Hello Skyline team,

I have some .d files containing .tsf that I want to analyze using Skyline. These are MS1 only files. I am using Skyline-daily (64-bit) 23.1.1.335 (6a818db84). And as soon as I import the data, Skyline will close.

I can share a file if that helps.

Thanks
Ho-Tak

Hello! I'm having an issue where I'm trying to copy-paste a list of 30 compounds into skyline and it's breaking apart some of the entries (quant and qual are separate). I don't know how to add them together, or insert one into the other, etc. If anyone knows what I should be looking in depth into in terms of excel syntax that would be super helpful.

Thank you!

Hello,
I use Skyline to process my MS data of 13C-fluxomics approaches. I study the 13C-incorporation inside my metabolites of interest.
In my transition list I associate different m/z to one compound. For example, for glutamate analysis I need to integrate:precursor,[M+1], [M+2], [M+3], [M+4], [M+5]. The number of m/z depends on the total number of carbon inside the molecule.
After loading my transition list, the order in the targets panel is not kept and the [M+1] is always the last m/z of the list for all metabolites.
How can I change that and put the [M+1] juste before the [M+2]?
thanks a lot,
Maud

I want to quantify a compound with the calibration curve of an other (with structural similarities). I didn't find the way to do it even with the tutorials I found.
Is it possible? If yes, could you help me?
thanks a lot,
kind regards,
Maud

Hi Skyline Team,

Can skyline export to Agilent method in a segmented manner? I could only export to Agilent method with a static SRM method but I wish to know if I want to have multiple segments in the method, such as for time 1~10 minute, do MS2 scan, and for time 10~20 minute, do a transition table, and how do do this from skyline and export? I have a lot of transitions and methods to export in this way. Many Thanks!

Hello --

When a new document is started (i.e. - set molecule/trans settings, transition list imported via copy/paste from spreadsheet, raw data imported as Agilent .D files or Thermo .RAW files) the isotope prediction algorithm does not include the isotopologue of highest abundance (what we might think of as the “main” monoisotopic signal for each imported compound). To be clear, these are high-res MS1 data. Therefore the idotp does not get calculated correctly. I can fix this by simply saving, closing, and re-opening the Skyline file, which isn’t such a big deal, but this happens every time I start a new skyline file. Happens to others in my lab also across different computers, and it's been happening across version releases. I think it occurred in version 22, continues in the latest v23 release, and also in the most recent Skyline Daily release (attempted and replicated issue on 7 Dec 2023).

I have attached a PPT file with screenshots and some more description of what we see. As mentioned, if I close the file and re-open it, the issue is gone forever (in that file, but comes back when we make a new one). There is some (rather ugly) data shown in the file, but it illustrates my specific problem.

Has anyone else seen this issue? If it was a one-off bug I would have just thought nothing of it, but it's been happening routinely for at least a few months.

Thanks
-Doug

Hi Skyline Team,

Is current Skyline software compatible with MacOS?

If yes, could you please link or notes, how to install it in MAC, PC.

Regards
Dilip

Is there a feature export for protein sequence coverage? Like percent coverage or coverage map?

Hello I m working with Masslynx sofware but the latest version 23.1 is not working corrctly with MSI 9Masslyns Skyline interface. Where can I get the older skyline 22 version
thanks

Hello,

I am using Skyline-daily (64-bit) 23.1.1.318 (6096a599b) on Windows 10. I've tried to upload a zip (wphipps5_prm.zip with the Skyline documents and raw files) but I can't tell if it went through.

I am getting the following error message trying to import PRM data that previously successfully imported into Skyline:

Failed importing results file 'C:\Users\wphipps5\Desktop\New folder\STMS_20230613_heladigest02_PRM.raw'.

pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\wphipps5\Desktop\New folder\STMS_20230613_heladigest02_PRM.raw'.
---> pwiz.Skyline.Util.AssumptionException
at pwiz.Skyline.Util.Assume.Fail(String error) in C:\proj\pwiz\pwiz_tools\Skyline\Util\Util.cs:line 2041
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.get_ChromIds() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 556
at pwiz.Skyline.Model.Results.ChromCacheBuilder.<GetChromDataSets>d__38.MoveNext() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 614
at System.Linq.Enumerable.<SelectManyIterator>d__172.MoveNext() at pwiz.Skyline.Model.Results.ChromCacheBuilder.CalcPeptideChromDataSets(ChromDataProvider provider, List1 listMzPrecursors, HashSet`1 setInternalStandards) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 525
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 375
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256
--- End of inner exception stack trace ---

The file above in the error message is already in the document because 6 months ago it would successfully import. I am experiencing this error with newer samples and thus was retrying older files. Some older files work, others do not.

Thanks for any help
Bill Phipps, UW

How can I add a modification of amidation for synthetic peptides.

This is a loss of one O and a gain of one NH. Net loss of 0.9839 on the COOH terminus.

I tried making the modifications in peptide settings but it doesn't accept them.

