Since they are all very good scans from the raw file, I don't understand why Skyline is considering less than 10 of them. Is there any way to make Skyline import all the scans present in the raw file?

Thanks in advance for any answer and suggestion you may provide,

Best regards,

Ulisse Cucchi from Nerviano Medical Sciences (Italy)

Help! Help! Could you please help me out ?When I try to use skyline to process my Agilent QQQ 6460 LCMS data, It cannot show chromatogram correctly. When I click on the lysine or any other molecule, it cannot show chromatogram. I don't know what cause this.
I attached the screen shot for what I get from skyline and what I get from mass hunter.
I also attached my transition list.
Thank you,

Hello Skyline team,

I am currently doing targeted proteomics on Altis with FAIMS and Skyline Version 20.2
I was wondering if it is possible to have a optimization library with both information about the optimized CE and optimized CV values or if I can import two separate libraries, one for CE and one for CV

Currently I can only use either CE or CV optimization library but not both...

thank you for your support

best,
Chi

Hello, I am working on a PRM method with stable-isotope-labeled internal standards. Technically it is "SureQuant" where the SIL peptides are monitored to trigger acquisition of endogenous peptides. MS2 scans for the heavy SIL peptides are carried out in an orbitrap at a resolving power of 7,500 at 200 m/z. However, MS2 scans for the endogenous/light peptides are carried out at a resolving power of 60,000 at 200 m/z. Right now I am using 7500 at 200 m/z for both the light and heavy peptides. However, I would like to enforce a separate and more stringent threshold for the endogenous MS2 scans that reflects the different instrument settings used in my method. I believe that on account of the thresholds being too low for the endogenous (in skyline), some of the endogenous transitions appear to have a lot of interference. At the moment is it possible to change this in skyline?

Hi Skyline team,

I just started to learn using Skyline to process SWATH data acquired by Sciex 6600. Library was created by multiple DDA runs searched by ProteinPilot software. Following your instruction, input FASTA files and spectra library. More pep/Proteins than number in library are shown in Skyline. How to explain this?

thanks,
Heyi

Hi Skyline people

We are performing a CE optimization on a SMR experiment run on a QTrap5500 for small molecules, and following the Tutorials. Experimentally, everything worked fin.
We exported the Transition list into the .dam files with multiple the CEs, and reacquired data on known standards. When reimporting the wiff files into Skyline, we get the expected 'curve' for the CEs.

The Replicate graphs show a legend for the colors and CE Steps (-10,-9. etc).

Is there a way for Skyline to show the nominal CEs used by the instrument in the graphs instead of the Steps? Or, is there any parameter in the Document grid to show the Nominal CEs??? All the parameters with CEs I select show either calculated values, or Steps.

Hope I made myself clear!

Cheers

Alejandro

Is there a way to set up retention time decimal places? I use a windows computer and it displays only one decimal place. ..like 14.3. Can I set up Skyline to display 14.32 or something like that?
I use RT +/- 0.02 min between the analyte and heavy labeled standard, and it's making the comparison a bit difficult. I know the skyline displays two decimal places when I export the report in Excel.

Thanks!
Muluneh

The library.tsv file from EasyPQP is in OpenSWATH format. But when loading it in Skyline, it says "the file F:\dev\msfragger\test_skyline\library.tsv is not a supported spectral library file format.". According to https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild, it should support OpenSWATH tsv file.

I put an example of library.tsv in the attachment. Could you please take a look when you have time?

Thanks,

Fengchao

Hello Skyline team, hello Skyline community,

Is there any tutorial etc. which deals with Skylines “Comparing Peak Scoring“ function? I would like to understand the plots (e.g. the different y-axis options) as well as its application better. The tutorials 14, 15 and 18 cover peak model building in general but I didn’t find anything on the “Comparing Peak Scoring” option.

Briefly about my situation: In my targeted MRM assay I am monitoring peptides across multiple organs (9x) and cell lines (5x). For data analysis I am using mProphet peak scoring models. I was now wondering if it is best to use for each organ type its own peak scoring model, or if a mixed-organ respectively mixed cell line peak scoring model can be used for the individual organs. Anyone with any experience in that? Any suggestions, any hints ?

Best, Peter

Dear all,
I am trying to install MSstats but it is not possible to intall de R packages.
I attached the error
Thank you for your help
Irene

Hi, We recently purchased a 7500 and were wondering what support there is for the 7500? Ie in method generation, parameter prediction, data review etc.

Thanks
Dawn

Dear Skyline team

I was wondering if Skyline support DIA files with FAIMS (multi CV) annotation acquired on the Exploris 480.

Thanks in advance,
Sudip

Hi Skyline Team,

I have a use case where i would like to use the same set of stable-isotope labeled molecules both as iRT standards as well as surrogate internal standards for the purposes of quantification. If i set the molecule as an iRT standard (i.e. the little "clock" icon comes up on the molecule) then when i go to the right-click menu to set that molecule as a surrogate standard the selections are greyed out. I can't think of any practical reason why this would not be allowed. Am I doing something wrong, is there a functional reason this is the case, or is this a bug?

Representative Skyline file attached. IF you look in the document grid, you do have the surrogate standards assigned to different molecules, but this is only because i did this part before applying the iRT correction.

Help as always is appreciated.

Cheers,

Will

Dear,

I am trying to build a spectral library in Skyline using an msp file based on predicted spectra. Since we work with histones, we derivatize our peptides using propionylation, so all unmodified lysines and N-terms are blocked with a propionyl group. Therefore, Propionyl (K) and Propionyl (N-term) are fixed modifications, unless another variable modification is present. However, Skyline does not seem to recognize the modification "Propionyl" from the msp, although I also defined it as a modification in the peptide settings. All other modifications (e.g. Acetyl, Dimethyl,..) that I defined, are used correctly in the library. After some research, we figured out that Skyline recalculates the precursor m/z for each peptide (based on its sequence) and doesn't use the one that is stated in the msp. So for a peptide that carries a propionyl group, the m/z is recalculated as if the peptide would be unmodified because Skyline does not recognize the propionyl.

