Is there a quick way to refine the number of transitions used for PRM quant. I want to only use the Top 4 ranked transitions per precursor? Thanks

Dear Skyline Support Team,
We have just finished a PRM experiment comparing different treatments and time points, in total, 396 samples.
I have already uploaded all the files in Skyline and now, as I usually do, I would like to confirm the peak picking by manually checking all the peaks (correct peaks at expected RT and peak boundaries).
However, as I have 90 target peptides and 396 samples that can be endless. Is there any tip or suggestions from your side to deal with such a huge set of targets and samples?
Many thanks in advance.
Kind regards,
Carmen

Hi there. Hoping this will be a quick issue.

I've encountered this error a couple of times now, where I go to open my most recently saved file and there's some error. In this case...

• Positive mode: "Failure opening ........\pos_07Oct.sky. The file contains an error on line 16933 at column 9."
• Negative mode: "Failure opening ........\neg_07Oct.sky. The file contains an error on line 10862 at column 9."

Now usually I'm pretty good about doing a "Save As'" with the current date so if something gets ganked up I can go back to the prior date. I didn't do that last week, and now the file's broken and I'd like this to not happen again.

I'll upload the files shortly. Thank you so much!

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

Hi Brendan et al,

I am using a uniformly 15N labeled protein internal standard in my DIA experiment. When setting up my Skyline document, I know how to add a 15N heavy label for each residue on a peptide, and this works fine for the analysis.

My question is about the user interface/display. If I navigate to View-->Modifications from the menu bar, I can change the way that the modifications are displayed in the peptide list on the left. When there's just a single modification in a peptide (whether that's a PTM or a heavy residue), it's pretty straightforward, for example: "LVNEVTEFAK[+8]" when displaying just the mass shift of a heavy Lys, or "LVNEVTEFAK[Label: 13C(6)15N(2) (C-term K)]" when showing the full name.

However, when selecting a 15N label for every residue, the peptide list can get pretty hard to read: "LVNEVTEFAK" becomes "L[+1]V[+1]N[+2]E[+1]V[+1]T[+1]E[+1]F[+1]A[+1]K[+2]” or "L[Label: 15N]V[Label: 15N]N[Label: 15N]E[Label: 15N]V[Label: 15N]T[Label: 15N]E[Label: 15N]F[Label: 15N]A[Label: 15N]K[Label: 15N]".

I would like to be able to list a peptide as uniformly 15N-labeled with one label or a shorter labeling scheme - is this option available and I just don't know how to display it? Something like "[U-15N]LVNEVTEFAK" might work and wouldn't make it seem like only the residue to the left is labeled, as it might with putting a label pertaining to the whole peptide after the last residue, like "K[Label: 15N]", for example.

I do get that I can choose View-->Modifications-->Not shown, which omits all modification labels, identifies modified residues with bold+underline, and displays heavy peptides in blue bold. That is useful, but I am actually dealing with a lot of different modifications on the endogenous light peptides, and so without any labels I would have a whole list of light peptides which all look identical. So I would like to be able to display the modifications for identification at a glance, rather than having to hover over each peptide to see what particular modification is present.

Thanks for your help, and thanks for all your work on what has been an indispensable software tool in my research.

Josh

Hi Skyline team,

I am using Skyline to process SWATH data from 150 LC runs. Attached please find the 1 page of report data. My question is why peptide, ELSD.. and ELDTV... have multiple rows results which have same Q and Z scores and different areas. Thanks,

Heyi

Hi,
we have encountered a very strange thing in our quantifications of metabolites. In these cases we used stabile isotope-labelled internal standards for each analyte and exported the "ratio-to-standard". We have opened the same file on two different computers and exported the "ratio to standard" both with the setting "SUM" and "MAX". On one computer the exported values are identical (and plausible looking at the peaks), but on the other computer the "SUM" setting leads to exactly 2-fold increased ratios while the "SUM" setting yields the same results as the other computer.
As the ratio-to-standard is only a single value, I expected identical values for both settings (as they are on my computer), but the other computer consistently yields two-fold higher ratios, also for other analyses files, if we use the "SUM" setting.
I hope that we are just missing something, some setting we may have altered, but two different results depending on the computer from which you export from the same file would be next to the "Xerox bug" for me...
Best
Joerg

Hello Skyline Team,

we encountered a strange issue, and did not find anything similar by using the serach function in the forum.

We are using a Shimadzu GC-MS QP 2010 SE for targeted FAME analysis.
As skyline does not import the GC/MS file format we export them as MZXML files.
We use the newest version of skyline available 21.1.0.278, also treid it with an older version before encountering the same problem.

Various methods of a naming scheme have been tried and can be sen in the Fame1ms1 image.
highlited there is that one of the chromatograms is just a flat line but the all transitions get picked for all molecules.

If Into the same Transition settings the same file but only ms2 levels, the chromatogram looks how it's supposed to, but i loose the chromatograms for most of the molecules, as seen in immage Fame1ms2.

If I only use a single molecule for analysis on ms 2 level everything looks like its working perfekt, as seen in image Fame2ms2, wich is a solution but due to the amount of samples and moluces not a solution we would prefere.

Thank you for all your work on the Software
Greetings from the Proteomics Pirates

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 Skyline team,

I'm trying to extract ms1 scan from my 300+ raw files with explicit retention times of 100+ compounds fro quantification purpose. But skyline would often extract and integrate the wrong peak, especially when the compound in a certain file is very low and Skyline would integrate another peak, which can be 10 min far away from the Explicit retention time. It would be impossible for me to do manual integration for all the peaks...
It seems that Explicit retention time window only works when exporting method for SRM or PRM. I'm wondering if there's a way to set the extraction window?

Yunyun

Thank you!

I input 575 peptides from a DDA into Skyline to build a spectra library and a PRM precursor list. After examining the data, I deleted the other peptides from the Target Window and was down to 199 peptides, 201 precursors. However, when I went to export the PRM precursor list, Skyline still exports all 575 peptides. Is there a way to export only the ones that I have in the target window?
Thanks,
Duc

Dear Skyline team,

I am working with DDA results and have 12 samples that I am trying to compare and see differences between. I have been noticing that my protein list changes depending upon how many samples I choose to search or display in skyline at a time. Is there a reason for this? I find it curious, because if I search my serum sample independently I only observe ~70 proteins while if I search it with all of my other samples I see 200 proteins and I still see strong protein abundances for the other ~130 proteins that I did not originally observe when individually searching these samples. Is there something I am missing here and a correct way I should be grouping these samples if they are not replicates or very similar?