Dear Skyline Team,
I have searched the MS data using Proteome Discoverer 3.0 and I wanted to import the peptide search data to Skyline and analyse the peptide peaks. I need to import the data including low number of synthetic heavy labelled peptides (in some cases the file should include a single peptide).
I found out that it is not possible to use the data when the PSMs are validated using Fixed Value PSM Validator because the q-value is absent. It is needed to use the Percolator. However, using of Percolator is not good for me in case of such a peptide poor data. Is it possible to solve this and use the data validated by Fixed Value PSM Validator somehow? In some cases, the synthetic peptides which I measured and I know that they are present could not be seen in Skyline when I import the data validated with Percolator but they are seen in Proteome Discoverer search validated with Fixed Value PSM Validator. In case of files which include only a single peptide I was not successful with importing the peptide search at all, see the attached printscreen.
Additionally, I have one more question regarding importing the peptide search data. I need to analyse some specific peptides which were previously identified by our shotgun proteomic analysis and should be further studied. These peptides are relatively long (20 - 25 AAs) and contain some miscleavages. In this case the Skyline showed that the shorter version without miscleavages is present and did not show the longer version with miscleavages despite the fact that the longer version with miscleavages is present for sure and the shorter should not be present at all. Is there some solution how to see the correct peptide which was synthetized for this purpose?

Thank you very much for your comments,
Jana Vaclavkova

Hello,

I'm fairly new to Skyline and have a problem concerning the visualization (and maybe also integration?) of MRM data. I'm doing LC-MS/MS and metabolomics.
I attached an image of a compound's XIC peak (precursor; m/z 361.2010) below. As you can see the peak is interrupted. The high points are MS/MS scans of the actual compound. The low points are MS/MS scans of a precursor ion with the mass 363.2166 Da.
Now there is an overlap of monitored transitions at that retention time with the compound eluting directly before that having a mass of 363.2166 Da. I figured that there could be some "interference" on the software level, but that would be weird, because there are two more transitions being monitored at that time (precursors m/z 651.7973, 777.6940) and they aren't seen in the XIC peak of m/z 361.2010.
Furthermore, every other XIC peak looks normal, no matter how many transitions might be overlapped at their respective retention times or the masses of these. Except for a similar case of a precursor ion of m/z 315.2319 showcasing the same interrupted XIC peak pattern and a compound eluting before that with a mass of 317.2475. The XIC peaks of m/z 363.2166 and 317.2475 don't have these interruptions.

The question would be: What is happening? Why is the XIC peak of one compound interrupted by another compound and why so selectively? Is there maybe an issue with some isotope recognition option ...? Is there a way to adjust the XIC's mass width?

Thank you!

Dear Skyline team, when building a skyline compound library, I'd like to use the file/import/document function to combine skyline files. The trick is that when combining the files I want Skyline to merge the compounds that are the same, but not re-integrate all the other runs for the compounds that are different (because, those compounds don't actually exist in many samples). However, using 23.1.1.268 I can't seem to get this to happen; when I use the import/document function it always interrogates all the files for all the compounds, no matter which options I select. Can someone take a look at this using the two files I have attached? To summarize, functionality I want:

Combine Files
Collapse same/shared analytes
Perform no additional re-integration (leave compounds that are unique to each file alone, and don't try to find peaks in the other raw data).

Is this possible?

Cheers

Will

I found the following issue:

• In small molecule mode,
• when extracting fullscan + DDA or fullscan + PRM data,
• where we extract at least the precursor and one fragment,
• where the precursor has the exactly same mass as the fragment

this results in no trace being extracted for the precursor.

For example,

• adding a precursor at 192.0768 and a transition at 192.0768 -> no precursor trace extracted.
  In contrast:
• adding a precursor at 192.0768 and no transition -> precursor trace successfully extracted
• adding a precursor at 192.0768 and a transition at 999 -> precursor trace successfully extracted (even though there is obviously no peak at 999)
• adding a precursor at 192.0768 and a transition at 192.0769 -> precursor trace successfully extracted
• adding the same molecule with formula C9H9N3O2 -> precursor trace successfully extracted, but note that the precursor is maybe not precisely at 192.0768.
  Notably:
• adding a precursor at 192.0768, and two transitions at 192.0768 and 999 -> no precursor trace extracted.

Steps to reproduce:

• Download https://skyline.ms/_webdav/home/support/file sharing/%40files/20231108_Skyline.zip?contentDisposition=attachment
• Extract
• Install fresh Skyline 23.1
• Open fragment_check_empty.sky
• Settings -> Import -> fragment_settings.skys
• File -> Import -> Results; STD12.raw

The precursor is successfully extracted for "Carbendazim", "Carbendazim-wfragment999", "Carbendazim-wsmallshift", "Carbendazim@45" but not successfully extracted for "Carbendazim-wfragment192".

There seems to be some masking going on?
Note: While it is probably usually not so good to use the unfragmented precursor mass in the MS2 for anything serious, it is a convenient workaround when I want the MS2 extracted but don't know any fragments already. I kind of prefer this to using an arbitrary mass.

Hi,

I am sure this is a very basic question, but I have spent half of the day searching and couldn't find the answer, so thank you ahead for being patient with me.

I am given a skyline template for 20 peptides. I found the EIC plot (attached) that was shown when importing results were in progress very useful to have an overall idea of the runs, especially when each peak has a different color.

These are my questions:

1. I assume this is an EIC plot, that extracted the m/z of the 20 targets in the skyline template. Is that correct?
2. Is there a way I can revisit the plot and export the information, such as the area/signal intensity, RT, and correlated m/z or peptides sequence of a peak?

Thank you so much!

How do I revisit the EIC plot?
And is the EIC plot of the targets m/z in the files?

