Hello,

Just wanted to again request that a future version of Skyline be able to go directly from document to method on an Agilent QTOF. It's very helpful for our Agilent QqQ and we would get a lot of use out of the ability to do it on our QTOF. Thank you.

Hi,
i want to import SIM-data from our GC-MS from Agilent. However, i repeatedly get the following error message upon import of the runs:

Failed importing results file 'C:\Daten\Skyline\GC\Allopregnanolone\10µmolar A -sim.D'.
Invalid chromatogram ID SIM SIC Q1=215.15 start=5.087066667 end=14.497416667 found. Failure parsing m/z values.

Could it be that my transition list is flawed or i this a problem of the import file structure?

Many thanks in advance!

Joerg

I have 224 peptides into which I spiked a pierce iRT standard mix and ran it as 3 methods to cover my retention time calibration. When I go to add the non-iRT peptides to the iRT calculator I get an error saying I have insufficent correlation and no peptides will be added to the iRT database. This is PRM data and I am not using a library at this point, so I do not have any fragment correlation but the dotp for the precursors all looks pretty good. The tutorials are all based on triple quad with transitions calling the peptides. Is this something I have to do? I ran these all back to back on the same column with the iRT peptides spiked in so I don't know why there wouldn't be correlation. Is my data bad or am I doing something wrong??? I am attaching a screen shot...is this a different procedure with PRM than MRM data? I do see my intercept for my peptide mix is lower than the intercept for my iRT standards...could this be my issue?
Thanks,
Bob

Hi Brendon,

I have search engines results as mzid file which I import into Skyline and create a spectral library. I then map the peptides to proteins and export the library as a OpenSWATH report. Is it possible to do all these steps using the command line? I have several of these and interested to automate the process of library generation using Skyline.
Steps:

1. Import mzid and clean (probably bibliospec does this)
2. Calcualte iRTs using standards
3. Map peptides to proteins
4. Import into project and export as a report.

Any help appreciated.

Srikanth

Skyline ( 64-bit) 19.1.0.193
Masslynx V4.2 SCN895/ Waters Xevo TQD
Windows 7 professional 64 bit

Skyline is installed at the same computer as the instrument . I was trying to export CE optimization using the File/ Export /Method/Multiple methods but received an error ( screen shot attached). However, I was able to export into the .csv format using File/Export/Transition List

Waters Tech Support was contacted but they don't have a solution at the time. Masslynx security update isn't installed either.
Wonder if anyone else observed this issue and was able to resolve it.
Thank you,
Tran

Hi Brian, Brendan et al

Please see attached to this message a reasonably detailed account of a pretty bad bug in peak area reporting which seems to occur when non-quantitative transitions are included in Skyline documents. It affects reported total peak areas, such that external measurement of results (such as stats or against calibration curves) will not match up what is returned by Skyline.

As always, let us know if you have any questions.

Cheers

Will

So I am trying to build a scheduled method for 224 peptides which are all +2 charge. When I go into build the multiple methods to get the scheduling I selected 30 precursors maximum per run thinking this should be about 8 injections, but under Thermo Fusion it says I need 172 methods to get there. If I switch it up to QE or Agilent QTOF it says 8. I did a test export and I found 172 .csv files some with only 1 peptide in them. Is this a bug? Should I use the QE settings? What is the most isolation precursors I should use in method building for a Lumos instrument? In any event 172 methods can not be right..do I have some weird setting I am missing? This is with fixed carbamidomethyl and all +2 charge so it should be exactly 224 precursors.
Thanks
Bob

Dear Skyline Team,

in the attached skyline daily I want to highlight some things I would be happy to get your feedback.

When exporting mProphet features unfiltered it seems to list all potential peaks for a single peptide at various retention times and with different z-scores and p-values, however, the q values for all peak candidates are the same despite different p-values, is that a bug?

Also, the checkbox to filter for highest scoring peak can be highly useful but also lead to errors. Problem here is that when correcting peak picking (by hand or with Avant-Garde) this export still reports the highest scoring candidate but not the actually picked peak. Having this filter ( only integrated peak instead of highest scoring or both) would be very nice as it would deliver desired results for both automatic and manual peak picking.

Best,
tobi

Dear Skyline-Team,

I am using a PRM method with multiplexing (MSX) for targeted measurements on a "Q Exactive HF-X" mass spectrometer. Here everything works well and I can observe precursors as well as transitions. After transferring the method onto the "Orbitrap Exploris 480" platform and loading of the raw files into skyline (and Skyline daily) I still see the precursors, but no transitions. When I open the raw files with "SeeMS" I can see both, the MS1 and MS2 scans. Since the skyline method worked for the HF-X runs I am a bit lost why it is not working for the Exploris runs. Do you have any suggestions how to solve the data import problem?

Best,
Fynn

Dear Skyline team,

I find the way of numbering amino acids of a protein in Skyline somewhat confusing: The first amino acid is given as position 0. This behaviour is especially misleading, when trying to locate point mutations or PTMs, where the Skyline position is reported to be one less than the normally reported one.

An example: if, say, a deamidation is detected on the first N of F2QME5 (see attached picture), it would be reported to be at position 3. But the N is actually amino acid #4.

Is this a bug or a feature of Skyline?

Greetings,
Florian

Hi Skyline team,

I am trying to add q value annotation so that I can export the q value in Skyline report. But I do not find the Add a value annotation check box in my Reintegrate form. I am running Skyline(64-bit) 4.1.0.18169 on Windows 7. Do you know how can I fix the reintegrate form issue? I attached the expected reintegrate form and the actual integrate form in case these are useful.

Thanks,
Leah

I try to import GC MS Data (SingleQuad) to skyline, as I really like the software. The approach presented by Pawel Sadowski for Shimadzu and Thermo does not work for Agilent Data.