In the attachment you can find a short example of a predicted msp and the Skyline document with the corresponding library I built with this msp. In the msp you can see that the Propionyl group in the header is stated in the exact same way other modifications are, so I don't understand where the problem is.

Thank you in advance for helping me out!

Best,

Laura

I'm trying to install in a windows 10 computer and I'm getting this error:

" Setup has detected that the file 'C:\Users\ADMINI~1\AppData\Local\Temp\VSDA0E1.tmp\DotNetFX472\NDP472-KB4054531-Web.exe' has changed since it was initially published.

See the setup log file located at 'C:\Users\ADMINI~1\AppData\Local\Temp\VSDA0E1.tmp\install.log' for more information."

Hello

Is this possible?

I was hoping to automate a workflow that involved it.

thanks

Lee

Hi Skyline team,

Just want to know details about different types of skyline files, e.g document, view, skyl and chromatogram data. if I want to upload a result to panarama, I need to upload all 4 files, or just document file. thanks,

Heyi

Hello,

First of all, I am quite new to the MS field so there might be some obvious error I just don't know any better about. I am working on Windows 10 Pro with Skyline 20.2.0.343.
I am trying to analyze PRM data measured on a Thermo QExactive Plus, including heavy peptides for quantification. Doing identification with Proteome Discoverer using our Mascot server, it can find the native peptides (unfortunately only 10-80 PSMs depending on the sample) as well as the labelled ones (more than 1000 PSMs per sample).
When trying to build a library with the msf or PD result files and importing the data to Skyline, the peptides do not show up as targets at all. I went back and tried to import some of the Mascot dat files, which then gave me the error "Peptides had multiple ambiguous peptide matches and were excluded". I redid the library build and importing, checking the box "include ambiguous peptide-spectrum matches", and now I can find the peptides in the library (only with the dat files, not the msf or PDresult), but it still doesn't show up as a target peptide. I could then manually add them as library matches to the target peptides, but I don't really understand why it would not do this automatically. I played around with a lot of peptide and transition settings but nothing worked to make them appear.
What could be an explanation for this, Is there anything I am missing?

Thanks,
Susanne

Hello Support Team,
I want to analysis a data in skyline in which I want to match the spectral data obtained in an MRM experiment with the Prosit predicted library.
I could see that the Prosit is making predictions for my target peptides but dotp value is not shown.

However, if I monitor precursors ions also, then only dotp value is shown (but in my data, i have product ions for which I actually need the dot p value).
Could you please help me resolve this.

Looking forward to hearing from you.
Regards
Mehar Un Nissa

Hi,

I've recently upgraded to Skyline 21.1 (downloaded on 22/7/21) and I have the following error when opening a saved .sky file. This happens with old files from a different Skyline version but also with newly created and saved files.

---------------------------
Skyline
---------------------------
Failure opening U:\KTGS\Skyline test sample\Skyline test sample2.sky.
The file contains an error on line 81 at column 9.
---------------------------
OK More Info
---------------------------
System.Reflection.TargetInvocationException: There is an error in XML document (81, 9). ---> System.InvalidOperationException: There is an error in XML document (81, 9). ---> pwiz.Skyline.Util.AssumptionException: error reading mz values - declared mz value 325.13691 does not match calculated value 324.129085450091
   at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 2055
   at pwiz.Skyline.Model.Serialization.DocumentReader.ValidateSerializedVsCalculatedProductMz(Nullable`1 declaredProductMz, TransitionDocNode node) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1623
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionXml(XmlReader reader, TransitionGroup group, ExplicitMods mods, IsotopeDistInfo isotopeDist, ExplicitTransitionValues pre422ExplicitTransitionValues, CrosslinkBuilder crosslinkBuilder) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1606
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionListXml(XmlReader reader, TransitionGroupDocNode nodeGroup, ExplicitMods mods, ExplicitTransitionValues pre422ExplicitTransitionValues) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1466
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupXml(XmlReader reader, Peptide peptide, ExplicitMods mods) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1335
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadTransitionGroupListXml(XmlReader reader, Peptide peptide, ExplicitMods mods) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 1261
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideXml(XmlReader reader, PeptideGroup group, Boolean isCustomMolecule) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 908
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideListXml(XmlReader reader, PeptideGroup group) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 843
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupXml(XmlReader reader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 813
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadPeptideGroupListXml(XmlReader reader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 654
   at pwiz.Skyline.Model.Serialization.DocumentReader.ReadXml(XmlReader reader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Serialization\DocumentReader.cs:line 619
   at pwiz.Skyline.Model.SrmDocument.ReadXml(XmlReader reader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1998
   at System.Xml.Serialization.XmlSerializationReader.ReadSerializable(IXmlSerializable serializable, Boolean wrappedAny)
   at Microsoft.Xml.Serialization.GeneratedAssembly.XmlSerializationReaderSrmDocument.Read1_srm_settings()
   --- End of inner exception stack trace ---
   at System.Xml.Serialization.XmlSerializer.Deserialize(XmlReader xmlReader, String encodingStyle, XmlDeserializationEvents events)
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass742_0.<OpenFile>b__0(IProgressMonitor progressMonitor) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 328
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_21_1_x64\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_21_1_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_21_1_x64\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_21_1_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.SkylineWindow.OpenFile(String path, FormEx parentWindow) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 345
---------------------------

Any help would be appreciated!

Cheers,

Tony

Hi there,
My small molecule analysis methods produce some pretty ugly peaks for a few analytes. Skyline does its best, but I do not expect any integration algorithm could figure out some of my data. What I have been searching for to fix the problem is some place to specify explicit integration boundaries. In reading back on support, I understand that "explicit retention time" and "window" will get me close but these are more "suggestive" to skyline (and in fact do not work for my peaks). Integrating a peak and then rt-clicking and hitting "Apply Peak to All" also seems more suggestive than explicit and does not work. I would like to have skyline integrate all peak area between two specified RT's without any consideration to inflection points or anything else and then apply this setting across the whole batch for that particular molecule. Is that possible at this time? Thanks!