Dear Skyline Team,

I am analyzing lipids using a timsTOF Pro. The Bruker PASEF-timsTOF files were recalibrated using Data Analysis (Bruker) after measurement.
Is there any possibility to retrace whether Skyline is using the recalibration after measurement or the calibration that was done before the measurement both for the m/z values as well as for the ion mobility dimension?
I compared both files (no recalibration called "raw" and the recalibrated one called "recal") and could not observe any differences which led me to the assumption that Skyline is not using the recalibration. However, I would need to work with the recalibrated data.
Could you clarify for me if Skyline is able to access the recalibration or not? And if it is possible, how to ensure that it took the recalibration?

I uploaded a Skyline file containing the raw file and the recalibrated file (raw_vs_recal_C13_50uL_neg.sky.zip) and the data files (data_tims_calibration.zip).

Thanks for your help!

Best,
Felina

Dear,
We are trying to add a Prosit library to the attached Skyline document but we keep getting the error message indicated in the ppt file. Could you please help us troubleshoot this issue? Thank you very much. Best, Daniela

Dear Skyline Team,

I designed the following PRM experiment: 10 antibody-derived peptides (500fmol) were spiked into a human cell line lysate (500ng). The 10 peptides were targeted using a Orbitrap Ascend and an OT-OT (MS1-MS2) PRM method. I ran the RawFile through Maxquant and all 10 peptides were identified. When I import the RawFile into Skyline, I get the following results (see attached File for Setup):
Transition Settings > Full-Scan > MS/MS filtering > Acquisition method: PRM
--> Transitions can only be detected for some precursors, even though they are present in RawFile
Transition Settings > Full-Scan > MS/MS filtering > Acquisition method: DDA
--> Transitions are being found for all precursors

Question: Why don’t I find Transitions for all Peptides when using the Acquisition method: PRM settings?

Please find RawFile and Background Proteome here:
https://uwmadison.box.com/s/4lbnv7hs6yfloh2p6f3zkwihmk613xms

Thank you and have a great day
Marcel

Dear Skyline Team,

I have downloaded the skyline software over our computer. It was installed perfectly. what happened another day, I was trying to open the skyline, instead opening, it asked me to dowload and install again and again.

Please suggest me about the remedy!

Regards,
Jitendra

I am aware that I can plot pressure traces (at least from a IQX/Vanquish setup). For small molecule analysis this can be very useful - mainly for QC-ing runs....and indeed, the feature is listed under QC: Transitions->QC->Pump Pressure.
Is there a way to have, for example minimum and maximum pressures/values written to the (Skyline) Report ? I believe I have tried all options but with no luck.
Example of pressure trace attached (not sure about the shown pressure units ).

cheers, henrik

Hi,

I was wondering if there is a way to sort peptides within a protein in the targets pane? E.g. I want to put all the peptides containing an acetyl together.

Best,

Laura

Hi,
I am trying to build and export a scheduled PRM method (in a form of isolation list) from Skyline. When I'm done with the process I'm unable to export the method since "results for all peptides are not imported" (see "scrn1.jpg"). And no, I do not want to use RT predictors. It seems I am doing something totally wrong - but what?
I of course checked Skyline tutorials, but there is close to no mention of how to achieve my goal. Basicly, I've tried to build a spectral library using DDA data and PD results that I already have. I checked if the spectral library I'm using contains RT data - it does (see "scrn2.jpg"). It seems that Skyline wants results to let me export a method, but as I understand it, results should not be required since I've build a spectral library from DDA data already and that information is used to select candidate peptides for PRM analysis. Importing results onto candidates from this spectral library also results in missing/nonsatisfactory data (yellow and red dots next to peptides/transitions). Anyone has any idea of what to do to get start / stop times into the exported list automaticlly?
In the end I would like to use DDA data from samples to select candidates and build a scheduled PRM method - how can I achieve this?
Thanks!

Dear Skyline team,
I have two requests/questions:
I am working for a pharma company that produces peptide therapeutica. In my job I have to quantify impurities, that come along with the production. So far I can can get very nice results with this taks using the Skyline software.

1. Would it be possible to please add the option "No digestion" in the digestion settings of the peptide settings? Now I am doing a workaround to using a lot of missed cleavages.

2. Also sometimes the synthetic production goes wrong and we get something that we call " abortion sequences". That means on the end of either terminus (but most often on the C-Terminus) a couple of amino acids are missing. Would it be possible to add the option "unspecific cleavage" on the c- or n-terminus? So that the software take into account that every amino acids at the ends of the peptides can be cleaved?

Thanks a lot
Jana

Hi Team,

Can the concentration multiplier field be updated from the command line? Looking to import/update a document (example attached) with Concentration Multiplier values from the Molecule Quantification report/document grid but I'm not sure if this can be done from command line or not. Please advise.

Cheers,

Will

I'm getting this error when I try to import DIA data from a qTOF shimadzu LCMS-9030.

I have tried to directly import the .lcd data and also transform it into mzml data.

At 12:01 PM:
Failed importing results file 'D:\JC\Shimadzudata\20231026\20231026_P450Sec_DIA_001.lcd'.
error reading spectrum scan=1

I don't see this problem with DDA data, have not tried MRM yet.

thanks a lot.
Juan Carlos.

Hi,

we are using Skyline on HRMS / PRM data. We want to quantify based on MS1 area of the precursor. Then, we would like to use the ratio between fragment 1 and fragment 2 as a "Qualitative ion ratio".

Right now, I only see the option to set feature as either "Quantitative" or not "quantitative", i.e. it is either in the numerator or in the denominator of the "Qualitative ion ratio". The "Qualitative ion ratio" is then calculated as the sum of "not quant" divided by sum of "quant". Unfortunately, this includes the precursor ion count. I would like to exclude the precursor completely from the "qualitative ion ratio" calc and get only f1 / f2.