So far my I was to create different adducts for the basepeak molecule, but this is not possible if you just want to create a mass additon that is not in the adduct list.
I tried [M(-5.0)+H] or likewise, it does not work with the actual version.

If I try to add more than one precurser, skyline starts to label them

I attach a file of the agilent data, maybe that makes it aesier to solve the problem...

Hi Skyline Team,

I have begun using the command line version of Skyline to automate the processing of a large metabolomics dataset. With the user interface, I am able to import the peak boundaries to make sure the correct peaks are being integrated; however, with Skyline runner I am unable to do this. Right now, I am able to import my transition list and all of my datafiles and generate a skyline document with Skyline runner, but then I have to manually go in and open the skyline file and import the peak boundaries and then export the final report of the peak areas. If I was able to import peak boundaries through the command line, I would not need to open the interface at all. I don't know if this is a simple fix on your end, but it would help me out a lot!

Thank you!

-Ethan

Hi,
I am currently working on a project designed to discover peptide bio-markers using SRM. I observe some peptides peptides with five well represented transitions, other peptides with less transitions. Based on the changes observed between samples, a few peptides show one transition better than other transitions selected in the method. I have a few questions related to this:

1. What is the minimum number of transitions recommended to use in the data analysis?
2. For peptides with maximum of three transitions selected, one or two transitions are not located under the main peak area (the area under the transition with the highest intensity), is it recommended to delete those "poor" transitions and use only two or maybe just one transition for some peptides?
3. When transitions in actual samples (please see the figure attached) do not line up with the transitions found in the heavy "spiked" peptide, should I extend the boundaries to cover the area of all transition selected in the endogenous peptide in the sample, or should I place the boundaries based on the transitions observed for the heavy "spiked" transitions?

Thanks

Abdullah

I hope these questions are not redundant. Whenever I add to my transition list, everything I add, is not getting analyzed. I need to re-insert a new list and import the RAW files from scratch in order to quantify a new compound in my small molecules list. Are there any re-analyze option ?
Also, when I export the transition list, it shows that there is no CE chosen (because I'm doing MS1 quant) and it export it as a generic format. When I import the same file again, I get an error. Any idea?
I'm using version 19.10.193, small molecules installation.
Thanks

Hey Everyone,

I have a targeted steroid assay which quantifies roughly 35 compounds. The compounds contained in the calibration curve have different concentrations based around their reference range. Is there a way to individually assign concentration values for each compound in a calibration standard instead of a blanket value for all compounds?

Best,
Gregory
West Coast Metabolomics Center
UC Davis
Oliver Fiehn Laboratory

Hi All,

I more often measure peptides that have a predominant breakage point (mostly proline) and thus the most abundant ions are formed from the same ion but in two different charge-states. Can I store the CE value of multiple product ion charges from the same peptide ion fragment (y6+ and y6++ for instance) in the library?
Similarly, but less relevant can I do the same for a peptide sequence with different precursor charge?

I was not able to find this anywhere in the tutorial or support forum. Hopefully this is possible without changing the software too much. I suggest one or two additional columns with precursor z and product z. Or is it possible to provide the charge in the "product ion" column?

Greetings,
Erik

Hello,

I have installed both MSstats and AvantGardeDIA with all the R versions and libraries successfully. However, I am facing some issues while running both of these external tools right now. Such as;

MSstats - As you can see in the attached file. The MSstats function exports the results to a Temp directory with all the basic column information in it. But, the Fragment Ion column contains both fragment ions and precursors identified in the DIA search with respective "Area". According to MSstats, precursor information is not required for DIA analysis and how to exclude precursors from being exported by the MSstats as MSstats input file?

AvantGardeDIA - Whereas here in AvantGardeDIA, this tool is not able to change the working directory. I do not know what exactly is the reason here. I have also attached the screenshot of the current set working directory for AvantGardeDIA\Run_AvG_Module\GlobalRefinement below. Why this tool is not able to change the working directory and how can we resolve this issue?

Thank you,
Chinmaya

I am analyzing Q-Exactive HF PRM Data with Skyline.
In Skyline after importing of raw data, for compound G, the Skyline XIC is totally different from what I can see in Xcalibur.
For 78.9585, the height in Skyline is 880, while in Xcalibur it is 5.02e4.
For 492.9919, the height in Skyline is 75346, while in Xcalibur it is 1.98e4.
What is the cause of the error and how can I improve it??

I have attached the skyline file, transition setting and the XIC, MSMS snapshot from Xcalibur for your reference.

Hello Skyline Team,

I am trying to perform DIA analysis with 2 DIA raw files against a quite large spectral library having 581197 precursors corresponding to 477997 peptides using Skyline 19.1 in our Windows workstation (Please find the attachment for details).

However, I find Skyline hardly utilizing CPU and RAM for any small changes with occupying all the available disk space (Currently the disk containing this skyline document has 1.8 TB space).

It would we great if there is an option in the skyline to set the number of processors/cores to be used, I have not seen any.

Thank You,
Chinmaya

Hi Brendan,

I am glad that I found the question that I wanted to ask you too. We recently performed an MRM analysis using 6495 LC-MS Triple Quadrupole (Agilent). We optimized the MRM transitions for the quadruply charged precursor ions of the light and the heavy peptides. Here, are our transition settings used in the method: 545.3 (precursor, +4) and the transitions are 509.0 (b, 4+); 640.0 (b, 3+); 678.3 (b, 3+). The Skyline recognized the two of transitions such as 508.5155 and 639.67. The third transition of 678.3 in our method was 677.6849 in Skyline transitions giving 0.6151 Da difference. Unfortunately, this was the most intense peak but wasn't accepted by Skyline because I couldn't extend the method match tolerance above 0.6.
The data look very good and I was wondering if there is a way to extend the method match tolerance, so I can include that transition for accurate quantification?
I hope I was clear and was able to describe the problem.
Thank you in advance,

Karine

Dear Skyline team,

I am trying to extract MS1 quantification from timsDIA PASEF data. I have 4 raw files (.d folders) that were processed using FragPipe/MSFragger/Philosopher. Which, by the way, works great for these data.