Best Regards,
Karsten Poulsen

Dear Skyline team,

I recently acquired in collaboration with Bruker some lipidomics data on the timsTOF pro, but I cannot read it into Skyline. I'm getting the following error:

At 13:42:
Failed importing results file 'H:\Data_Bruker\21010929_Celegans_ttPro\HESI - RP\neg bbCID\HESI_Celegans_MTBE-2_150ul_bbCID_4ul_neg_19_1_3206.d'.
[Reader_Bruker::createInstrumentConfigurations] no case for InstrumentSource 18
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'H:\Data_Bruker\21010929_Celegans_ttPro\HESI - RP\neg bbCID\HESI_Celegans_MTBE-2_150ul_bbCID_4ul_neg_19_1_3206.d'.
[Reader_Bruker::createInstrumentConfigurations] no case for InstrumentSource 18 ---> System.Exception: [Reader_Bruker::createInstrumentConfigurations] no case for InstrumentSource 18
bei pwiz.CLI.msdata.ReaderList.read(String filename, MSData result, Int32 runIndex, ReaderConfig config)
bei pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\pwiz_x64\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:Zeile 197.
bei pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:Zeile 291.
bei pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:Zeile 188.

Thanky you very much for your help.

Best regards,

Michael

Hi all,

Apologies if this questions has been already answered, but I cannot find an answer.

Some of the analytes will not be displayed in Skyline no matter how hard I try to match with the Precursor Name with the Precursor Name I used when creating it in Masslynx.
I took great care to match capitals, spaces, dots, hyphens and so on, but mysteriously some Precursor Name I can rename to anything and will work, while with other Precursor Name even a space difference in the Precursor Name will make it "vanish". IT doesn't seem to correlate with the "complexity" of the Precursor name as you see on the screenshot, hexoseamine (misspelled originally...) is a simple name while other more elaborated naming works just fine.
I hope this question makes sense, attached a screenshot of the problem.

Any idea?

Thank you!

Best regards,
Virag

**
Skyline: daily
Waters TQ-S with latest Masslynx Software

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!

PC running Windows 10 Pro for Workstation
Version of Skyline: 21.1.0.146 (9b557b3b8)

Hello all!
I am looking for help trying to decipher this error that occured when trying to run the DIA-Umpire process in Skyline

System.IO.IOException: Error occurred running process.
Command-line: C:\Users\Erin Jeffery\AppData\Local\Apps\2.0\339HZP6L.A8J\H0P5GENV.PDC\skyl..tion_e4141a2a22107248_0015.0001_b2be94ac8cfe2cb4\msconvert -v --32 -z --mz5 -o "C:\Users\Erin Jeffery\AppData\Local\Temp" --outfile 3wdl2dwq.iuq.mz5 --filter "diaUmpire params=C:\Users\Erin Jeffery\AppData\Local\Temp\fyciffec.5ha.params" "C:\Users\Erin Jeffery\Downloads\SkylineDIA_210927\210901_Jurkat1_tryp_DIA_4hr_Fr4.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

Is Skyline looking for msconvert that is in a different path? I don't have much experience with troubleshooting error codes. Any help is appreciated!

Hiya,

I wanted to ask why Skyline and Mass Lynx are showing different M/Z values for the same peptide.

It's a peptide with the sequence: ADCLTQCAAR. In skyline, the heavy peptide comes up as 588.2622 but in Mass Lynx, its 588.3.
Is there a way to change the M/Z value in Skyline to match the one in Mass Lynx? As I have the results form Mass Lynx using the M/Z 588.3 and when I bring those results into skyline, the chromatograms are empty.

I have attached some screen shots to explain. The M/Z in question is the one tagged "BCR5 ISltd" in the Mass Lynx screenshot.

Thanks,
Shimon

Dear Skyline team,

I am having difficulty to open results files from a DIA experiment from a Bruker impact II .d files. When I try to import a DDA experiment it works well but once I switch to DIA the .d file is not recognized as mass espectrometry format. I would like to ask your help if possible.

Best regards
Joao Luz

Hi,
I am looking at some peptides over multiple replicates and I find that Skyline is not constant in its assignment of the peak range of each peak from replicate to replicate.

Please have a look at the images attached. For replicate 4 in the image attached, skyline selects from 25.8s to 26.27s but for replicate 5, it only selects from 25.8s to just over 26s. Is there a way to make Skyline more constant in the way it does this?

I could manually go around adjusting each peak but that would introduce a lot of bias in my work. I also plan to use Skyline for a method involving 116 peptides across 85 samples, so manually adjusting each peak would be a lot. Moreover, if you look at the "Peak Area- Replicate Comparison" graph, you could easily make the mistake in thinking that a peptide is not being properly detected in a particular replicate (e.g. replicate 5) as its peak area appears so small in comparisons to the other replicates but that's not actually the case, a smaller peak area is just being selected by Skyline.

Final question, is there a way to change the dwell time in Skyline when I export a method? I am exporting methods for a Xevo TQ_XS.

Thanks,
Shimon

Hello Skyline Team,

Thank you for your continued development and user support for this great tool! We have been using Skyline as our primary tool for processing LC-MS data for several years. Unfortunately, we have recently encountered a problem that is effecting a seemingly random subset of our data. When we import raw data from an Agilent 6545 QTOF where both profile and centroid data were collected into Skyline with the "centroided" option selected as the precursor mass analyzer in the Full-Scan tab of the Transition settings, with some files, the MS1 spectra are lost and flat chromatograms appear for all transitions (see screenshot_centroid_1). When looking at the individual spectra within Skyline, it shows only SIM spectra (see screenshot_centroid_2). With other files acquired around the same time and with the same instrument (I included one example that worked and one that did not), this is not a problem. I have checked the raw data in other software and the centroided spectra are contained in the raw data of these problematic files. In troubleshooting this issue, I tried converting the file to mzML (with MSConvert) with CWT centroiding the data imports correctly into Skyline and the data looks fine. When I used MSConvert with vendor peak picking, the problem is not fixed. I also tried loading this file into Skyline with the "TOF" option selected as the precursor mass analyzer and 10,000 resolving power and the data loads correctly for all files. This issue seems to be isolated to centroid data. I have included two Skyline documents (one centroid, one profile), the raw data, transition list, and screenshots. Any help or advice on next steps on resolving this problem would be greatly appreciated.