Is there any trick to do this with the current version of Skyline? E.g. exclude MS1 data from "qualitative ion ratio" calculation, so we could set one fragment to quantitative and use the other one as "qualifier"?

Hi Skyline team,

Thanks for building an amazing program. I have previously built a skyline file for low-res small molecule quant. This file was working completely fine, but I have since saved and closed it, and now have an error re-opening the file. I have attached the error message as a .txt file, but it appears as if a declared m/z value does not match a calculated value. Is there a way for me to fix this without opening the file in skyline?

Happy to upload the skyline files if needed.

This data was quite time-intensive to curate, so I'd rather not have to start again.
Thanks in advance.

Hello,

My data was generated from Thermo Quantis Plus and I used Skyline 23.1.

Some transition chromatograms were not visible. One interesting thing to note is that several compounds have the same chemical formula, but while some can be seen in the chromatograms, others do not appear to have the same Q1 and Q3 transitions.

Please see the attached files.

Thanks

Dear Skyliner's,

I'm trying to build a spectral library using PEAKS v.11 outputs (db.pep.xml or db.mzidentml.xml).

The data was acquired using ETHCD (b, y, c, z, z') to map 3 peptides with isotopic label

After importing the pep.xml file in peptide setting > library > build, I receive an error message. Information attached

Coudl you help me, please?

Best
Thalles

Hi everyone,

We are running targeted methods with isotopes in QQQ-LC/MS (Agilent) and we experience missing transitions when importing the results in Skyline.

I am attaching the transition list that we inserted, some raw data, and a screenshot of the Skyline interface.

As it is the first time I am trying to troubleshoot in Skyline, please let me know if you need more details.

Best,
Maria

Hello. Is there an option for 'last used folder' when importing data that I can enable? As my workflow includes comparing across experiments, it isn't convenient to store the Skyline document in the same directory as the newer (or older) data I want to compare. I admit eliminating the extra clicks to navigate are more of a convenience.

I searched the Settings and Tools windows within Skyline and a short search in the help files showed some requests in the late 20teens, but It looks like nothing more recent. If this feature is not available, is this on the roadmap moving forward? I am currently running Skyline-daily 23.1.1.268.

Thank you for your time, and Skyline is a critical part of my data analysis.

Hi,
I took the advanced small molecule course week before last and I've been trying to get a set of standards to calculate in Skyline following the instructions, I did not run them with internal standard, just trying to test a standard curve and get some idea of my calibration curve, however I keep getting 'Error: All of the external standards are missing one or more peaks. The selected replicate has missing or truncated transitions' no matter what settings I try. I've even tried setting everything as global standards, I've tried putting it on the peptide/molecule mixed, I've tried every quantitation setting combination I can think of, have no idea what I'm missing. You can see in the document grid I've got area counts for everything. Any help would be greatly appreciated.

I attended a intro to Skyline today and the instructor recommended I ask in support. I experience a consistent issue when going from using 2 monitors to one where the docking arrows will not reset to a single monitor unless I disconnect the monitor and force it to go onto one. Since my monitors are identical, it shouldn't be a frame rate or zoom issue. Happy to screen share the issue because I'm not exactly sure how to screen shot this problem

Hello to the Skyline team!

I have greatly enjoyed using y'alls software but have been getting hung up on an little issue involving my figures of merit. In some of my quantitative experiments (for small molecules) I have been unable to get consistent LOD values utilizing the 'blank plus 3* SD'. Although I have a handful of blanks within these runs, I believe my issue is due to the fact that there are no peaks within the blanks of my given RT window. I have used the bilinear regression to utilize the turning point as my LOD which I have greatly enjoyed but would like to be able to calculate my LOD with both approaches.

I am looking for some guidance as to how to have some numerical value for my blank signal (peak area?) that would allow me to get usable SD's.

I apologize if this question has been answered already and I greatly appreciate your help.

Thanks:)

Hello,
I would like to use autoQCloader with my TT5600 (on Analyst 1.7).
So I try ot export the isolation list from Skyline Daily. For that, I follow the tutorial "autoQC_PRTC".
It works if I select in Instrument type "Agilent Q tof" but it doesn't work with my instrument "SCIEX QTOF".
In attachment, this is a screenshot of Skyline Daily.
Can you help me?
Thanks
Virginie

Dear skyline

I want to find unique peptide including isoforms, but skyline doesn't support that function.

So I check excluding unique peptides overlapping with other proteins, but include isoforms one by one.

It's a hard, inconvenient work. I want to know if there are plans to add these features.
If not, I'm curious how I can effectively utilize the functionalities I mentioned.

Thank you for reading

Hello,
I have my own transition list that i would add to skyline. My usual process was adding the transition list through the menu but when added it is in gray and it does not allow me to use to report my results? not sure if this is because of a new update and theres a new way of doing it?

Hi,

when searching e.g. for molecule names in the Spectral Library Explorer, the search currently looks for text only at the start of the string. I.e. the filter "benzo" finds "benzophenone" and "benzothiazole" but not "1H-benzotriazole". I tried using * or such, but this doesn't seem to be working. Especially for molecule names with some prefixes (e.g D-Glucose vs Glucose, or prefixes like 1,1',2-alpha....- etc) this makes it complicated to find the molecule if the user doesn't know the precise database name.