I am not able to build a spectral library using the DDA-MS1 quant workflow, with Import - Peptide results etc for building the list form the search results. Neither using MSFragger pepXML files nor using interact-.pep.xml files after PeptideProphet work. When using interact-.pep.xml files, I get an error that it cannot find spectral files. When using pepXML, it finds the files to get the spectra (from file_calibrated.mgf files produced by MSFragger) but gives an error at a later stage.

However, I am able to trick Skyline into extracting MS1 quant from DDA files by going through the DIA workflow. When going that route, I am able to select pepXML files, Skyline finds the corresponding spectral files, and it can build the library. I can also select .d DDA files and perform quantification. However, with this trick, Skyline tries to quantify both precursor and y/b ions (because it is a DIA workflow), which takes a lot of time. I just need precursor quantification from MS1, but removing b,y ions from the list of ions to quantify gives me an error.
Also not that using interact-*.pep.xml files (instead of pepXML from MSFragger) still does not work when using the DIA workflow.

So, ideally, I would like to being able to

1. Load PeptideProphet pep.xml files, and not just the MSFragger pepXML files.
2. Being able to use DDA-MS1 quant route rather than DIA route when working with DDA data.

Admittedly, I am an inexperienced Skyline user. So I may be doing something wrong. However, Brett Phinney seemed to confirm the errors I am getting with DDA files.

I could get someone in my team to work directly with the Skyline team to resolve these issues, or to learn how to do this analysis properly.

Thank you,
Alexey Nesvizhskii
University of Michigan

Hey Skyline Forum,

I have a peptide with two labels. One is a N15 labelled full protein that is added to monitor digestion, the other has a single amino acid label. They share the same light peptide, but I've had to "duplicate" it to have both modifications present in Skyline. Is there a better way to do this? The issue ends up being if I change the integration window for one, it ends up having a different light peak area than the other.

I've attached a screenshot of what this looks like in my work space.

Thanks,

-Zach

Hi, everyone. I met a problem for MSstats analysis with both DIA and PRM data. Here is the error messages:
** Reading the data for MSstats.....
** Peptides, that are used in more than one proteins, are removed.
** Truncated peaks are replaced with NA.
Error in SkylinetoMSstatsFormat(raw, removeProtein_with1Feature = TRUE, :
** Please check precursors information. If your experiment is DIA, please remove the precursors. If your experiments is DDA, please check the precursor information.

Can't finish analysis.

Skyline version is daily(64-bit)19.1.338 and Msstats is install as external tools.

Thanks.

sandre

Hi, everyone
I've got the problem about MSstats download.
Once I go to Tools> Tool store
The error was occurred as "Error connecting to the Tool Store: The remote server returned an error (407) Proxy Aythentication Required.

Is there anyone know the solution, please

Thank you
Supitcha

Dear all,

I wanted to report some strange behavior of Q-values and z-scores as shown in the attached screenshot and the uploaded file ,,mProphet scoring on removed peaks". (I expect it to show up pls let me know if not)

When working with DIA and peptide identification in skyline, when removing peaks manually (right-click, remove peak) and via Avant-Garde DIA, those peptides yield Q-values and Z-scores as if nothing happened. I even checked with a different scoring model and see values changing aka refreshing, but peptides where peaks were removed (see RT as well as dotp = 0) still show values from prev. existing peak boundaries. I would expect something like #N/A.

Could you please take a look at this? I guess a workaround would be to filter peptides where dotp > 0.00 or where RT fails.

Can you please also think of extending the document grid for exporting all intermediate values of the scoring? Or is there already a way to score Skyline curated data with Percolator? (When decoys might not be normally distributed etc.).

Looing forward getting in touch with you.

Best,
tobi

Dear Skyline support,

I've noticed a couple of times that the values of idotp can change drastically if certain precursor transitions are turned off and on again. In the attached Skyline document, the idotp values are all around 0.3-0.45 (pretty low for how they visually look) yet if I disable and then re-enable the monoisotopic precursor transition, my idotp values increase at least two-fold (0.84 - 0.94).

The final idotp values look to be correct based on the workaround, but I could imagine this could cause some trouble for people that don't know about it and automatically filter their transitions based on idotp.

Cheers,
Chris

When it is necessary to change the boundary limits of a selected peak across all replicates, this is very time consuming, and it seems like this could be automated pretty easily. It would be great to have a place where you could type in an upper or lower boundary (or both) and have it apply to all. Or even better, apply to selected replicates. This would cut down on hours of QC work done by hand currently within this software, where the automated peak selection was not working well.

Hi.
When trying to import a FASTA protein database with some sequences in lowercase, Skyline fails to process the database, but gives no error message. It took me some time to find out what the problem was. Converting all sequences to UPPERCASE solved the problem.

It would be great, if you could provide support for lowercase libraries or at least give some meaningful error message.

Greetings from Bavaria,
Florian

Dear Skyline team,
I am a new user and I have a problem with extracted ion chromatograms in Skyline. I run BSA sample on Q-TOF Impact II instrument (Bruker) using MRM method. When I analyzed raw file on DataAnalysis program I see ideal coeluting peaks corresponding to EIC from precursor ions 740 and 820.
Why I don't see the same coeluting peaks in Skyline? I used the tutorial recommended for the Bruker instrument.

Thanks in advance,
Arman

Dear Skyline team,

I am preparing a GUI that helps improve accessibility for users to perform design experiments and analyze data in the small molecule mode of Skyline. I would like to have it as an interactive tool, so I've read the documentation that has been written but noticed that the documentation is quite dated (2013 and 2015 for external tools and interactive tools, respectively). Are there any updated versions of these documents? I would like to know if there are new commands that can be used with the SkylineTool.dll and if these tutorials are relevant for small molecule/mixed modes.