Raw data and Skyline documents will be uploaded separately and linked.

Error has been reproduced on Skyline Daily (21.1.1.223) and Skyline (21.1.0.146) running on Windows 10.

Many Thanks!
-Ethan

Hi,

We just started to use Skyline to process DIA data. One questions is how we can export peptides confidently present in the data.

thanks,
HY

Hi Skyline Team,

Please see attached a document which is giving some odd behavior when using iRT in small molecule mode. I built a retention time predictor using 11 analytes (stable isotope labeled spike-ins) in the sample, then used a few runs to build an iRT library for about 50 other compounds. This seems to have gone really well. I then imported additional data and told it to import only 1 minute around the predicted RT, which it did. However, there are still a lot of cases where Skyline integrates ('picks') the incorrect peak, even though there is a very nice peak directly at the 'predicted retention time'. This is easily visualized because you have the nice option to show the Predicted Retention Time in the chromatogram window.

Document attached. Hoping there's just a button i neglected to check somewhere that tells Skyline to actually use the predicted retention time that it has access to, to help pick the right peak. Otherwise, i just can't understand why Skyline wouldn't be using the information available. Right now it seems that even though the predictor is enabled, it's pretty much doing the same thing it would if you gave it no RT information at all.

Thanks!

Will

Hi,

I am having some troubles when importing SWATH data as results. I want to compare histone peptidoform expression across several conditions (cell lines) using peak areas. To do this, I made a QC sample (mixture of all conditions) as a baseline. However, when importing the SWATH raw data of these QC samples into Skyline, it seems like something goes wrong when reading it. The XICs look a bit strange. I tried reimporting results several times and made a new Skyline project, but nothing helps. You can find a screenshot of some XICs in the attachment (the QC samples are in the bottom left). Hope you can help me out :)

Thanks in advance!

Kind regards,

Laura

Hi, I installed skyline on my laptop. but I am not able to import raw files. the error info is pasted below. I tried to copy all the raw files to local drive it also failed. Can you please help with this issue?

At 3:23 pm:
Failed importing results file 'Y:\Collaboration_Services\Raw_files_FPL\Fusion Lumos\2021\Guo Xue\Ub\210923_Whole_titration_A549_Raw\210923_GX_Ub_A549_W_M2_100pg_spike.raw'.

pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'Y:\Collaboration_Services\Raw_files_FPL\Fusion Lumos\2021\Guo Xue\Ub\210923_Whole_titration_A549_Raw\210923_GX_Ub_A549_W_M2_100pg_spike.raw'.
---> pwiz.Skyline.Util.AssumptionException
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.Results.SpectrumFilterPair..ctor(PrecursorTextId precursorTextId, Color peptideColor, Int32 id, Nullable1 minTime, Nullable1 maxTime, Boolean highAccQ1, Boolean highAccQ3) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilterPair.cs:line 57
at pwiz.Skyline.Model.Results.SpectrumFilter..ctor(SrmDocument document, MsDataFileUri msDataFileUri, IFilterInstrumentInfo instrumentInfo, Nullable`1 maxObservedIonMobilityValue, IRet/entionTimePredictor retentionTimePredictor, Boolean firstPass, GlobalChromatogramExtractor gce) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilter.cs:line 267
at pwiz.Skyline.Model.Results.SpectraChromDataProvider..ctor(MsDataFileImpl dataFile, ChromFileInfo fileInfo, SrmDocument document, IRetentionTimePredictor retentionTimePredictor, String cachePath, IProgressStatus status, Int32 startPercent, Int32 endPercent, IProgressMonitor loader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 121
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1264
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 233
--- End of inner exception stack trace ---

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,

When I save a file, skyline usually creates and saves a MIDAS format as well. For some reason the files are no longer saving this data format. Is there something Ive accidentally clicked off? Im running Skyline version 21.1.0.146

Thanks!
Tatiana

Dear,
We have been starting to optimize DIA method in Exploris 240, and we are evaluating the gain or loss in points per peak based on different isolation windows.

We would like to know:
1-) how does Skyline calculate points per peak of a peptide. Does it add the points per peak given by the MS/MS spectra and the MS1 full scan acquisition? Or does it account only for the MS/MS spectra?
2-) In addition, can you share with us the best parameters to extract the chromatograms from DIA acquired data?
Thank you very much in advance. Best, Daniela

Morning Skyline team,

I have some questions want to get help.

1. How can I export mProphet p value and q value in report? I tried, but can't find p value. Only find detect Q value, is it the same with q value in mProphet ?
2. what redundant library have? only contain redundant peptide info? could we delete them?

thanks,
Heyi

Dear all,

I follow the tutorial Small Molecule Method Development and CE OptimizationPrint
and it does not work. I cant get the information needed.

I just wanted to report this.

Best regards,

Hi all,

I was wondering if there is a way to import a sample list (e.g. csv file) to Skyline Batch with the exact files I would like to download? Sometimes a general expression won't solve everything (?) or perhaps I just don't know how to use it fully.

Or maybe an interactive box that allows selecting which files are desired after visualizing the contents in a server space (e.g. PXD project)?

Sincerely,
JC

I have a peptide started with K on the n-terminal, when biotinylated the peptide, this n-terminal K will be double labled with biotin, is there a way we can put multiple modification on the same amino acid like this situation? Thanks.

Hi,
I'm new to Skyline. Trying to use quantify a handful of peptides identified in PD but can't seem to figure out how to do that. Also, because it was a targeted analysis, for some peptides I have >1000 PSMs. What do you recommend importing as library? Thanks.