1. is there already a way how to use wildcards in the filter string? (So I could type "*benzo" and find "1h-benzotriazole")
2. would you consider adding wildcards and/or always searching in the entire string?

Thanks!

Hi,

I made a spectral library in Skyline after manual curation of DIA data from a standard. I exported this library as a blib file and now I want to use this to facilitate annotating the peptides from cell lysates. I loaded it in a new Skyline document and I can see it also includes the retention times, however, I can't see the blue lines showing the MSMS IDs from the library on the chromatogram. I remember I used to right click the chromatogram and click show MSMS ID but I'm unable to find that function. Is this because the library was built using DIA data (and not search results from DDA data)?

Thanks!

Best,

Laura

Hi,

I recently upgraded to the new version of skyline and I have been trying to load some DDA search results. For some reason, the chromatogram seems not to load when I do this on the new computer. So I decided to use an old skyline document I created with the old version of skyline that had the chromatogram. The issue now is that whenever I try to load the ion mobility library using this document, skyline crashes. My goal is to use the document to create a PRM method and without the ion mobility library, I can't create the method for the Bruker TIMS tof.

I am following the steps on the attached file to create the mobility library

I just updated to the new version of Skyline (23.1) and now whenever I import files, the chromatograms are not showing up. I tried raw files that have previously worked and I tried uploading old files to older Skyline (.sky) analysis files. I then deleted the new version to restore the older version of Skyline and the software still didn't work, so I uninstalled and reinstalled the entire software and it still isn't cooperating. The chromatograms are all blank whenever I try to upload any raw file. Any ideas of how to fix this?

Hi everyone,
I have just started using Skyline after finishing the fantastic online introductory and small molecules course (2 days each).
Working in the forensic toxicology area dealing with small molecule quantification, I gained experience with SCIEX Analyst and Agilent Masshunter.

So here is my question: Is it possible to assign the regression weighting for the calibration curve for each analyte individually (e.g. amphetamine 1/x, methamphetamine 1/(x*x), ketamine none)? This is a common thing in forensic toxicology and would be crucial for a switch e.g. from SCIEX Analyst to your amazing software.

Thank you for your help in advance
Hannes

For batches with a large number of replicates and/or targets, the "peak areas" become less useful, as it becomes hard to visually evaluate the differences and the replicate / target labels are collapsed.

It is easy to zoom in on a subset of samples with ctrl-mouse wheel on the "replicate axis". It is also easy to quickly go through many samples with Ctrl-left and ctrl-right on the keyboard. However, when leaving the set of replicates onto which we are zoomed, the selection simply "leaves the window", i.e. we continue navigating through EICs but we don't see them in "peak areas". Also, selecting a replicate's EIC doesn't bring that replicate into the "zoom range" of the "replicate comparison" window.

It would be great if the range of visible samples would follow the selected replicate in a "moving window" kind of way. Also, if there was a scrollbar to move the window of selected replicates without having to zoom out and back in.

This is very hard to describe. Maybe I can make a screencapture to show what I mean, unless you already understand...

Note: the same applies to all of these replicate/molecule comparison views, like retention time, mass errors...

we are trying to identify a protein in a biomatrix. If the protein is exogenous, I want to find a specific surrogate peptide for this protein without interference from the matrix endogenous protein. Is there any tutorial for this?

For the calibration curves one can check linearity with the R2 value. However, methods like EPA Draft Method 1633 are moving away from this for checking linearity. Instead, they are using the calculation of relative standard deviation or relative standard error. Is there way to have the RSD or RSE for a calibration curve calculated and displayed like the R2 value on the calibration curve panel?

Currently, you can define regression options for the document in "Settings > Molecule Settings > Quantification" that apply for all targets. However, the response of individual targets is not the same, thus you might need for example a linear regression without weighting for molecule 1 and a quadratic regression with a 1/x weighting for molecule 2, and so on.

After the last Skyline MS Online event, I was told that this is currently not possible in Skyline, and encouraged to report a feature request. It was suggested that these settings might be implemented in the document settings as "Explicit" molecule option. Since I do not have permission to open a new issue, I use this forum for this request.

If this request needs any more clarification, please let me know. I would be grateful for the implementation of this feature, since it is the only obstacle to replace commercial quantification software in our lab.

Hi,

currently, when importing spectral libraries as MSP, there is no validation on the InChIKeys for small molecules. I.e. more or less any string passes.

This is fantastic for me because it allows me to bypass the "one spectrum per inchikey and adduct" limitation (by appending an ID to the inchikey). I just hope you never change this; and if you do, make the validation optional?

Thanks
-Michele

Hi Skyline team,

We are currently setting up an Quantitative bottom-up method for a therapeutic protein. We apply different forms of the protein to investigate the parallelism. Because one form has lower molar weight than the other, more surrogate peptide will be formed per µg of protein. We apply a dilution factor to correct for that. Skyline calculates a corrected concentration as required, but the accuracy is not corrected. Could you please advise how to generate accuracy's corrected by the dilution factor?
We apply a calibration curve and a global standard as internal standard.

Many thanks and with kind regards,

Jeroen

We are doing an upgrade from 21.1 to 22.2. There is the following improvement 'Improved default peak scoring model training to allow features that have some unknown score values to be used.' Our primary use of Skyline is targeted PRM work, would this have an impact on this application? Thanks

Hi Skyline Team!

I am analyzing Orbitrap Exploris 480 PRM data with an MS1 scan in each cycle. When I export the TIC from Skyline ("Total Ion Current Area") the numbers do not seem to represent the real TIC. I also tried Export -> Chromatograms but it is the same problem (although the numbers are different).