Cheers,
Chris

Dear Skyline,

I am trying to import MS data acquired on Waters Synapt G2 Si and I keep on getting the following error:

"The file *.raw does not contain SRM/MRM chromatograms. To extract chromatograms from its spectra, go to Settings > Transition Settings > Full-Scan and choose options appropriate to the acquisition method used."

I set all settings in Full-Scan to none and I still get the same error.

I also tried to import DDA data, it was also unsuccessful, I did change my Transition Settings to DDA/TOF and got:

"No scans in *.raw match the current filter settings."

Does it mean that Skyline can't match spectra to the predicted transitions? Or am I doing something wrong?

Thank you!

Dear all,

thank you very much for integrating Prosit into Skyline. I noticed that the dotp in the new library match mirror view does not treat y ions from deferentially labeled peptides as equivalent. Is it possible to extract the Comparison dotp via results grid for all peptides at once?

Best,
tobi

I am analysing some small molecules data with skyline. However, they are in .mzdata.xml format which cannot be recognized by skyline. How do I convert these files to a supported format in skyline?

Hi Skyline team,

We are trying to export the peptide optimization methods using Skyline and the methods will be used on a Thermo Altis. For some particular peptides, with repeated amino acids there will always a "TNG" error showing up and the whole exportation procedure will quit. Attached please find a screenshot demonstrating the details of the error. Can you help with some solutions?

Hi,
I'm using Skyline for screening of recombinant proteins for method development on our SRM instrument (TSQ Altis).
In this workflow, the method export has worked really well most of the time. I have a Skyline document with all my targets in the target list and then export my methods using One method per protein, since my proteins are in independent samples. Occasionally, when I have longer peptides, the collision energy which Skyline tries to write to the method files exceeds the collision energy possible on the instrument (<65). This is especially a problem when doing the CE optimization. This causes the entire methods export to abort and I cannot even retrieve files for the proteins which don't cause the error.

Would it be possible to introduce an upper limit in regards to collision energy?

Best,
/Andreas

Hey Skyline team, I’m loading some diaPASEF into skyline. It’s super fast!!!

One question. How should I make the library? What formats/ search engines are you using? Does the library use the ion mobility?

I’ll be happy to share some data with everyone

Cheers

Brett

I am try to load data from Bruker Amazon SL using mzML (raw) and .dat (MASCOT). Having issues. Appreciate someone can help me with this. I am attaching files of interest with this. Thanks !!
Rohana

Dear All,

Is there a way to import denovo peptides with no enzyme or protein ID to skyline? Useful in quantifying small peptides (2-4 AA).

Thank you,

JM

Hi, Skyline team.

I find the mirror plots very helpful when comparing synthetic to endogenous spectra but would love to see the mass error (in PPM relative to theoretical m/z) above the fragment ions. Is this possible? If not, would you please add this feature?

Thanks in advance,
Scott

Hi,
Following up on David's comment below, in the latest Skyline release there seems to be a bug with library building from MaxQuant's msms.txt file as well. It seems like the scan number which is used in the library is actually the one after the identification scan as it appears in the msms.txt file (Skyline library scan=msms.txt scan+1).
Thanks for your help

Hi All,

I hope this message finds you well. I am having a recurring problem building spectral libraries or importing search results from Comet/percolator output.

In peptide settings when I try to create a spectral library or import results, I am given an error informing me that the pep.xml file is not from one of the recognized file format and it references the first line in the search results within the pep.xml file. I am running comet from the crux interface in Cygwin.

crux v 3.1.windows.amd64, percolator v 3.02.0, Skyline v 4.1.0.11796, Comet version "2017.01 rev. 4"

I cannot find another example in the support forums.

Thank you,

Tim

Dear Skyline Team,

I was wondering if there is a way to export the protein abundance in Skyline based on the average of the peptide areas.
Thanks,
Benoit

Hi Skyline team,

we are experimenting with the QuanDirect (aka IS-PRM) method on the Orbitrap Lumos using heavy spike-in peptides and everything seemed to have worked fine so far. When importing the data into Skyline (daily 19.1.1.309) I run into the problem, that Skyline extracts all MS2 scans, i.e. also the ion-trap scans which are only targeted at the trigger y1 ion and which do not contain any other ion information. When combined with the MSMS Orbitrap scans that actually contain the fragment ions of interest this results in sawtooth-like peaks (see picture attached). How do I tell Skyline to only import/extract the hi-res Orbitrap PRM scans?

Many thanks,
Markus

Hi,

I am trying to upload measured results to build a libray. I measured my data with a Q Exactive HF 2. It seems that Skyline cant read my raw files. Am I doing something wrong?

Thank you very much for your support!
Melanie

When I attempted to import "Peptide Search..." from MaxQuant search results, I got the attached error. What should I do? Thank you.

Hi,

I am trying to compare the peak areas of my different proteins among 16 different samples. I can now see the different peptides for each protein as well as the spectra corresponding to every replicate. However, I cannot see the "library" peak area, only the peak area for each replicate. I had a read through the forum and found a suggestion that said to "right click" on the replicate comparison panel and select to see the library peak area, however, this option does not come up when I "right click". Would you be able to help me? The library I am using in peptide settings is a reference pool sample of all the 16 replicates. I attach a screen shot as well in case it is any help.

Thanks in advance,
Monica Espinosa

Hi,

recently I tried the EncyclopeDIA from your group, I want to use the library built by spectronaut.
However, I got nothing after conversion. So could you give me some advice about the format
and details on this ?

Thanks a lot.

Sunberg

Hello Skyline team,

I tried to Upload PRM results to Panorama and failed. Because I had identical analytes in different experiments, I erased the data in one of them and imported the new PRM data. This second file I cannot upload. Obviously, because I uploaded the file with the previous experiment (Although file name and PRM content are different).