Yan

One of my coworkers is working on SWATH data with skyline and says you're able to process data on skyline without needing to use retention time calibrators. Are retention time calibrators necessary, and if so why?

Thanks,

Tatiana

The error message stated that the mzid file contains an unsupported score type.
Please advise.
Thank you.

Nick,
Attached is the zipped file with Skyline files with two raw files, both of which are DDA LC-MS analyses of the Pierce PRTC mixture (plus two peptides). The first file has peptide RTs ~20-30 min., and the second has RTs ~10 min. longer. I cannot get Skyline to show the full RT range. Thank you.
Mark

Hi Skyline team,

I am trying to study the coverage of a biopharmaceuticals with Skyline. The goal is to digest the recombinant protein present in formulation and see if a full coverage can be obtained. The instrument used is a xevo qtof g2-xs and the data is acquired in MSE mode.

I would like to steer away from Biopharmalynx and use Skyline instead, but I find it hard to set parameters that can give me confidence in peptide identifications since I do not have a spectral library. I thought I could gain additional confidence with a predicted library using Prosit, but since the MSE mode does a ramp up of collision energy, a predicted library with a fixed CE was not helpful.

Do you have any suggestion, tutorial or paper I can refer to?

Thanks,

Ruma

Hi Skyline team

I am new to using Skyline for data analysis and am trying to generate PRM calibration curves, only using heavily-labelled versions of my peptides of interest. I have been following the data processing and analysis outlined in Skyline Webinar 17 in the first instance.

I generated a spectral library of my peptides of interest pooled together, and with data acquired using DDA mode on a QE-HF. I then spiked in mixtures of my 22 peptides of interest into a complex background at different concentrations and acquired PRM data using the appropriate inclusion list with the QE-HF, with the same LC parameters as the DDA run.

I imported the raw data, and filtered for my peptides of interest using Edit>Insert>Peptides. There are transitions showing up for the majority of the peptides I am interested in, apart from two, for which I get a message saying "Chromatogram information unavailable". These two peptides have the same primary sequence aside from the fact that one contains deamidated asparagine and the other contains asparagine linked to N-acetylglucosamine. These two modifications are included as variable modifications in within the appropriate settings.

I can see that both of these peptides are present within the spectral library, and when I make XICs of my RAW files using FreeStyle, they also seem to be present at the correct m/z.

I wondered whether you might be able to help me figure out why I am unable to see the peaks using Skyline, please. I have had a look on your forum, but I can't work it out.

Is there a way I can send screenshots privately to you, if you require them?

Thanks,
DanCam

Hey,
I need to use skyline daily and tried installing the latest version but was not able to install it so downloaded skyline daily unplugged version from the URL: https://skyline.ms/wiki/home/software/Skyline/daily/page.view?name=install-disconnected-64 but, getting an error something like this(mentioned below)
ERROR DETAILS
Following errors were detected during this operation.

• [9/17/2021 3:48:29 PM] System.ArgumentException

• Value does not fall within the expected range.
• Source: System.Deployment
• Stack trace:
  at System.Deployment.Application.NativeMethods.CorLaunchApplication(UInt32 hostType, String applicationFullName, Int32 manifestPathsCount, String[] manifestPaths, Int32 activationDataCount, String[] activationData, PROCESS_INFORMATION processInformation)
  at System.Deployment.Application.ComponentStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
  at System.Deployment.Application.SubscriptionStore.ActivateApplication(DefinitionAppId appId, String activationParameter, Boolean useActivationParameter)
  at System.Deployment.Application.ApplicationActivator.Activate(DefinitionAppId appId, AssemblyManifest appManifest, String activationParameter, Boolean useActivationParameter)
  at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivation(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl, Uri& deploymentUri)
  at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
  --- End of stack trace from previous location where exception was thrown ---
  at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
  at System.Deployment.Application.ApplicationActivator.PerformDeploymentActivationWithRetry(Uri activationUri, Boolean isShortcut, String textualSubId, String deploymentProviderUrlFromExtension, BrowserSettings browserSettings, String& errorPageUrl)
  at System.Deployment.Application.ApplicationActivator.ActivateDeploymentWorker(Object state)

COMPONENT STORE TRANSACTION DETAILS

• Transaction at [9/17/2021 3:48:29 PM]

• System.Deployment.Internal.Isolation.StoreOperationSetDeploymentMetadata

• Status: Set
• HRESULT: 0x0

• System.Deployment.Internal.Isolation.StoreTransactionOperationType (27)

• HRESULT: 0x0

could you please check and tell me what could be the issue?

How can I increase the RT range in the peak profile window to view peptides that have shifted in RT beyond the range that I guess is expected by Skyline. In other words, how I can make the RT range the full time range of the LCMS analysis and then pick the peak?

Dear Skyline team,

I tried to install Skyline21.2 (64 bit) on a computer with win 7 and got an error.
could you please tell me what's wrong?
Thanks,
Niyatti