Here is an example why it does not make sense. I have 4 samples and a blank in between but the blank has the same (or even higher) TIC.

When I check the raw files in Freestyle the blank is a flat line at 2E7 while the samples are 2E10.

What am I doing wrong? I would be happy to send files if needed.

Many thanks,
Markus

OS is Windows 11, running Skyline-daily (64-bit) 23.1.1.268 (c58f1f56e).

Hello,
I am trying to go through the tutorial for targeted method editing and I cannot get past page 7. Every time I try copying and pasting the fasta contents into Skyline I keep getting an error stating, "This operation has added 35 new proteins with no peptides meeting your current filter criteria. Do you want to remove all empty proteins before showing the document?" I say to keep them, but then the protein names are listed but they are empty; no peptides. I have tried manually selecting the peptides for the proteins, but only the ones that I can see through the tutorial since I have no idea what the others are. Then, when I try copying the same material under "Insert > Proteins" or "Insert > FASTA" and paste the material into the table, it looks very chaotic and nothing is where it should be. I have even tried manually inputting a couple of entries and that still did not work for that part. I have watched online videos and have read the tutorial for this, but I have not gotten it to work. Is there someone I can talk to who can walk me through the process, maybe I am missing a step or inputting something incorrectly. Also, I have the most recent version of Skyline.

Hello!

I am trying to import a spectral library generated using Fragpipe v20 to use for the analysis of DIA-PASEF data acquired on a timsTOF HT system but I am getting an error message (below). I saw other requests with a similar issue that were solved by having both the library.tsv and corresponding .speclib file in the same folder but this doesn't seem to help in my case. Any assistance would be greatly appreciated! The spectral library files are >50mb so I've uploaded it to my google drive here: https://drive.google.com/file/d/1sVtwVrs4ksSC63cc36mAUyBkSMNDORlM/view?usp=sharing

Thanks again,
Vincent

Skyline-daily (64-bit) 23.1.1.268 (c58f1f56e)

System.IO.IOException: ERROR: unable to determine DIA-NN report filename for 'Human_VR_SCPC9_MOLT4_MCF7_Plasma_HpH_48Fr.tsv.speclib': the TSV report is required to read speclib files and must be in the same directory as the speclib and share some leading characters (e.g. somedata-tsv.speclib and somedata-report.tsv)

Command-line: C:\Users\vincent.richard\AppData\Local\Apps\2.0\OV8TV4EZ.5Y5\Q4A3KQV0.GGZ\skyl..tion_2e441fc3bf6adc7f_0017.0001_e4c70d6dfe97aa96\BlibBuild -s -A -H -v warn -o -c 0.95 -i DIA_PASEF_test -S "C:\Users\vincent.richard\Downloads\DIA-PASEF_test.redundant202310050527.stdin.txt" "C:\Users\vincent.richard\Downloads\DIA-PASEF_test.redundant.blib"
Working directory: Z:\CBORCHERS_Users\Vincent Richard\DIA_spectrallibrary
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\pwiz\pwiz_tools\Shared\CommonUtil\SystemUtil\ProcessRunner.cs:line 181
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\pwiz\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 433
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161

Hi,

I want to upload the PRM data (manually curated) that I analyzed in Skyline into the MassIVE repository. The MassIVE repository requires the result files, such as mzIdentML and mzTab. How do I export the Skyline results in mzTab format compatible with MassIVE?

Thank you for the help!

Marco Hadisurya

Hi, Skyline Team.

I'm running into issues installing the Avant Garde tool in Skyline. I've tried to install it through both the Tool Store and as an External Tool, but I keep running into issues with specific R packages not installing. The two packages that won't install are stringr and ggplot2. This is happening on several different computers running the most current version of Skyline Daily.

Do you have any suggestions about how to remedy this? I tried installing the specified versions of ggplot2 (3.2.1) and stringr (1.3.0) outside of Skyline through R (v 4.0.3) but there were issues with the tool when I did that. I attached the error message I got related to the tool stoor failed installation of ggplot2 and stringr.

Scott

I'm sorry if this has been addressed elsewhere, but I can't find the answer. When I use Skyline to export an overlapping window DIA isolation list for Thermo Fusion, the output looks like this:

m/z z t start (min) t stop (min) Name Isolation Window (m/z)
404.433719 8.003638
412.437357 8.003638
420.440995 8.003638
etc.

I am using a Fusion Lumos, which wants a single column isolation list that looks like this in the default XCalibur 4.2.47 method:

Calculated m/z Window
400.4319-408.4355
408.4355-416.4392
416.4392-424.4428
etc.

How can I get Skyline to export an isolation list in the format that XCalibur wants?

I'd like to export a report that contains the candidate peaks for EVERY compound. I'd like to do it from the command line. A report exists but (see screenshot) ONLY for one file at a time and with no command line access (unless I misread the docs).

Format would be the same as in the screenshot PLUS extra columns for identifying the compound: "File Name", "Compound Name", "Product Mz", and "Precursor Charge" (see below screenshot). Each row would be a candidate peak.

In the data from a Waters Xevo G2-XS QTof with lockspray applied, the lockspray signal is stored as a separate channel. When the data is imported the data from the lockspray channel are included as datapoints. Since the compound of interested is not present at the point of lockspray, the peak or baseline is shown as zero when the lockspray was applied. This creates holes in the created chromatograms, see attachments. Is there a way to import only one channel and exclude the lockspray channel?