The error message is:
Failed attempting to create sharing file C:\Users.......sky.zip.
An item with the same key has already been added.

Can I solve that somehow?

Thanks a lot!

Cheers
Ingo

I have tried to install version 19.1 of Skyline in my Windows 8.1 Pro laptop. I also tried Skyline daily. Both are not compatible and there is an error installing it. I have attached the screen shots of the error for your reference. Please do suggest the best version that is compatible with my OS.

I am working in establishing a scheduled PRM method of the Pierce PRTC standard mixture using a QEHFX.

I am noticing that there is an issue with the precursor for most of the peptides (do have signal for transitions). I’m assuming I have a global setting that is wrong?

Hello Skyline team,

I have spent the past while searching previous posts for my particular error and could not find it. I am trying to upload MSe proteomics data acquired on a Waters G2. When I try to import the .raw files I get the attached error. Unfortunately, these data files cannot be shared. The error occurs for any attempted import of these .raw files.

I'm using Window 10 Pro with 64-bit operating system. I'm using Skyline (64-bit) 19.1.0.193.

I do apologize if this is a trivial fix.

Thank you!

Hi,

Our institution acquired a shining new Orbitrap Eclipse with a FAIMS source module. The FAIMS module need optimization for targeted analysis of our analytes, in order to obtain the best compensation voltage for transmission of the ion of interest.

It would be great if Skyline could facilitate this optimization step, similar to the optimization of collision energies that is already done. In fact, Skyline already seems to include a very similar optimization feature "Compensation Voltage', which seems to be intended for use with the SciEx Differential Mobility Separation. Can this feature be adapted to be used with Thermo FAIMS Compensation Voltages, or is this something that is intended for implementation in a future release of Skyline?

Regards,

Martijn van Duijn

Hi Skyline Team! I wanted to "bump" a thread entitled “Optimizing FAIMS on new Thermo instruments” from Martijn van Duijn back in September. We are now in the same boat (acquired a shiny new FAIMS device for our Lumos!) and at this point we just want to incorporate the data (say, 3 different CVs across 7 different peptides) into Skyline from our normal system suitability PRM acquisitions. I see where compensation voltage is a selectable unit under the ion mobility settings, but it doesn't look like Skyline is able to extract data for a single ("best") CV in the chromatogram view. Is this possible with current Skyline Daily (19.1.1.309) yet?
Thx,
Erik

Hi everybody,

During development of my HL library sometimes software (MQ, PD, etc) does not detect peptides even though they can clearly be found in the spectra. Is it possible to load my PRM runs into Skyline and manually select the spectra that should be used for the library by using the Skyline chromatograms?

Thank you!

Hello,

Do you have tutorials on optimizing MS/MS parameters for small molecules?
Note: I only have the precursor m/z info, fragments ions are unknown. It seems like the small molecule tutorials available in Skyline website are only for for both precursor and fragment ions are pre-determined already.

Thanks!

Richard

I'm assuming that the 'subsequent' in 'apply peak to subsequent' refers the result files lower in the file list. Is that true?

If either case, when I use it I get an error:

Top bit of the error window says "Failed to apply peak. Specific argument was out of the range of valid values. Parameter name: count:". More info gives this info:

System.Reflection.TargetInvocationException: Specified argument was out of the range of valid values.
Parameter name: count ---> System.ArgumentOutOfRangeException: Specified argument was out of the range of valid values.
Parameter name: count
at System.Linq.Enumerable.Range(Int32 start, Int32 count)
at pwiz.Common.PeakFinding.FoundPeak.SetBoundaries(Int32 startIndex, Int32 endIndex) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\PeakFinding\FoundPeak.cs:line 97
at pwiz.Common.PeakFinding.PeakFinder.GetPeak(Int32 startIndex, Int32 endIndex) in C:\proj\pwiz_x64\pwiz_tools\Shared\Common\PeakFinding\PeakFinder.cs:line 52
at pwiz.Skyline.Model.Results.ChromatogramInfo.CalcPeak(Int32 startIndex, Int32 endIndex, FlagValues flags) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\Results\ChromHeaderInfo.cs:line 2579
at pwiz.Skyline.Model.TransitionGroupDocNode.ChangePeak(SrmSettings settings, ChromatogramGroupInfo chromGroupInfo, Double mzMatchTolerance, Int32 indexSet, ChromFileInfoId fileId, OptimizableRegression regression, Transition transition, Nullable1 startTime, Nullable1 endTime, PeakIdentification identified, UserSet userSet, Boolean preserveMissingPeaks) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\TransitionGroupDocNode.cs:line 2710
at pwiz.Skyline.Model.SrmDocument.<>c__DisplayClass162_0.<ChangePeak>b__0(TransitionGroupDocNode node, ChromatogramGroupInfo info, Double tol, Int32 iSet, ChromFileInfoId fileId, OptimizableRegression reg) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1721
at pwiz.Skyline.Model.SrmDocument.ChangePeak(IdentityPath groupPath, String nameSet, MsDataFileUri filePath, Boolean loadPoints, ChangeNodePeak change) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1774
at pwiz.Skyline.Model.SrmDocument.ChangePeak(IdentityPath groupPath, String nameSet, MsDataFileUri filePath, Transition transition, Nullable1 startTime, Nullable1 endTime, UserSet userSet, Nullable1 identified, Boolean preserveMissingPeaks) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1719 at pwiz.Skyline.Model.PeakMatcher.PeakMatch.ChangePeak(SrmDocument doc, SrmTreeNode nodePepTree, TransitionGroupDocNode nodeTranGroup, String nameSet, MsDataFileUri filePath) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\PeakMatcher.cs:line 418 at pwiz.Skyline.Model.PeakMatcher.ApplyPeak(SrmDocument doc, PeptideTreeNode nodePepTree, TransitionGroupDocNode& nodeTranGroup, Int32 resultsIndex, ChromFileInfoId resultsFile, Boolean subsequent, ILongWaitBroker longWaitBroker) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Model\PeakMatcher.cs:line 150 at pwiz.Skyline.SkylineWindow.<>c__DisplayClass1198_2.<ApplyPeak>b__0(ILongWaitBroker monitor) in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineGraphs.cs:line 2111 at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 232
--- End of inner exception stack trace ---
at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Util\Util.cs:line 1909
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\pwiz_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 180
at pwiz.Skyline.SkylineWindow.ApplyPeak(Boolean subsequent) in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineGraphs.cs:line 2111