The following properties have been set:
Property: [AdminUser] = true {boolean}
Property: [InstallMode] = HomeSite {string}
Property: [NTProductType] = 1 {int}
Property: [ProcessorArchitecture] = AMD64 {string}
Property: [VersionNT] = 6.1.1 {version}
Running checks for package '.NET Framework 3.5 SP1', phase BuildList
Reading value 'SP' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v3.5'
Read integer value 1
Setting value '1 {int}' for property 'DotNet35SP'
The following properties have been set for package '.NET Framework 3.5 SP1':
Property: [DotNet35SP] = 1 {int}
Running checks for command 'DotNetFX35SP1\dotNetFx35setup.exe'
Result of running operator 'ValueGreaterThanEqualTo' on property 'DotNet35SP' and value '1': true
Result of checks for command 'DotNetFX35SP1\dotNetFx35setup.exe' is 'Bypass'
'.NET Framework 3.5 SP1' RunCheck result: No Install Needed
Running checks for package 'Microsoft .NET Framework 4.7.2 (x86 and x64)', phase BuildList
Reading value 'Release' of registry key 'HKLM\Software\Microsoft\NET Framework Setup\NDP\v4\Full'
Read integer value 528049
Setting value '528049 {int}' for property 'DotNetFull_Release'
Reading value 'v4' of registry key 'HKLM\SOFTWARE\Microsoft\NET Framework Setup\OS Integration'
Unable to read registry value
Not setting value for property 'DotNetFull_OSIntegrated'
The following properties have been set for package 'Microsoft .NET Framework 4.7.2 (x86 and x64)':
Property: [DotNetFull_Release] = 528049 {int}
Running checks for command 'DotNetFX472\NDP472-KB4054530-x86-x64-AllOS-ENU.exe'
Result of running operator 'ValueEqualTo' on property 'InstallMode' and value 'HomeSite': true
Result of checks for command 'DotNetFX472\NDP472-KB4054530-x86-x64-AllOS-ENU.exe' is 'Bypass'
Running checks for command 'DotNetFX472\NDP472-KB4054531-Web.exe'
Result of running operator 'ValueNotEqualTo' on property 'InstallMode' and value 'HomeSite': false
Result of running operator 'ValueGreaterThanEqualTo' on property 'DotNetFull_Release' and value '461808': true
Result of checks for command 'DotNetFX472\NDP472-KB4054531-Web.exe' is 'Bypass'
'Microsoft .NET Framework 4.7.2 (x86 and x64)' RunCheck result: No Install Needed
Launching Application.
URLDownloadToCacheFile failed with HRESULT '-2146697208'
Error: An error occurred trying to download 'https://skyline.gs.washington.edu/software/Skyline-release-64_21_1/Skyline.application'.

Hi, I am trying to import peptide search data into Skyline for dia analysis. The library was generated from an 'open search' of dda files (timsTOF) that were converted to mzML. When I try to use the individual .pepXML files (15,000 - 20,000 KB) for each sample or the 'interact.pep' file (177,609 KB) Skyline tells me that it cannot find any spectra for the library. I also tried using the library.tsv.speclib file generated but this also caused an error as 'has version 3 bu BiblioSpec only supports up to version 2'. These dda and associated dia files were previously searched in Spectronaut software with no issues. Would you have any ideas as to why Skyline cannot read any spectra from the output files generated from the fragpipe search? The objective of this search is to identify PTMS which is why I wanted to use output from a fragpipe open search

I have been searching thermo .Raw files to see proteins that were observed. I have been successfully doing this on two different computers, but I have failed every time on this computer no matter what. It is my desktop computer at my office and would look to be able to work on Skyline while in the office. It always fails after running the percolator. Any ideas on what is causing the search to fail? I have uninstalled and reinstalled skyline twice. I am using skyline 64 bit version and I am working on windows 10 64 bit version.

Thanks for your time!

Dear Skyline team,
I know how to do normalization just by one compound normalized by one heavy compound by skyline. However, Do skyline have the ability to normalize 17 compounds by 3 heavy compounds? Not every compound has heavy compound as internal standard. If yes, How should we process this in transition list? I attached my transition list in the attachment.
I really appreciate if you could help with this.
Thank you,
Katie

Dear Skyline team,

I am wondering how Skyline divides transitions when exporting into multiple methods? I have 740 transitions which I exported across 3 methods but I can't see any trend in each method to indicate how to decision was made.

Thanks!

I have raised this issue with Fragpipe dev as well https://github.com/Nesvilab/FragPipe/issues/453 but maybe it is Skyline/timTOFpro setup issue? I am trying to follow the tutorial https://fragpipe.nesvilab.org/docs/tutorial_skyline.html where DDA-Search import seems to have worked (attached
image) but it gets stuck at building-molecular-search-library stage?

I have loaded the IO files at https://drive.google.com/drive/folders/1ILiqR2mvWeMWIpeM55qyj9jehPebWBrF which includes the logs, wondering if you can take a look/identify the issue and tell me how to proceed?

Hello Skyline team,

For prm-PASEF
May I ask if the targets per frame graph corresponds to the max, the average or the median number of targets per frame within an MS cycle?

Thanks

Dear MacCoss Lab, I'm working on enriched phosphopeptides and would like to import the identified phosphopeptides (filtered) from MaxQuant to Skyline to visualize the phospho spectrum or phosphosite spectrum. so is there any way I could do that in the skyline? If, yes then could you please help me out with the same.

Hi all,

A colleague has performed a scheduled PRM, including a full ms scan in each cycle. Then he has created a Skyline project to analyze the targets both at the precursor and fragment level. Now we have a question about the y-axis values displayed when the EIC chromatograms of multiple peptides are displayed (when you highight a peptide list or a protein) as in the image attached. Are the values the summed intensity of all transitions? Precursor or fragment transitions? Is it only the intensity of the highest transition?

I hope all is going well and hope to hear back from you.
Sincerely,
JC

Hello,

I've created a transition list in Skyline 64 bit (v 21.1.0.146 (9b577b3b8)) that I would like to export as a method or transition list to use on my Waters Xevo TQD system. Working from the instrument computer, when I choose 'export' and 'method' and select the Waters file provided in the 2021 small molecule online workshop (VerifyETemplate.exp) as the template field I get an error: System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

I tried this with other .exp files and receive the same error, and the full text of the error is attached.

Thanks for your help!
Claudia

Dear Skyline Team,

I was just trying to set up a configuration for Skyline Batch. I added the path to the R script in "R script file path" then clicked "OK". However, I did not see the path appears afterwards. I have attached some screenshots to illustrate the problem.

Am I missing some steps? Is there anything I can do to fix this problem?

Thank you in advance.
Shimin

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

Good morning,

First of all, thank you for your work. I really appreciate the program and its multi-platform versatility.

I would like to suggest if the MRM transitions list that are contained into the RAW files of triple quadrupoles instrument (see attached pictures) can be automatically imported into Skyline. To my knowledge, this function is not present and the user need to manually create a transition list.
I am currently using a Waters instrument (TQ-XS) and exporting the MRM transitions is very laborious and not automatable.

Thank you and best regards.

Gioele Visconti

HI,

I will contact you because I try to find the linearity range of my peptide for quantification assay. So I Injected 10 different quantities of synthetic heavy peptide and then I looked calibration curve (generated by Skyline). I also export total areas (from result grid) and analyse with excel.