Hi Skyline team
I was wondering if Skyline support MS1 filtering on data acquired using Boxcar on the Exploris.
Thanks a lot
Diego

Hi,

I am getting an error when trying to install Skyline on my PC. Screenshot and details attached.

Have you seen this before? Can you offer any guidance? Thanks

Hello Skyline team,

My name is Jiwon and I am currently a graduate student at Korea University.

I have been using skyline for a few months, but couldn't find a solution for a very time consuming/repetitive job which lead me here.

I wish to save the image files of the transitions: 3 files (total, light, and heavy) for each peptide with a specific pattern name.
For peptide sequence XXXX,
I wish to save the total image file as XXXX_a, light as XXXX_e and heavy as XXXX_s in a designated directory (they can all be in the same directory) in .jpg format.

Currently, I was only able to save the files through right clicking > Save image as one by one, and was wondering if skyline is capable of saving all the images at once.

And also, would it be possible to leave the x-axis and only auto adjust the y-axis based on the selected region only?
An example file is attached below (skyline_adj : wanted scale, skyline_no adj: default image)

Thank you in advance!

Sincerly,
Jiwon Hong

Hi Skyline team,

I would like to confirm how skyline treats a fragment ion that is produced from multiple precursor ions of the same peptide when the data is acquired with MSE. Without the use of IMS it is not possible to discern from which precursor the fragment ion is being produced due to the lack of quadrupole isolation.

When the integrated fragment area is summarized for the peptide and a transition for the same fragment exists in two (or more) precursors, is the area of this "repeated" transition summed twice (or more) or does Skyline recognize it is a repeated value and only counts it once?

As always, thank you for your time and help.
Sincerely,
Juan C.

Hello Skyline team,

Perhaps this matter was already asked but I could not find any answer after I drastically limited my search to "export fasta".

I have done a DIA analysis of some special tissues from an model organism. The basis for the analysis was a rather large fasta database of not so good quality which I used as the background proteome. The analysis of the run data was done with Umpire/Fragpipe and imported into Skyline for visualization and curation. I did quite a bit of manual refinement and came up with a status in which I erased a lot of identified by the search engines proteins that do not match my quality restriction. Besides the generation of a spectral library I would like to also create a fasta file of either all or a specified fraction of the proteins remaining after the curation of the data. This fasta coould be used for other purposes or as a core background proteome that could be further expanded by additional data.

Is there any chance to export the proteins remaining after curation as a fasta file from Skyline. If there is no direct way, can you propose an other tool or procedure to do so from any Skyline generated report.

Best Karl-Heinz

Hi, Skyline team

Can I get an explanation about how Skyline determines peak boundaries?

-Minyeong

Hi. I have a Skyline document of something like 5000 protein entries and I'd like to filter it based on the amino acid sequence length of the proteins in the document. Specifically, I want it to only be the proteins under 150 aa. I can obviously export results to a table and do it there, but I'd like the Skyline document to only include this subset. Is that possible?

Hi Skyline Team,

I am using the "show ion rank and type" feature for full scan targeted MS/MS analysis (=PRM). The feature makes my life a lot easier than before, thanks a lot! I have, however, a question: for a peptide that was detected quite confidently, I see a color code in the MS/MS that is perfectly matching my PRM XICs on the left (see first screenshot). When I switch to the show ion rank and type view, I am only getting a few matches for the MS/MS (see second screenshot). My library ion match tolerance is 0,5 m/z and what I have here are high resolution MS/MS orbitrap data. I wonder, why is my ion matching so poor when matching ion rank and type?

Thank you!
Roman

Hi, Skyline team

Is the value of max height of a peptide coming from the highest point in the borderline I set?
I thought the max height in the picture attached was around 1.2e5, but when I checked in the csv file, it was 4.3e4

-Minyeong

Dear Skyline Support team,

With a larger team of bioinformaticians and developers we are prototyping data processing and analysis workflow based on Skyline. In one of the use cases we performing targeted MS1 feature extraction using from the TimsTOF Pro data. We already successfully tested the workflow on Windows, both via GUI and command line and we are right now at the stage of transferring it to containerized environment on our Linux cluster.

As a first stage we are just using official docker image (https://hub.docker.com/r/chambm/pwiz-skyline-i-agree-to-the-vendor-licenses), but we experiencing a high number of import failures with the following exception:

Unhandled Exception: System.AccessViolationException: Attempted to read or write protected memory. This is often an indication that other memory is corrupt.
   at System.Collections.Generic.GenericArraySortHelper`2.Swap(TKey[] keys, TValue[] values, Int32 i, Int32 j)
   at System.Collections.Generic.GenericArraySortHelper`2.PickPivotAndPartition(TKey[] keys, TValue[] values, Int32 lo, Int32 hi)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.IntroSort(TKey[] keys, TValue[] values, Int32 lo, Int32 hi, Int32 depthLimit)
   at System.Collections.Generic.GenericArraySortHelper`2.Sort(TKey[] keys, TValue[] values, Int32 index, Int32 length, IComparer`1 comparer)
   at System.Array.Sort[TKey,TValue](TKey[] keys, TValue[] items, Int32 index, Int32 length, IComparer`1 comparer)
   at System.Array.Sort[TKey,TValue](TKey[] keys, TValue[] items)
   at pwiz.Skyline.Util.ArrayUtil.Sort[TItem](TItem[] array, TItem[][] secondaryArrays) in Z:\pwiz\pwiz_tools\Skyline\Util\Util.cs:line 945
   at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.SortSpectrum(SpectrumInfo spectrumInfo, Int32 i) in Z:\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 997
   at pwiz.Common.SystemUtil.ProducerConsumerWorker`2.Consume(Object threadIndex) in Z:\pwiz\pwiz_tools\Shared\Common\SystemUtil\ProducerConsumerWorker.cs:line 185
   at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
   at System.Threading.ThreadHelper.ThreadStart(Object obj) 

When attempting to import a single file multiple times it fails 30-40% of the cases, at random stage during import (see example import log attached).
We invoke Skyline by:

docker run -it --rm -v /Users/m/surfdrive2/Glycopeptides/data:/data \
proteowizard/pwiz-skyline-i-agree-to-the-vendor-licenses wine SkylineCmd --dir=/data \
--in=output/clean.sky --out=output/with_data.sky --timestamp --memstamp \
--import-file=Controle_16_Slot1-36_1_3174.d --full-scan-precursor-res=40 \
--log-file=output/import_log.txt > data/output/import_console_log.txt 

Setting up options like '--import-process-count' or '--import-threads' doesn't seem to have any influence.