I have a PRM run with only one precursor (file attached). At the end of run I would ideally like to have the peak areas for the precursors. In the attached images the areas that are plotted are of the product ions (View->Peak Areas->Replicate Comparison). How do I get the area for the precursor in different runs. Also, in the area plot in red colors, what are those numbers (126, 129...) at the baseline of the peaks? And is there any way to print out the numbers or plot the numbers? If someone can point to a webinar that shows this, that will be great. Thank you.

Hi,

I imported some heavy labelled PRM results into skyline and for the light version of a peptide I am interested in, only 2 fragment ions were detected.

In the heavy version of the same peptide, all 3 fragment ions were detected.

Can I assume from this that there is close to 100% labelling, in the sense that the light peptide is no longer present in the sample? And its only the heavy version left in the sample?

Thanks,

Shimon

Hello! I am still a user in my Post Doctoral lab, but now I have my own lab and am an administrator for a new Panorama project that will house my independent projects. When I try to upload to Panorama from Skyline, I only see my old lab's folder and not my new folder recently made of which I am the administrator.

I've tried resetting Panorama log in within the Tools > Properties window, but nothing is working. Can you please advise how I can make my Skyline program talk to my new Panorama folder?

Thanks!

Sarah

Hi there,

Recently installed Skyline 19.1.0.193 on Win10. Trying to create a background proteome with no luck. I can successfully create the .protdb file containing 10,000 proteins from a single FASTA file; however, when I select my enzyme it fails to digest this background.

Any help would be appreciated.

Many thanks,

Gordon

Hi Skyline Support,

We observe that if Skyline detects maxima at different retention times for the different charge states, it appears to use an Average value for the method for all the charge states. This results into incorrect Scheduled retention times in the MRM method generated by Skyline.

Please advise if this is a feature limitation for the current version? ...

Best Regards
Imran

Hi, I am trying to define the analyte concentration for samples defined as QCs in the Document Grid under replicates. I am unable to define separate analyte concentrations for different peptides - it applies the last defined concentration to all peptides. Any advice on how to correctly approach this?

Thanks in advance

Hello,

I am wondering why you cannot generate a scheduled MRM transition list using just a .clib file from a Panorama Chromatography library. I have uploaded multiple proteins from multiple runs/documents into the library and can export one concatenated clib file that I thought would allow me to generate a scheduled method with all of the validated targets. I pull the .clib file into a new document and when I attempt to generate a scheduled method it throws an error that I need scheduling information from a data file to generate a scheduled method/transition list.

The clib file contains the RT, Precursor, and Y ion information for all the peptides in the library. Why can it not be used to generate a scheduled transition list without importing data.

Thanks!

Kyle

I'm working with skyline (v19.1) to do some peptide based absolute quantification. All experiments are from MRM files (Instrument: 6500 QTRAP). As a general example, let's say the skyline file contains 2 proteins that are in different concentrations in my cal curve :

Protein A
-Peptide ABCDE
=Peptide FGHIJ

Protein B
-Peptide KLMN
-Peptide OPQR

I am very likely missing something basic, but don't see how to specify unique concentrations for the calibrators if Proteins A and B are in my cal curve at different concentrations. It seems like I can only specify analyte concentrations for 1 protein only and those values are repeated for all other proteins in the skyline file (i.e. Calibrator concentrations for Protein A will also be the concentrations for B).

How do I specify different concentrations for the proteins in my skyline file so that cal curve data can be evaluated appropriately?

I'm sure I'm overlooking something simple. If this is true, just point me to the right tutorial.

As always, thank you.
Bryan

Hi!
I am analysing DIA data and have a spectral library that I am searching my DIA data against. Some of the transitions are of very poor quality (indicated with a red dot) and I would like to remove these; however, I have a lot of data and it would take a few days to do this manually. I have been looking at some of the filtering options in edit->refine->advanced and in the results tab, I have set a cut-off on the 'Min peak found ratio' to 0.8. Can you describe to me the meaning of this setting as well as the other settings (max transition peak rank etc).
Thanks a lot!

Hi Skyline team,

For importing srm/mrm assays nowadays it becomes more regular that together with Q1/Q3 coordinates also the iRT is provided.
Is it possible to import a list of (oxi/phospho modified) peptides with their iRT?
I know you can import an iRT peptide database to an iRT calculator but this is only possible when you have measured the peptides yourself and export them as an iRT database .
So ideally when you first want to run an SRM assay with >100 peptides you can already let skyline predict the RTs based on iRT and export a method to measure the data with wide RT windows. It would be nice to be able to import an excel list with two columns [modsequence] [iRT] like you can also do with Q1/Q3 coordinates.

Hopefully you have a solution so I do not have to run many unscheduled runs first to let skyline know the iRTs of my peptides of interest!

Kind regards,
Erik

Hi Dear

I'm wondering if it is possible to get rid of peptides that had not been consistently detected in both light and heavy channels (or its noisy in some) by filtering the data using rdotp values. In addition, has the signal to noise/background been implemented in Skyline?