I found that calibration curve is different because in case of skyline, it takes peak areas (Max height) and in my case I look total areas and linearity range is not the same between both calibration curve.

Can you help me to understand ?
Thanks in advance

Priscillia

Dear Skyline team,

one major pain point for my workflows manifests as follows:
When one enables further transitions for a precursor that already has a peak picked, those transitions would not have any extracted data, yet.
So I then reimport the MS data (Orbitrap full MS2, centroided extraction) in order to extract the data.
The transition is then displayed in the XIC successfully.

Yet, none of the quantifications or dotp information for the peak will adapt to the new data. In my understanding, the (manually picked) peaks should stay in terms of positioning, but all the extracted area measurements, dotp etc should update according to the new XIC data.
The same (presumably) effect comes into play, when one adjusts the extraction ppm limit, which could have major effects on the XIC (as is visible), but the summary data again is not updated.

This forces me to either run full peak reintegration, losing all manually picked peaks, or delete the .skyd, which causes deselected peaks to be picked automatically again. Both of these options are quite burdensome with fully curated files.

I am using Skyline 21.1.0.146

I just tried to set up a minimal example using a simple small molecule setup.
I am attaching a minimal skyline and rawfile. There is only one transition with a manually picked peak (just some noise for this example).
If you open that file, you can take note of the transition area (17608) extracted at 1 ppm.
You can adjust the extraction ppm to 1000 and attempt reimport (via edit -> manage results).
This to me doesn't update the XIC at all (a separate bug?). But upon saving and reopening, the XIC is updated to reflect the 1000ppm.
The transition area now is still 17608 and only updates when playing around manually with the selected peak limits.

I feel that this is likely very much unintended behavior and hope that this description will be helpful.
Please let me know if any further info is needed.

Best regards,
Jonas

Hi,

I tried to install Skyline Admin 64 bit 21.1.0.146 (current)

The installation happens and is seen in Control panel->Programs and Features.

However the icon for Skyline 64 bit is not seen in Start->Programs
Hence cannot open sky document.

I have tried to uninstall Skyline daily and Skyline Released version which was installed before. And also cleaned up the folders in the path C:\Users\Administrator\AppData\Local\Apps\2.0 before installing admin version of skyline.

Please suggest a solution.

Thanks,
Shobha

Good Morning,

I am using Skyline for several years and mostly for metabolite analyses.
It often happens that peaks are automatically not correctly selected. In most cases this is the case when there are several peaks in the same time window. Even when I use the function 'Apply peak to all', it doesn't help me in most cases. Still the wrong peaks are annotated and often also different ones. This is a little bit frustrating, as I have to spend a lot of time in manual peak annotation.

I was wondering if you can implement the following function into Skyline-daily:
When you right-click into the intensity window, you get the function to manually enter a time frame which is used for peak annotation no matter what/if there is a peak. This function would force to use a selected time frame, e.g. 5.45 - 5.55 min. This would be fantastic and I can imagine that others have similar problems.

I would appreciate an answer. Thank you very much!

Kind regards,

Stephan

Hello Maccoss lab!

My name is Lucas, and I am an RS3 in the hoofnagle lab. I need to integrate all chromatograms over an explicit retention time window for each peptide. Is this possible in Skyline?

Thank you very much!
Lucas

I am doing prm-pasef with heavy peptides but the integration in the IM dimension is off (similarly to https://skyline.ms/announcements/home/support/thread.view?rowId=48885) for method generation from dda-pasef.
Is there any plan to have it adjustable like peak boundaries? How can I manually edit the IM windows?

Thanks!

I'm hoping to see if there is a solution to a formatting issue with importing a CSV transition list for small molecules.

I will attach my working list to this message. The error messages that I've been receiving are:

"Failed reading the file...Line 'Organic Acid and Derivative' has 1 fields when 5 expected."

"Failed reading the file...Data not found in first column"

I am trying to calculate masses of metabolites with corresponding adducts, and have been able to in the past (for isotopically labeled molecules), but I'm not sure what has changed about how I'm formatting the document. Please let me know if there is a way to fix this issue. Thanks!

-Jana

Dear Skyline team,

Thanks a lot for your help. We just started to use skyline for targeted proteomics. Everything is fine till we reach where we have to calculate the linear equation for CE. Although we can see nice peaks with good intensity but the graph is not up to the mark. I have attached the screen shots of our skyline and the graph. Please find it attached. We are using Skyline (64-bit) 20.1.0.155 version on windows 7. Please let me know if you want to have some more informations.

The other thing I would like to ask that there is no internet in our computer. Do you have some system to upgrade our skyline version offline?

Thanks in advance,
Looking forward hearing from you
Best regards
AMK

Dear Skyline,
I got some trpysin digested peptides for a protein for which I used PREGO. I got directed to a webpage which has given scores and ranks for each of my peptides. Can you tell me what the scores and the ranks are about? Are they giving any information about the detectability of the peptides in a mass spectrometer? I got the following output from PREGO.

Rank Sequence        Score
1       GGLDLR  0.16867737160352636
2       GDLSLR  -0.06754151848263762
3       GESLFQGR        -0.17631845487837808
4       GEPLQVER        -0.6208215989137332
5       TLIYYLDEIPPK    -0.9140796672874998

I am new to proteomics and it would be really helpful if you guys could help me out :)

Thank you.

Hi There,

I had a difficulty in installing Skyline to my office laptop. It always says that "Application installation did not succeed. Cannot locate application files on the server. Contact the application vendor or your administrator for assistance."

What would be the problem?

P.S I logged in with my personal email account, not my working email in the laptop.

Many thanks & best regards,

Xiaoxiao

Hi Skyline team,

We are using Skyline for DIA. We have a few .sky documents complete with libraries and iRTdb that are used as master templates for any project with the same sample types. Most of these .sky templates were setup on version 19.1.0.193 (administrator installation)

We have a new project with over 200 files and thought to run the import on a new computer that has 21.1.0.146 installed. Unfortunately there is an issue with forward compatibility, our master templates are "broken" when opened on the new version and all imports fail. We have also tried to setup the templates from scratch on version 21 but this did not work either.