We already tested this using docker on Linux and OS X hosts with similar error rates.

We do not see this issue when running the same workflow on Agilent Q-ToF data, which may point out this is somehow linked to reading Bruker data.

Despite high failure rates we are able to import our 40-sample test dataset in the end (with up to 9 re-tries) and the final results are identical as on Windows.

I am wondering if you could help us with fixing this problem. Is this a Proteowizard issue or Wine? Should we contact proteowizard support directly? Our software engineer is eager to get involved, but would need some support and feedback to get started.

We will appreciate any help you might provide.

Marek

Hi,
I regularly perform screening of peptides where I look for all potential +1 and +2 product ions from the different precursors I have selected. However, I often end up with cases where there are multiple identical precursor ion-product ion pairs, e.g. a b3 ion with the same m/z as a y3 ion and so forth.
This is unfortunately not limited to identical m/z, but also includes product ions with small differences in m/z that after rounding by the MS control software end up with the same m/z.

Would it be possible to implement something similar to the "Remove Repeated Peptides", but for transitions in Skyline?

Dear Skyline Team:

Recently I'm going to build a glycoproteome library from Byonic's DDA searching result. I have put files of .mzid and .MGF in same place. However, when I added the file and started the building, the message of error came out.

Here's the message:
System.IO.IOException: ERROR: No spectra were found for the new library.
Command-line: C:\Users\張崑皓\AppData\Local\Apps\2.0\LK9XBYRP.8XT\EGDNLVVT.38L\skyl..tion_e4141a2a22107248_0015.0001_b2be94ac8cfe2cb4\BlibBuild -s -A -H -o -c 0.95 -i stepHCD_stHp_20210629 -S "C:\Users\張崑皓\AppData\Local\Temp\tmpA3BF.tmp" "C:\Users\張崑皓\Desktop\skyline\Library\stepHCD_stHp_20210629.redundant.blib"
Working directory: C:\Users\張崑皓\Desktop\實驗室\實驗\20210529_stHP_HCD_EThcD_DIA\stepHCD
於 pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) 於 C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs: 行 65
於 pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) 於 C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs: 行 201
於 pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) 於 C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs: 行 157

Please let me know if there's anything should be refined.
Best
Tomson

I'm unable to install Win 10 Enterprise new workstations that are being setup, but it keeps trying to install .net 3.5 which is quite outdated. I can't get further and my IT doesn't want it on the workstation since it's so old. Is there a version of Skyline that can avoid this install?

Thanks, Dorothy

Dear Brendan and Brian,

I have a plea for a feature that would save us a ton of time, and I hope would benefit many and would be pretty simple to implement. I would like to ask for a "Apply Peak Boundaries To All" feature in the right-click menu on the chromatogram window. I don't know what the back-end functionality of the "apply peak to all" or "...to subsequent" is, but it seems to be hopelessly broken and we spend a ton of time manually curating some peaks that have odd shapes or have not great S/N. My vision for this new feature is that if i integrate a peak and click the "Apply Peak Boundary To All" button, Skyline would just blindly apply those peak boundaries to that analyte across the document. No questions asked, no peak detection, just update the start and end time for the integration for every replicate in the document. Currently, in order to accomplish this task we need to integrate one replicate, then copy the table for the given retention time out from the doc grid, paste down for the replicates we need (make sure to not go too many replicates down!), save as a peak boundary file renaming key columns, then import the peak boundaries. The whole process seems such a waste of time for something that would seemingly be very easy to keep within Skyline. As with many things, I probably overestimate the simplicity but I hope it can be done. This would save us potentially hours in curation per plate of multiplexed quantitative metabolomics data.

Your humble beggar of features,

Will

Hi,
I am setting up a DIA analysis workflow using DIA raw to build a library. I met a error message about MSconvert (error 1).
System.IO.IOException: Error occurred running process.
Command-line: H:\20210708-TPP-ZQN\msconvert -v --32 -z --mz5 -o "C:\Users\PC\AppData\Local\Temp" --outfile sz44e0ox.pqm.mz5 --filter "diaUmpire params=C:\Users\PC\AppData\Local\Temp\1afneglc.puq.params" "H:\20210708-TPP-ZQN\TPP-Huh7-1-0.raw"
Working directory:
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 65
at pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:line 139

Then I install Skyline in another PC, I met a new error (error 2) as followed:
System.ComponenModel.win32Exception (Ox80004005): The system cannot find the file specified
at sytem Diagnostics Process.StartWithCreateProcess (ProcessStartInfor startinfo)
at System Diagnostics Process.Start (ProcessStartInfor startinfo)

Please advise ? it is so difficult to solve the errors by myself. thanks

Hello Nick,
I tried search results using FragPipe and raw files. However, when I tried to import the search results in skyline, there is an error report showing that I need to have mzxml files instead for import.
I am not sure how to solve this. I think the reason is because I have run mzxml format files before and it saves the previous cached data. I am not sure if I need to remove previous cached data because they are from a separate skyline file. At the same time, I really need skyline to import raw files instead so that I can start a ion mobility library (if mzxml files are imported, ion mobility library will show no result.)
I have attached the error report below. Thank you so much!

Best,
Linwei

I am using Skyline (64-bit) 20.2.0.343 and want to determine the sequence coverage for all my proteins.
If I hover over the protein name in the Targets pane I can see which peptides were found for each protein but is it possible to get the % coverage of the peptide and export a list of the covered peptides from Skyline?