Best regards,
Leandro

Hello,

I have been using skyline smoothly to run a quantitative small molecule assay with a calibration curve and check standards and the whole nine yards for a few months now and it is great!!! Thank you all so much for your hard work in bringing small molecule functionality into this great platform!

Now the issue.
Skyline works fine for 1 analyte with 1 standard curve. I have recently tried to add a second analyte with a separate calibration curve to the same skyline document and I am only able to input one value in the "Analyte Concentration" column of the document grid for each replicate that is set as a "Standard". (I have multiple analytes in my calibration curve and their concentrations are different in the CAL levels). I can only load up one analyte curve worth of values and then they repeat over and over again in the grid even if the "Molecule Name" or "Precursor" are set to different values.

Is it possible for skyline to have multiple calibration curves set in the same document? Can you add a custom sample type like "Standard2", "Standard3", etc. and assign separate calibration values there?

My current workaround is to pull the data files into two separate skyline documents, with different analytes. I then import the annotations separately in each one and combine the calculated concentration data afterward using the replicate name as the index to join them on. Please let me know if there is another way to do this or if there is something in the works!

Again. Thanks so much for your work!

Kyle

Hi All,

Sorry for the bother, but I was wondering if there any way to set a threshold for peak intensity in small molecule mode in Skylines.

My group uses Skyline for a GC-MS data set that uses a surrogate standard for quantification. This method has a pretty noisy baseline, especially in our blanks and double blanks, as the lack of filtering causes a lot of product ions to be present.

The issue we have is with the double blanks. Due to the noisy baseline, Skyline picks out a very small peak for the internal standard and for the other molecules of interest. Due to the calibration curve being normalized to the surrogate standard, this causes incredibly high concentration values to be reported for our double blanks. Currently we deal with this problem by deleting the Surrogate Standard peaks for double blanks, but we wish to automate this process and to make sure that peaks are not picked out when they are not actually present. Is there a way to set an intensity threshold for Skyline to pick peaks?

Allison

Dear Skyline team,
I have two questions:

1. I have a set of data created by skyline that needs to be normalized. In my exported transition list, I have included total area normalized and I use normalized to global standard in my peptide setting. I am wondering how this normalization method was performed. What is the "mathematical formula" used to calculate total area normalized. Please find a screenshot showing the total area normalized for one of the peptides in the attachment.
2. I have also attached another screenshot showing one of the heavy peptides that is peaked twice and are located adjacent to each other, with similar transitions, peak areas but at different retention times. Could this be a dimerization issue?
   Thanks in Advance!
   Abdullah

After importing files, I am not able to see the product ion chromatograms. Also noticed that the file size for the skyline chromatogram file is much larger than the skyline document file, when it should be the other way around.

Hi guys,

I have a very general question regarding error distribtion in quant proteomics experiments. Often the standard deviation and therefore the p-value is calculated using log values of the extracted ion chromatogramm areas. By using the log values instead of the original values you will get a completely different distribution and a much lower relative standard error! Why do people use the log areas instead of the original values? In my very small understanding of statistics, this artifically improves the test statistics and makes the results better than they appear! I would be very happy about an explanation because we are using an in-house developed cross-link quant software which also does it in that way and I would really like to understand the background!

Thanks a lot in advance

Julius

Hi Nick,

I posted a last question in our recent conversation regarding error calculation based on log or non log values? Did you see this? Do you have an answer to this?
Thanks a lot
Kind regards
Julius

Hi Skyline team,

is there a way to specify the extraction of y-ions with a phosphate loss? I checked that the 'Phospho (ST)' modification on the peptide of interest has the 97.9769 neutral loss specified, but neutral loss transitions are not defined automatically.

(The context of this question is that I observed a y-98 ion as a site-determining ion for a phospho-peptide that is in my library. Now I want to see if I can detect site-determining ions for isoforms that were not identified, i.e. are not in the library.)

Thank you,
Michael

Dear Skyline Team;

I have a question regarding the DIA Isolation Window Scheme output when using Skyline Daily 64-Bit 19.1.1.283 the output table contains "Start and End m/z" with the ability to show "Margin" and "CE Range" and has the "optimized window placement" feature but there is no "Centre m/z" similar to the EncyclopeDIA Scheme Wizard? Older versions of Skyline and presentations show "Generate Target " toggle that would show a centered target m/z in the output list that could be exported as a csv into the Thermo Fusion MS2 targeted list. The current output is just start and end m/z. Do you suggest manually calculating the "centre mass" for the DIA acquisition as a work-around.

Also I have noticed the masses generated when using margin widths of 0.2 if using Skyline and EncyclopeDIA window generators give different values. I have seen Searle's bitbucket documents describing Skyline's settings for DIA windowing acquisition settings.

Is there a formal document explaining the windowing scheme settings MSX,Overlap, Overlap MSX, Fast Overlap Experimental and what these settings are? I see many presentations for Q-Exactives but not much for Lumos or Fusion.

Thanks for entertaining my questions

_Darryl

Dear Skyline team,
I am getting an error while adding an “on-column” spectral library (blib generated directly from Protein Prospector) to an iRT calculator (Pierce iRT set supported by Skyline). The error is “1 run was not converted due to insufficient correlation.”

I’ve uploaded a dummy Skyline document to the Upload folder. It contains two libraries, both of which are generated from the same DDA data, just two different searches (one focused on identifying the iRT peptides and the other on other peptides in the samples).

Could you take a look at the error and let us know if it is something that needs to be fixed on the Protein Prospector end?
Thanks!
Anatoly

Hi,

I`m trying to set up a targeted PRM method by applying an RT-predictor on a sample where I only measured the iRT peptides (Biognosys iRT-11) in the calculator.
When I go to Export: Isolation list and try to select 'scheduled', I get:
"Retention time predictor is unable to auto-calculate a regression. Check to make sure the document contains times for all of the required standard peptides."
All 11 iRT peptides are detected with good signal and when I look at only the standards in View> Regression> Score to Run, the regression looks fine.