Our files were acquired on Thermo HFX. We have eliminated file corruption as the cause, as test imports on a few raw files with the .sky master templates work on other computers with version 19.

Will it be possible to give us access to the 19.1.0.193 installer file as a temporary solution?
Edit: I have found the link to the archive.

This is the error that we're getting on version 21.

At 2:46 pm:
Failed importing results file 'C:\Users\FPL\Documents\Liyan\to_import\20210722_Liyan_SFN_EvoSep_HCS001.raw'.

pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\FPL\Documents\Liyan\to_import\20210722_Liyan_SFN_EvoSep_HCS001.raw'.
---> pwiz.Skyline.Util.AssumptionException
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.Results.SpectrumFilterPair..ctor(PrecursorTextId precursorTextId, Color peptideColor, Int32 id, Nullable1 minTime, Nullable1 maxTime, Boolean highAccQ1, Boolean highAccQ3) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilterPair.cs:line 57
at pwiz.Skyline.Model.Results.SpectrumFilter..ctor(SrmDocument document, MsDataFileUri msDataFileUri, IFilterInstrumentInfo instrumentInfo, Nullable`1 maxObservedIonMobilityValue, IRetentionTimePredictor retentionTimePredictor, Boolean firstPass, GlobalChromatogramExtractor gce) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectrumFilter.cs:line 267
at pwiz.Skyline.Model.Results.SpectraChromDataProvider..ctor(MsDataFileImpl dataFile, ChromFileInfo fileInfo, SrmDocument document, IRetentionTimePredictor retentionTimePredictor, String cachePath, IProgressStatus status, Int32 startPercent, Int32 endPercent, IProgressMonitor loader) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 121
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CreateSpectraChromProvider(MsDataFileImpl dataFile, ChromFileInfo fileInfo) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 1264
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 233
--- End of inner exception stack trace ---

I was playing around with some small molecules, and a few are analyzed in negative polarity. They were imported to Skyline with -1 for the precursor and product charge states. When exporting the Isolation lists for Thermo Fusion, this polarity is not handled correctly. The charge state z field is set to -1 for these compounds, which the Thermo method editor highlights as an error. Instead, the z field should be written as a 1, and there should be a column with the Polarity header, with the string Positive for the positive compounds, and Negative for the ones with negative z. This is handled correctly for exporting transitions lists to the Thermo Altis, however.

I was trying to optimize some collision energies by hand, and running into the fact that one cannot optimize parameters for Isolation lists. This ability would be very useful for small molecule work.

Thanks
Philip

Dear Skyline team,

thank you for providing us with the possibility to get numbers representing protein abundance per replicate directly from Skyline, which is also supposed to be part of the next main Skyline release. I intend to use this option rather frequently in my research and would like to understand it a bit better. I am mainly interested in the normalization case against heavy (each light signal must have a heavy counterpart to be considered).

In the release notes, you state that this protein abundance value is the same that the Group Comparison (GC) framework uses. I thought that, as default, on protein level GC sums up all transition areas that belong to the same protein (also across different peptides) that are quantitative and have a heavy counterpart and then normalizes this sum to the sum of heavy signals. This is slightly different compared with calculating normalized ratios transition by transition and then averaging those ratios, since strong signals dominate the sums of values, as previously described on several occasions. However, the explanation in the reporting window for the "Protein Abundance" feature now states that "The Protein Abundance is calculated by taking the average of the normalized areas of all of the Transitions under the Protein." This now sounds to me exactly as the latter option to do it and the question is, whether protein abundance numbers really are exactly what the GC framework uses?

Thanks a lot,

Roman

Dear Skyline team,

One of my calibration curves won't show up (error is "The selected replicate has missing or truncated transitions") but all other calibration curves for all other analytes look fine. Any idea what is going on? Can I send you a shared file privately to see if you can pinpoint the problem file?

Thank you!
Laura Dubois

Dear Support Team,
I was using Skyline for analyzing an MRM based data and also using a spectral library. During analysis, for some of the peptides, I noticed that the dotp value shown in 'peak area view' and that in 'target window' are different. However, for most of the other peptides, it is same at both the places.

Could you please help me understand the problem. Looking forward to hearing from you.
Attached here is the screenshot for the same.

Regards
Mehar

Hello,
I am analyzing some FAIMS data to quantify heavy/light ratios but uncertain of the order of operations to obtain mProphet q-values.
When I apply ion-mobility settings to select the best CVs prior to adding decoy peptides, the mProphet model training does not fail and the decoy vs target distributions are clearly separated, but if I apply IM setting after adding decoys (followed by re-importing) it seems CVs are also picked for decoy peptides and the training model fails.

Any suggestions on what to do?
Thank you!

Using DDA peptide search with the latest Skyline release

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

Hi,
I did a targeted analysis in Skyline using a library build using Prosit as a data source (Library --> Build --> Prosit). All worked out great, but as a final quality control and as a figure in a presentation, I want to compare MS/MS spectra from my runs against the library spectra, following the old View --> MS/MS spectra option. However, when I use View --> Library Match I can only generate plots between Prosit and my library (conveniently, I get a perfect dotp score as my Prosit-build library spectrum is compared with, well, Prosit).
Nevertheless, such a mirror plot with a measured MS/MS spectrum against the library would be great, although I would also settle for the old style without the mirror option, but it seems to no longer exist or I am just unable to find it. Search function sadly does not help as the old View --> MS/MS spectrum document is still online under "Tips", so maybe someone could point me in the right direction...?

Thank you in advance,
Jan

Is there a plugin (similar to MSstats for proteomics) to help with downstream statistical comparisons for small molecules/lipids data analyzed within Skyline?

Thanks!

Dear Skyline team,

I hope you can help me with two short questions:

1. I am double-checking if it is somehow possible to plot the "rdotp" value for a precursor above the bar showing its peak area in the "Peak Areas - Replicate comparison" graph? I can plot "dotp" in my Skyline version (4.1.0.11796), but not "rdotp".

2. Could I simultaneously have two "Peak Areas - Replicate comparison" graphs opened within one Skyline document? For example, one grouped by "Replicate" and showing non-normalised peak areas while the other grouped by "Condition" and showing CVs of pear areas normalised to heavy?