For this workflow I used IDA from a SCIEX QTOF 5600 system which were converted to mzXML format and as search engine Comet (pep.xml) was used (both out of the Trans-Proteomic pipline software suite).

Best regards,
Simon

Hi,

I am setting up a DIA analysis workflow using DIA raw to build a library. I follow the tutorial using your dataset or my dataset but I obtain the same error message. I tried on 2 different computers. Please advise ?

Skyline

Error

OK More Info

System.IO.FileNotFoundException: Le fichier 'C:\Users\franc\AppData\Local\Temp\gyo1ftie.mkn.mz5' est introuvable.
Nom de fichier : 'C:\Users\franc\AppData\Local\Temp\gyo1ftie.mkn.mz5'
à System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
à System.IO.File.InternalMove(String sourceFileName, String destFileName, Boolean checkHost)
à pwiz.Skyline.Model.DiaUmpireDdaConverter.Run(IProgressMonitor progressMonitor, IProgressStatus status) dans C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\DiaUmpireDdaConverter.cs:ligne 148

Hello,
I use the Prosit prediction, but when I turn on the mirror everything disappears and I see only an empty window saying Spectral infmation not available.
What could be wrong?
Thanks
Gerhard

When importing certain files from our Xevo G2-XS QToF Skyline crashes.
It crashes directly, without displaying an error, just a windows popup that the program has stopped working. The files open in MassLynx without problem.
The samples are system suitability checks for small molecule work. Skyline is updated to the latest version.

What kind of information would you need more to help with this?

Hi, there:

I am new to skyline and have problem opening spectra that were collected elsewhere. Just wondering if you have a step-by-step instruction? Do I need any other file besides the spectrum RAW data file? I watched your educational online videos but still could not get it to work.

Thanks.

QJ

I am confused that how I can export the modified site? like S206

Hi, Skyline team.

I found out that some transitions are excluded in report file although they were shown in profiles.
Also, those excluded transitions don't show green dots in the list.
I attached a figure showing this problem.
How can I export all the transitions?

-Minyeong

Hi team! I've been banging my head against this one for a little while, so I thought I'd reach out to see if you can save me from any more headache!

Context: histone PTM analysis with D3-acetyl chemical derivatization. This means peptides will have biological (structural) modifications (including "normal" acetyl), and then any unmodified lysines and peptide N-termini should be D3-acetylated.

I did a paired DDA+DIA for each sample. Searched the DDA with Byonic (PD), built a spectral library with Bibliospec (Skyline), and added the detected peptides to the Skyline doc for quant by DIA XIC (Library Explorer > Associate proteins > Add).

Okay here's where I'm getting turned around. I am getting the craziest permutations of modification + D3 in the target list, and can't for the life of me interpret them! Some of these peptides have the same residue doubly-modified, e.g. a lysine with both structural "Acetyl" and then also isotope heavy "Acetyl:2H(3)". They all have a library spectra associated with them, so is this perhaps Skyline forcing all lysines to be "Acetyl:2H(3)" in addition to any other structural modification they might have?

I have no idea what I did wrong, but this is admittedly the deepest foray I've ever made into PTM analysis and I'm probably hacking some features together to get this to work. Is there some other way I should set up my Peptide Settings > Modifications? Any ideas or help would be greatly appreciated! Minimal Skyline doc example attached.

Thank you!
Lindsay

Hi all !
I glanced through the MCP guidelines for reporting Targeted proteomics data but could not find any mention of the minimum number of peptide(s) per protein required for reporting PRM data.
Would be great if anyone can throw some light on this or perhaps share a document that explicitly provides information on this !

Hello,

I am investigating Skyline's small molecule interface for our metabolomics data processing workflow. I am new to Skyline, so I apologize if this question has been asked and answered.

I am interested in importing an assay library (or transition list) with retention times and relative fragment intensities. So far, I can successfully import a transition list with these fields: PrecursorName, PrecursorFormula, PrecursorMz, ProductMz, ProductCharge, PrecursorAdduct, PrecursorRT, PrecursorRTWindow, PrecursorCE.

What is the best way for me to import this component of our spectrum data for my metabolites to Skyline? Is there a field name that would allow me to attach relative intensities to these entries as well? I noticed the LibraryIntensity field was present in the "Skyline format.tsv" import file for Webinar 10 (proteomics) but not present in the list of column names for small molecule transition lists in a previous support ticket (2018, see link below). If I'm trying to use the wrong tool, could you suggest an appropriate alternative for me in creating a Skyline-friendly library?

Thank you for your time and your assistance,
~Stefanie Kairs

Reference links:
Webinar 10
2018 Support Ticket "small molecule transition list error"

Hi skyline team,
I ran into an issue that for some reason the software calculate the LOD much higher than the LoQ. I am using labeled standard and choose "normalize to heavy" for the quantification and I have loaded three solvent blanks and denotes those as blank. I am not sure if it is because the labeled standard or some of the setting I have was wrong so that my Lod is higher than loq. Can I get a LoD in this condition?

My quantification setting is below
Regression fit: linear
Normalized method: ratio to heavy
Regression weighting: 1/x2
MS level: all

Max LOQ CV: 30%
Calculated LOD by: blank +- 3 SD
Qualitative ion ratio threshold: 30%

Thank you

Hi,

After upgrading Skyline Daily, I have lost functionality in Lipid Creator for exporting to Skyline. See attached error message. Any help resolving this issue would be appreciated!

Good afternoon.
I have a couple of problems in using Skyline for metabolomics.

1. The software does not allow me to simultaneously monitor all the transitions present in the method (more than 400). I need to create multiple methods to analyze a single sample.
   When I try to upload a sample by inserting all the target molecules in the skyline, I get this error message:
   "Failed importing results file
   An item with the same key has already been added. "
2. Even if I prepare methods containing fewer transitions, for some molecules Skyline does not allow me to evaluate the TICs even if by using the Analyst Software (Sciex) I am able to see and integrate that transitions and TICs.

Can you help me?