The complete file is attached.

Thank you,
Michael

Hey Skyline

I'm using skyline to analyze SmallMolecules. Back in the days I had QC data files that only consisted out of two traces (FUNC001 and FUNC002) whereof the first one is the data of the sample and the second one the data of the lockmass. For a while I added a third trace into the data acquisition method (FUNC003) that consisted of the data from a PDA detector. These data traces are huge but didn't botter skyline to function as it did before (it was just very very slow). My workaround was opening that FUNC003 trace in notepad and deleting a bunch of data points in the middle before I processed the data in skyline --> slow in the beginning, fast in the middle and slow in the end).
I've been running without PDA for over a year now, but since short I need it again and my workaround doesn't seem to work at all anymore --> skyline stops working (even when I don't delete a bunch of data points in the middle).

Are there settings that I can change so the FUNC003 trace is ignored like it was only a file with two FUNC files?
In attached I've already shared a .raw file to clarify my problem.

Steven Vds

Dear Skyline Team,

filtering for 1% FDR by mProphet model is easy in Skyline by filtering for Detetction Q value <= 0.01. However, we have some issues with that and would like to export data of target and decoy peptides, filter the data ourselves and then perform the 1 % FDR cutoff.

What would be the most feasable way to perform the 1% FDR filtering (Q value calculation) outside of skyline with only z-scores available? Is it a simple formular or more complex? In case it requires complex code, could you please link it? (Noncoder here and I already checked the tutorials and cited paper but did not find q value mentioned seperately)

A short comment is highly appreciated.

With best regards,
tobi

Dear Skyline Team,

I am trying to import 4 wiff2 files into a Skyline peptide search but one of the files keep giving me the “Failed importing results file” message, picture attached (Pic1). All wiff2 files are fine when I open them in BioPharmaView so I don’t think that the wiff2 file is corrupt. It does not help to just import a FASTA file as my library and then import the wiff2 file directly.

Could you please help me to figure out what the problem is?

I also attached the problematic wiff2 file.

Hi Skyline Team,

Tools used: Skyline 19.1.0.193 and MaxQuant 1.6.5

I realized (after hours of questioning my sanity :-) ) that there is a bug when using this new feature where Skyline takes the MaxQuant result and goes back to the raw file to extract the raw scan (great feature, by the way).

When one uses the MaxQuant msms.txt as is, the scans that are extracted by Skyline look nothing like they should look like, because the wrong scan is extracted. I tested this on multiple instances, however not MaxQuant versions (will follow). What happens is, that Skyline always takes the scan subsequent to the actual scan-number (ScanNr + 1) it should extract. Probably an indexing error or the difference between the scan number and the internal counting when accessing the raw file?

A working quick-fix for this issue is to edit the scan number column of the msms.txt in Excel and subtract 1 (ScanNr - 1) from the respective scan number. Skyline will then extract the correct scan.

Can you reproduce this or is my MQ version bogus? I will try an older one tonight.
Best
Daniel

Hi there,
I am running MS1 filtering for a bunch of peptides, I have a standard with high concentration and every peptide's peak is properly picked. Then I am loading some data with much lower concentration of those peptides and I find that skyline will pick wrong peaks at a very different RT.

How do I tell skyline to just look at 1 min RT window within the peak in file A for all the other files?

Thanks!

Hi Skyline Support,

I was wondering if there is a possible way to perform an automatic transfer of data to Skyline just after the acquisition.
That would be very helpful especially for large number of samples.

Thank you very much for your help,

Best,
Benoit FATOU

Good afternoon,

I have watched Webinars 3 and 4 and found it very helpful but I have a question regarding spectral matching.
I conduct PRM on the Thermo Q Exacitive and this is my current workflow:

• I choose the precursor and product ions of interest and export an isolation list for the Q Exacitive
• I get the Raw Data file from the Thermo and run it through PD (with Percolator) which produces a PD result file
• I then import this PD result file as a PRM search (File -> Import -> Peptide Search) and match it with the same PRM Raw file by following the instructions in the Import Wizard
• This produces what I would call really good spectra. The chosen product ions are clearly detected with Peptide ID annotations.

However, when I import the raw result file ( File -> Import -> Results), I don't get the same level of detection. In most cases, none of the product ions detected when I do the workflow above are seen in this result file approach.

Can you please explain why there is this discrepancy? And if my current workflow is correct?

Thanks,

Shimon

Hi,

I tried to use Skyline(v.19.1) for MS1 filtering + spectra library building, by importing a mzIdentML file from PeptideShaker (v.1.16.42). While the spectral libraries were built, I got stuck in the peptide search import at the Extract Chromatograms part. The MGF files referred to in the mzIdentML file were missing. SEE ATTACHED.

When I tried to help Skyline locate them by clicking on the Find button, and navigating to the MGF file location, nothing was displayed. Apparently, Skyline does not support the MGF spectra files referred to in the mzIdentML file.

Will MGF files be supported for chromatogram extraction in a later release? Then I could do MS1 filtering on the PeptideShaker result, something which I think would be a nice feature.

Best,

Marc

Dear Skyline Team,
I am getting an empty .blib file after exporting spectral lib from skyline. can you please help me?

I have generated the lib from PD2 msf file and everything was good, but when I try to export the lib. I am getting a 20kb empty file. I have tried to generate the lib again but that did not help. I have also tried to open the skyline file using skyline-daily and export the lib. but I am getting the same emtpy folder.

Best regards

Wael

Hello skyline team,
I'm trying to analysis proteome data with Mascot dat file.
Previously I succeeded to analyze other data.
However It doesn't work in this time.
Investigation of the reason of error clarified some files made "dat.cdb" file.
What is dat.cdb file and how do we analyze them?

Thanks for your assistance and effort.

Shin