Thanks in advance!

Hi all,
I am running MS Stats in a SRM experiment with heavy peptides but I think something is wrong because in the Comparison plot I see the log2 Fold Change instead of Log 2 Ratio (L/H). Maybe the label type is not correct (light instead of L, heavy instead of H) but I do not know how to change it.
I share the skyline doc
Thank you in advance
Irene

We got a new error when trying to load in raw data:

At 3:10 PM:
Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
System.IO.FileNotFoundException: Could not load file or assembly 'pwiz_data_cli.dll' or one of its dependencies. The specified module could not be found.
File name: 'pwiz_data_cli.dll'
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache()
at pwiz.Skyline.Model.Results.ChromatogramCache.Build(SrmDocument document, String documentFilePath, ChromatogramCache cacheRecalc, String cachePath, MsDataFileUri msDataFileUri, IProgressStatus status, ILoadMonitor loader, Action`2 complete) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromatogramCache.cs:line 618

Any ideas on how to fix this for import?

Hi,
I cannot figure out why my Skyline document is missing results for some fragments, and Skyline's behaviour appears somewhat inconsistent to me.

The setup is a bit unusual, but I hope the following makes sense.

• I am interested in peptides with two K residues and these can be either 13C or 12C. I have therefore applied a variable isotope label and used Refine -> Permute Isotope Modifications -> Complete. This results in 4 precursors per peptide (both lysines labelled, no lysine labelled, the first lysine labelled, the second lysine labelled).
• For other reasons, my cells were grown on 13C glucose, all other amino acids therefore carry fixed structural modifications to make them 13C
• Samples were measured on a Sciex QTOF 6600 with a targeted PRM method generated by Skyline.

The problem is as follows: The two precursors with exactly one labelled and one unlabelled lysine have exactly the same precursor mass, but they do have unique fragments (fragments which contain exactly one lysine). I would therefore like to use these for quantification to distinguish the two label states. When I import my .wiff data, Skyline shows no result for some fragments. And it seems to somehow recognise which fragments are shared and unique between the two precursors. Sometimes it shows no data for fragments which are shared (which is weird but OK for me), but in other cases it excludes all unqie fragments (which is bad).

Do you have any insight why it is not extracting results for some of these fragments? I realise this is a somewhat off-label use of Skyline but hopefully there is a workaround. I have attached a Skyline file with two peptides. The interesting thing is happening with the heavy2 precursor where some fragments are not quantified. For the first peptide the unqiue fragments are not quantified, for the second fragment, the shared fragments are not quantified.

Thanks a lot for your help,
Stephan

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

Hi -

I plan to run a verification study of label free results and will be targeting 50 peptides. I have heavy labelled peptides that I plan to include in each sample. I would like to quantify with Skyline using a signal point curve. Does Syline have this quantification option?

Thanks,

Danie Schlatzer

Hi all,

I am looking to use Skyline to speed up the analysis of a target directed DCC run. For those not familiar with the experiment, you take a library of reagents (in this case aldehydes and hydrazides) which react to form a library of products (here acylhydrazones). The reaction is an equilibrium which can be modified by Le Chatelier's principle, in this case by the addition of an enzyme. The idea is to find the products in the equilibrium which are amplified by adding the enzyme, which indicate potential binders to the target in question. See the following article for an example : https://pubs.rsc.org/en/content/articlelanding/2021/sc/d1sc00330e#!

I am wondering if I can use Skyline to help with the analysis of the data coming from these runs. We run a blank experiment with no enzyme and one with enzyme and compare the formation of the products in both.

I created a transition list (M to M+H (not sure if this is what I should be inputting but it seems to work as I intend)) with all possible products of the equilibrium, and imported the LC-MS data from both the blank run and enzyme run. Using this I can easily figure out the retention time of the product and compare its presence or absence in each run.

However I Have the following issues/questions

• Skyline seems to be only reading the MS channel from the .raw file imported from the HRMS machine we use to perform the runs. Therefore the integration of the peaks is done on the MS channel. In an ideal world, I would be able to use the integration from the UV trace that is measured in parallel during the run to compare amplification of certain compounds by comparing peak areas. I realise this could also be done with the MS channel, but due to greatly varying ionisation potentials within the library of compounds, I have my doubts whether this will compare well with the more well-established. Therefore, is there anyway to find the masses of the products in the MS channel but use the UV trace to do the integration analysis? Or would this have to be done in separate software?
• I don't seem to be able to find a way to export the peak areas to a table to compare the values for the two runs, I can only generate the chart using the "Peak Area - Replicate comparison" feature
• I was wondering if there is a way to subtract two chromatograms generated by Skyline using the transition list to only see the difference between the blank and protein-containing runs.

I attached a sample picture of the extracted chromatograms with all the compounds shown.

Thanks in advance for your help, and I apologise in advance for any incorrect terminology I use, MS is not my strong suit.

Cheers,

Tony

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

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!

Hello,
I am using the Skyline version 64-bit 20.2.0.343. I was wondering if in the future an option could be added to change the background color of the Skyline chromatography profile and peak area/retention time graphs from white to another color, especially a black or gray option. When I use Skyline for hours at a time it would be nice to have a background color that is a little less harsh on the eyes. Not urgent, just something that would be nice to have!

Best,
Julianna

Hi Skyline Support team,

I used a converted library from Spectronaut for the DIA analysis.,by importing the transition list, other steps referring the instruction here https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/DIA-QE-20_1.pdf. The mProphet capture is in the attached. It seems to me a failed search with greyed RT difference and dot product. We got a very poor CV by replicates, low cross-correlations by retention time and quantification comparing with other DIA search results. The results are much better on other datasets.

What do you think could be the problem ? My guess would be 1) the neural loss ions or other ions are setting not properly from the converted Spectronaut library 2) retention time alignment failed (there displayed the RT in the library ) 3) the size of library is too large (300K precursors) 4)Is the Skyline Batch relevant for the DIA analysis?

As the library is very large, any operations took a long time. I would like to know your suggestions in how to quickly probe into the problem in quantification.

Thanks for your support

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, 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

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