Thanks
Anna

Hello Skyline team,

Recently, I have been performing a real whole-proteome DIA analysis of which the raw data was acquired on a QE-HF-X MS instrument both in DDA and DIA mode a week ago. According to your provided DIA tutorial, I started doing this by using Import Peptide Search wizard under "Import Results" "One at a time". At the very beginning, because of the very large dataset (35*60min run, a total of ~25 GB), I think, Skyline v21.1 presents a very low performance in extracting chromatograms for 9795 proteins including 6 transitions per peptide with automatical mProphet model training at the same time.

At present, after nearly 20 hours raw data importing, this step was still not completed yet, which clearly makes me astonished very!

My questions therefore come across in this way, they are:

• How to make Skyline work faster?
• Is Skyline-daily more helpful? In Skyline support forum, I learned that someone got good results after being changed form Skyline 20.2 to Skyline-daily. [https://skyline.ms/announcements/home/support/thread.view?entityId=6d9b9a8a-8e7e-1039-a0a7-e465a393a734&_docid=thread%3A6d9b9a8a-8e7e-1039-a0a7-e465a393a734]
• Is there an optimal workflow available? As an alternative approach, I think, I will try to import the raw data by Import Result one by one using default peak picking algorithm. Subsequently, I will train an mProphet peak picking model using the imported DIA dataset and apply it for the same dataset. Finally, if I am a lucky man, I would get an expected quantitative results which then will be used in R analysis. How about this idea?

Best,
Guihua Jia

Dear support,

I am having some issues with applying the IMS filter windows generated from the in-built predictor for DDA data with IMS separation. The data was generated with a Synapt G2-Si.

The steps I usually follow are:

• Load DDA data
• Filter reliable peptides
• Use IMS Predictor
• Re-import results to apply IMS filter
• (New due to change in settings) Set Window type as Resolving Power and set value of "15"; this was the optimal value found for my data in the past. BTW, why was this setting changed out of the library generation window? Shouldn't the resolving power settings be set prior to generating the IMS library?

This worked fine in version 20.1.0.155. However, I recently updated to the newer version 20.2.0.343 and every time I try to do the re-import I get an error message "An item with the same key has already been added.". Additional text (in German) attached.

I also tried removing the file, save, set IMS library created previously, set IMS filter settings, and then import the file again. This time the file loads, but no IMS filter is applied.

Can you give me some insight for this problem? What else could I share to help?

Sincerely,
JC

Hello Skyline team,

I searched and found that DIA-Umpire tutorial for TTOF is available (Attached figure), but there's no one that specifically for thermo QE instrument.
Where can I found that?

Best,
Guihua Jia

Hi Braden and Nick,

Did your guys have a chance to check the database I uploaded to see why it contains no score info.

https://skyline.ms/announcements/home/support/thread.view?rowId=51455

Thanks,
Heyi

Hello,

I am trying to open a file and keep getting the same error message - 'The file contains an error on line 4299 at column 10'

When I go to the line and column in notepad, I see the following (start at line 4298), line 4299 is the second line from top

   </transition>
    <transition fragment_type="precursor">
      <precursor_mz>1181.209019</precursor_mz>
      <product_mz>970.497127</product_mz>
      <collision_energy>0</collision_energy>
      <losses>
        <neutral_loss modification_name="Tmem5_take2" loss_index="9" />
      </losses>
    </transition>
    <transition fragment_type="custom" measured_ion_name="HexNAc Oxonium" product_charge="1">
      <precursor_mz>1181.209019</precursor_mz>
      <product_mz>204.086649</product_mz>
      <collision_energy>0</collision_energy>
      <transition_results>

Any help recovering this file would be greatly appreciated, thanks in advance!
Lily

I have a set of 16 synthetic peptides (heavy Cterm R/K) which I ran individually and as pool in DDA on a Qexactive and searched using MSfragger. I am now trying to import the results into Skyline to build targeted assays but it seems there are some issues.

I tried the usual import DDA search to build a library but once I load the pepxml files there are not a correspondent MGF file and an error. I cannot generate a library within MSfragger because there are not enough peptides across all samples for RT alignment.

Are there some preferred settings/tricks for import MSfragger output?

Thanks!

Hello Skyline team,

I tried to import MSFragger results into skyline. I followed the procedure given in tutorial on the following website by the develeopers of FragPipe.

https://msfragger.nesvilab.org/tutorial_skyline.html

I used the default method "DIA_Umpire_speclib" and the analysis by Fragpipe was successful. I can see the expected number of mzml, pepxml, calibrated.mgf, and interact....pep.xml files in the MSFragger subdirectory of the directory that also contains the raw files. This subdirectory also contains the protein.fas and tsv files generated by MSFragger.

The import of the interact...pep.xml files however failed with the error message shown in the attached file. For me, all the files described in the nesvilab tutorial are available and assume that here is either some file structure problem or some more syntax problem that I do not understand.

Best Karl-Heinz

Hello ,

We are working on tool which automates the manual process of creating MRM method using skyline and MassLynx.
we are using Water's Xevo TQ S instrument.
test was to find the min dwell supported to create write MRM methods.
Attaching the file where command passed to Skyline is shown and output of command runner also been provided.
Does Command runner splits the method into multiple experiment files?
Kindly suggest if anything is wrong with using command runner.

Thank you
regards
Shridevi

Dear Skyline,
Can we do LFQ analysis in Skyline?

Please suggest workflow.

Hi, I want to know about a few options in Edit Report.

What is 'Peptide Peak Found Ratio' and how 'RatioLightToHeavy' is calculated?

Regards,

Minyeong Ji.

Skyline-daily (64-bit) 19.0.9.190

Hi Skyline Team,

While attempting to install LipidCreator from Skyline Tool Store, we keep receiving this error. Any familiarity with this error? Any help to resolve this error would be greatly appreciated. Thanks!

Hi, I followed the guideline of working on the spectral library. However, it is not showing me isotope labels.
Attached screenshots

Hi,

I was wondering if it is possible to visualize modification specific reporter ions in the library match or if it is a feature that could be implemented.