Hi,
I have DIA files from small-amount samples. I did not spike-in iRT standards. Is there a way to get/see chromatograms for few candidate peptides? I am trying to load file in Skyline, however, since there are no iRT peptides, it is failing. I also tried CiRT but that also failed.

Thanks!

At 8:27 PM:
Failed importing results file 'F:\Users\rona\DIA-QE\DIA-QE\DIA\collinsb_X1803_171-A.mzML'.
The calculator test1-assay requires all of its standard peptides in order to determine a regression.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'F:\Users\rona\DIA-QE\DIA-QE\DIA\collinsb_X1803_171-A.mzML'.
The calculator test1-assay requires all of its standard peptides in order to determine a regression. ---> pwiz.Skyline.Model.Irt.IncompleteStandardException: The calculator test1-assay requires all of its standard peptides in order to determine a regression.
at pwiz.Skyline.Model.Irt.RCalcIrt.ChooseRegressionPeptides(IEnumerable`1 peptides, Int32& minCount) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Irt\RCalcIrt.cs:line 152
at pwiz.Skyline.Model.Results.ChromCacheBuilder.RetentionTimePredictor.CreateConversion() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 838
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 404
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

Dear Skyline team,

I encounter the issue described in the title during regular data processing, but have manually reproduced the issue on a small dataset to showcase it. The steps for manual reproduction are (1) deselect an identified transition, (2) reimport the data and (3) observe that the signal is not shown upon reselection until re-important the data.

I attached a power point containing two screenshots. Looking at the peptide for ADIPO you can see that the y9 fragment even if selected manually is not visible. Looking at the full scan spectrum, it is clear that this transition is identified though (as rank 3). Only after having selected the fragment and re-importing the raw file skyline shows this respective transitions is identified. I am happy to share the document and raw file via a link or email, but cannot publish it here.
Basically, when opening the transition selection menu I would like to see all available transitions from my spectrum independent of the preselected transitions as per the document settings.

Cheers,
Nadine

Dear Skyline

We are having problems with exporting methods with the new version.

See the message in the attached .png file.

Hi,

I have been working on a file for a over a few days and today when I opened it and tried to open the pick children window, skyline stalls, uses 20% of my CPU for maybe 5 seconds and then the window opens and instantly closes so that I cannot use it. The file is other wise able to see the data, can access the data to generate the full scan data for example. The Pick children window was working on this file earlier, not sure how it might have become corrupted but closing and reopening both the file and skyline does not help. The modify peptide window is functioning properly as well. I'm not sure but the performance feels very much like a bug.

Best,
Lance

Methods exported from Skyline-daily v 23.1.1.268 for Agilent 6495 (both scheduled and standard MRM) are invalid in MassHunter version 10.1 build 10.1.67. The methods are valid on a different Agilent 6495 running an older version of MassHunter (version B.09.00, build 9.0.9037.0).

Dear Skyline Team,

I hope you are doing well. This morning I tried to process some DIA raw file with EncyclopeDIA search. There is an error message that I share with you. Thanks in advance for your help.

Best Regards.

Hello Skyline Team,

I have measured endogenous peptides with their heavy counterpart by SRM. I have also spike a protein in my sample for normalisation.
I have some questions for translated results at protein level.
When I divide the intensity of endogenous peptide by their heavy counterpart it become a ratio and in many cases this ratio totally change the ponderation of each peptide for their contribution to the protein intensity. Typically, peptides representing the highest intensity (the one which would contribute the more to protein signal with the addition of peptide intensities) for the protein can, when divided by its heavy counterpart, be the one with the lowest ratio among the other peptide of the protein. In this case this will totally change the ponderation. I am not very confident with this phenomena because usually signal with the lowest intensity are those with the lowest quality (near LOD, lowest level of precision, lowest quality of the peak...). It means that with a division by the heavy peptides I will give more importance (not systematically) to signal with the lowest quality.
In fact their is different solution to combine the signal of peptide into protein signal. For instance:

-Signal of protein A= (Signal of endogenous peptides 1/ signal of heavy peptide 1) + (Signal of endogenous peptides 2/ signal of heavy peptide 2)
-Signal of protein A = (Signal of endogenous peptides 1 + Signal of endogenous peptides 2)/(signal of heavy peptide 1 + signal of heavy peptide 2)

As explained, the first formula can change the ponderation of the peptides contributing to the protein signal, the second one will not. We could imagine other solutions...Also, I have spiked a protein for normalising my signal and I do not know where I can put it in the formula, where would be the best place..
Do you have some advises for this problem ? In there some Skyline solutions ?
Thank you in advance for your help.