I have managed to show some modification specific neutral losses (Slide 1; e.g. y7-331), but there are several other ions that I can annotate to derivatization tag specific fragments that I regularly check and would be really nice if this were also highlighted for my validation steps and consideration for fragment transitions to track.

As always, thanks for the awesome work and support.
Sincerely,
JC

My lab is new to Skyline (and peptide MS), which we're using to analyze VERY simple peptide mixtures eluted from engineered MHC molecules as part of our reagent quality control.

I've asked members of my lab to capture chromatograms in a database as in the attached file. For database display, it would be helpful if all of the chromatograms had the exact same height and width (say 4" wide by 0.75" tall). I've tried to find this in Skyline and on the Support pages, but I haven't been able to find how to do it (assuming it's possible).

Help with this would be much appreciated.

John Altman
Emory University

Hi Skyline team,

i currently use skyline as open-source tool to analyse data that has been acquired on a Sciex 5500 qqq instrument. My research focuses on nucleotides and their modifications, that's why i often need to detect the same [M+H]+ mass during different retention times. Currently I only want to get a quick overview of the data, so i need the chromatogram, peak area and retention times. The problem i have is that skyline most of the time only recognizes one of my isobaric sMRM transitions.

I have acquired my data as full Range MRM scan and also as scheduled MRM scan. When creating the transition list for the full range MRM data i did not have so many problems - apart from adjusting the integration manually. But now when i want to use scheduled MRM data most of the time skyline does not find the corresponding chromatogram. Is there a way to assign this manually?

Attached you find a file for demonstration which includes 4 results, two of them are full range and two scheduled - you will see that some transitions are only detected within the full range files even though the peaks are present.

I'd really appreciate if you take a look into it

Kind regards,
Paul

Hello,
This is my first time working with peptides, so please forgive my confusion here!
So, I am trying to get the Precursor Ions for the following 3 peptides listed below. I am unsure if I have my sequences wrong, but the values that Skyline is returning don't seem to be correct. I'm using a Agilent Ultivo QQQ. And when I analyze, I don't see those masses. Also, a quick literature search leads me to believe they are incorrect.

Goserelin: XHWSYXLRP. Precursor Ion: 590.808319

Ipamorelin: XHXFK. Precursor Ion: 327.183703

Desmopressin. CYFQNCPRG. Precursor Ion: 601.2475

Also attached, is the full Transition List that Skyline returned.

Any feedback will be greatly appreciated!

Thanks,
Heather

Hi there,

Curious to the ability of Skyline to perform a peptide search using MSe data generated on a Waters Synapt G2Si. Reading some of the older posts, it sounds like PLGS was the preferred route prior to Skyline, just curious if this was addressed since you have added so many features as of late. Additionally, If this is indeed possible, would skyline be able to read the ion mobility data as well?

Regards,
Christopher

Dear Skyline team,

I have a question about the Linear in Log Space regression fit in Skyline. My initial understanding is that the equation of the calibration curve using this fit is Log(Y) = slope*Log(X) + Y-intercept (Y is RatioLightToHeavy, X is Analyte Concentration). However, when I tried to use this equation to calculate X from Y using this equation, the result does not agree with the Calculated Concentration, which is the quantification value given directly by Skyline.

I tried to do a similar regression fit (Log-transformed both X and Y, then do a linear regression fit) in another software and it gives the same slope but different intercept. When using the slope and intercept given by that software and the Log(Y) = slope*Log(X) + Y-intercept equation, I get the same quant value as the Calculated Concentration given by Skyline. Based on this observation, the problem seems to be related to the Y-intercept value given by Skyline.

I am trying to understand what equation is Skyline using for the Linear in Log Space regression fit. Could you provide some guidance on that?

Thank you,
Shimin

Dear Skyline team

Hi! This is Minyeong Ji from the Center for ProteoGenome Research in Korea University.

I'm using SureQuant Method but I want to customize it.

Let me explain about how I want to customize it first.

First, the SIL peptides are scanned with 7,500 resolution

Once the Quant Mode is triggered, the SIL peptides are scanned one more time but this time with 60,000 resolution.

I already gave it a try this way, and when I opened the raw file on Skyline, the profile looked like the image I attached.

So I want to know if I can give a filter with resolution so only scans with 60,000 resolution can be used for drawing profiles.

I tried to use the MS/MS filtering in Transition Settings but I don't think it filtered scans with 7,500 resolution.

Could you help me please?

Best regards, Minyeong Ji

Hi,

I have been trying to remove peptides containing specific AA, but I am having some issues.
I imported a msms.txt file from MQ then imported all the dda raw files.
I then went to the refine>advance> select all precursors/transition/peptides and I went to the filter tab selected> filter C/M added [Q|B|W] and then autoselect all matching peptides, but there are still peptides with C/M and also containing either Q, B or W.
Am I missing something obvious?

I asked about ms/ms filtering yesterday and I was asked to upload my skyline file here

so I uploaded my skyline document and a raw file.

Can you check on them please

Dear all,

I am exploring the software for small molecule work.

During the CE optmization I noticed the following issues

When I export transition list in csv value the precursor name is changed to something like Oxylipin.15-KETE.[M-]Ion [112_999451_112_999451][M-].CE_-17.0.light.

Another problem is that when I try to export a method the DP values are changing and therefore the data collect cannot be re imported in the software?

Why I am experience these and how can I solve it?

Thank you very much, Carlos.

Dear Skyline team,

I encounter a problem with the in silico digestion of my FASTA file with trypsin (KR/P).
(https://www.uniprot.org/uniprot/P0DTC2)
For unknown reasons, the first generated peptide starts at residue 77 where the first K residue is. But there should be many more peptides with the R cleavage site before this peptide.

Another problem I see is the count of the residue. Indeed, this first peptide K.RFDNPVLPFNDGVYFASTEK. Indeed, according to the fast file, the residue R is located in position 78 but skyline indicated in position 77.

I'm running the last version of skyline-daily.

Please find enclosed the skyline file I'm using.

Many thanks in advance.
Best regards,
Nicolas

Dear Skyline team,

for a MRM-assay I tried to generate a “reduced/tailored” spectral library covering only the peptides of interest using as input multiple, already existing *.blib-files (generated from DDA-data). To do so, I used the option: Filter for document peptides. The merged, resulting spectral library gets generated however it does not only contain the MS2 spectra corresponding to my peptides of interest. I.e. the “merged, reduced” spectral library (which got filtered for my 70 document peptides) comprised as many peptides as the original, full spectral library. No filtering takes place.

According to Kaipo’s post (https://skyline.ms/announcements/home/support/thread.view?rowId=25366) I understand the “filter for document peptides” option, that it will consider only the activated (ticked off peptides per protein) in the target window and it will ignore deactivated peptides, right ? I am using Windows 10, Skyline 21.1.0.146 (alpha), however I had the same problem also with your latest Skyline daily version. Thank you for your help.
Best Peter

Hello,

I'm having an issue where those transitions that are shown to be orange will switch which trace is red and which is green if new peaks are chosen on the red trace. It removes the peaks from the now red trace. This is an issue I've never encountered before but is now happening across all my skyline files. I have included the first file to have this issue.

Hello,

I am currently setting up a PRM method, but I am having some issues with one of my files. I have 42 peptides and set up a PRM run on a QExactive without full-scan or scheduling.
The first time the sample was loaded in to Skyline I was only able to detect peaks for 3 peptides. I was not sure if it is an instrument method setup issue, so I repeated the run this time only including 13 peptides in the inclusion list (with two detected in the previous run included), but this time I have no chromatograms for any of the peptides. Looking at the raw file there is no visible problem and the two runs look very similar. I also did a DDA run to see if I made a mistake with the setup, but it does not seem to be the case.

I must be missing something in the setup but I am not able to find where the problem is.

Please find attached the raw files as well as the Skyline document (Skyline version: 64-bit, 20.1.0.155).

Thank you very much for the help in advance!

Best,
Anna

Hi,

I wonder if there is a way to convert MSfragger generated splib to blib for use in skyline? I tried generating blib in skyline from MSfragger mzml, pepxml and calibrated.mgf output files but only get blibs that have proteins but 0 peptides/ precursors. and transitions - this seems similar to a previous thread report where the upgrade of Skyline 19 to 20 was reported to fail in including precursor spectra in the speclib. ANy suggestins would be appreciated.

Thanks,
Dietmar

Hello,

I'm having an issue where my file does not open once I have saved and closed out of the program. It keeps popping up with a window stating "The file contains an error at line 114 column 9". I have tried remaking the file with the same transition list only to see the problem occur a second time. I will be uploading both the file and the transition list (in excel format) under the file name KBD_RiboB. I believe the issue must be in the transition list but I used the same format for other samples (not the exact same list but the molecule name, adduct names, and adducts are the same as in other lists) and had no issue.

Dear Skyline Team,
I would like to use Skyline to analyze immunopeptidomics DDA, DIA and directDIA (DIA-Umpire) data. I'm using the wizard to do it but I'm stuck at the creation and importation of fastA file. We usually use "unspecific enzyme digestion" for normal Discovery approach and I don't know how to do it especially for directDIA (DIA-Umpire).
Your help to overcome this would be great.

Thanks in advance for your help

Best

Hui Song

Hi,

I was wondering if you could help me. I would like to export out my PRM transition list from Skyline and import directly into the processing method of Chromeleon. I was wondering if this is possible. I have tried all formats and nothing seems to be direct? If it is not possible is this something you are looking into?

Thanks

Andrew

Dear,

I was wondering if the new version of Skyline (v.4.2) allows to export SRM methods to MassLynx version 4.2. Last year I was unable to do so and I received a response that Waters was working on making it possible. As a solution to the problem I have been exporting the method to a computer not connected to the equipment (Xevo TQ-XS) and to an older version of MassLynx (v.4.1). Do you know if it is possible now to export directly to MassLynx 4.2?

Thank you very much. Best, Daniela

Dear Skyline team,

i remembered a newsletter indicating the wiff2 from Sciex can now be read with skyline.
However, I'am encountering difficulties to do so. Only solution i found is to convert the wiff2 in mzml using MS convert, but for some reasons, i have to convert the data one by one for the conversion fails (not all the chromatogram is converted in batch mode :( )
I have proteomic data from a X500r QTOF running SciexOS (version 2.0.1.48692).

Please find enclosed one of my file containing full scan data of a BSA digest.

Many thanks in advance for your help.
Regards,
Nicolas

Dear all,
I have samples of 5 different conditions
I have spiked heavy peptides into different amounts in each group of samples (to have a ratio light/heavy of 1 approx) but not a calibration curve.
What is the best results table to do the quantification and to see the significant differences?
Thank you in advance

Dear all,
I hope you are doing well.
We performed a discovery proteomics experiments comparing a WT and a KO bacterium proteome. We found very interesting differential proteins and we wanted to validate some of them by PRM. We chose some proteotypic peptides using Skyling but when we run targeted experiment we found that for a protein, just one peptide was detected in the run. We wonder if we can validate the difference in abundance of a proteins just the measurement of one peptide. I wanted to mention that the biological sample is very difficutl to obtain and we have not more material.

I would really appreciate your comments
Reagars,
Analia

Skyline team,
I wonder if there is a way to filter out contaminated transitions/fragment ions (on a large scale). I have run into many instances in processing DIA data that out of 6 top fragment ions some are clearly contaminated and often they are one of the higher ranked ones. Looking at mass accuracy they are clearly off (mass error >10ppm) while the rest of the fragment ions are spot on (<5ppm). Ranking clearly does not remove these. Sometimes they are just slightly shifted in retention or show a shoulder on the peak. I attach an example.

Thanks,
Tomas

Hi,
I am unable to build a spectral library or import results during my proteomics analysis. I had followed all the instructions from the tutorials as well. An error message shows up whenever I try to perform either of these tasks. Could you please guide me so that I can resolve these issues? I have attached a screenshot the two error messages that I encountered.

Thank you very much!