Nicolas Pierre

Dear Skyline team,

I hope you are well. I am new to skyline so apologise if I may have missed something, but last week I did some analysis and exported the files without having any issues. This was using the old skyline. The other day I received an email that a new was released so I downloaded the newest version. Just now I finished analysing some samples as was planning on to export them but the software doesn't allow me to. I don't know whether with the new version something may have changed
The way I have been instructed to export the samples is through the following:
File > export > report. In the report then I would then select edit list, select samples in columns which would prompt me import a file. I would insert the samples columns, press import and when I first did the samples in columns became highlighted. This time however, it fails to recognise the file.
Any suggestion on how to deal with the issue and export the files. Worst case scenario I was thinking of uninstalling and reinstalling but don't know whether that may help. I have also attached several pictures, hopefully that will be more clear.
Many thanks for your help and support
Best wishes

Federico Bernuzzi, PhD
Cancer Research Scotland

Dear all,

I have some data acquired using Fusion Lumos with the FAIMS installed and two CV voltages set. When I import the file without any spectrum filter, the data are imported correctly and one can see zig-zag style of the MS1 level intensities due to the the intensities from the two CV values combined into a single graph. When I set the spectrum filter in a way to select only CV values of -50 and -70, it gives me the following error:

At 12:05 PM:
Failed importing results file 'U:\712006-Proteomics\Instruments\Orbitrap_Lumos\Data\2023\230926_IS270_Lumos1\IS270_Lumos1_74IS_FAIMS(-50-70)_HCD-OT_DDA_HeLa_50ng_01.raw'.
Times (4672) and intensities (2336) disagree in point count.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'U:\712006-Proteomics\Instruments\Orbitrap_Lumos\Data\2023\230926_IS270_Lumos1\IS270_Lumos1_74IS_FAIMS(-50-70)_HCD-OT_DDA_HeLa_50ng_01.raw'.
Times (4672) and intensities (2336) disagree in point count. ---> System.IO.InvalidDataException: Times (4672) and intensities (2336) disagree in point count.
at pwiz.Skyline.Model.Results.ChromCollector.ReleaseChromatogram(Byte[] bytesFromDisk, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCollector.cs:line 120
at pwiz.Skyline.Model.Results.ChromGroups.ReleaseChromatogram(Int32 chromatogramIndex, Single retentionTime, ChromCollector collector, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCollector.cs:line 633
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Collectors.ReleaseChromatogram(Int32 chromatogramIndex, ChromGroups chromGroups, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1314
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, ChromatogramGroupId chromatogramGroupId, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 619
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, ChromatogramGroupId chromatogramGroupId, Color peptideColor) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 84
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, ChromatogramGroupId chromatogramGroupId, Color peptideColor) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 285
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 146
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 427
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256
--- End of inner exception stack trace ---

I have tested this on the freshly opened Skyline document with just a single iRT peptide in the list of peptides to import. It does work with the positive CV values used and also with the negative CV values for which there are no data actually present in the raw file. It simply returns no traces at this case. But when I set any CV value that is in the data (-50 or -70), I get the abovementioned error.

I have used 23.1.1.268 Skyline version.

Let me know if you would need any more data, I could share the raw file if necessary.

Thank you for double checking!

David

Dear Skyline Team,

I do targeted mass spectrometry for phosphopeptides. Since the same peptide can be phosphorylated at different positions, it is crucial to have a specific fragment ion for site localization. Is there a function in Skyline to somehow highlight a selected fragment ion in a peptide sequence? For example, highlighting amino acids with another colour or a rectangle (see attachment). This would allow a quick visual assessment of the role of the fragment ion for PTM site localization. Now I'm counting with my eyes and moving a pencil on the monitor, both suffer :)

I really appreciate any help you can provide.

Kind regards,
Nadzeya

Hi Folks!

I am utilizing Skyline Daily (23.1.1.268) to analyze my QQQ data, where I aim quantify relative abundance of different intracellular metabolites. During sample processing, I have included an internal, isotopic standards for relative & surrogate normalization purposes. With Skyline, I have successfully imported a transitions list with the appropriate heavy/light formulas, set up a normalization method, and specified which isotopic standard should serve as a surrogate. However, I'm curious how Skyline is actually quantifying the peak area ratio to the defined surrogate.

In my transition list, the majority of my molecules (including my isotopic standards) have multiple product ions and I have not specified as qualitative vs. quantitative. But, when I examine a Document Grid displaying the normalized areas, there is only a single value despite multiple transitions. Does Skyline have a default way of setting a quantitative ion that I'm overlooking? Or, without distinguishing qualitative vs. quantitative product ions, does skyline sum all available product ions and use this "total peak area" for the calculation?

Attached are screenshots to help clarify what I'm describing above. Let me know your thoughts & recommendations!

Thanks in advance,
Megan

Dear all,

not sure whether I am missing this feature being in Skyline already, but I would love to have an option to switch between different graphical layouts of the Skyline app, including e.g. different analyses grouping/shoving, different plots (like retention time comparison for replicates) displayed, positioned etc.

Or is this already possible please?

Might be related to this issue https://skyline.ms/issues/home/issues/details.view?issueId=270

Thank you in advance for your response/considering the feature request.

David

Hi skyline team!

I just imported my results (.raw files) after loading my fasta, but found out that my fasta was missing a pepitde... is there a way i can add this peptide now and integrate it for the result files? The loading of the files took about 4 hours, so i would prefer not having to start from scratch...

Best Shelley

Hi MS wizards,

I would like to request the addition of idotp as a metric that Skyline uses for deciding the correct center of the ion mobility window used for IMS-filtered EICs.

Attached is an example where the intensity maximization criteria (the only one used as far as I remember) leads to wrong selection of ion mobility values by co-eluting isobaric species. The wrong value was selected despite have identified the M+1 interference and marked it as non-quantitative.

Would it be possible to make Skyline to maximize both intensity and idotp (perhaps more weight on the idotp)?

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

Hi Skyline-Team,
thanks to your amazing support regarding my last question (https://skyline.ms/announcements/home/support/thread.view?rowId=62010) I could import my .mzml files converted from .wiff and .wiff2 files into skyline.
However, for some reason I can only see my MS2 transitions of interest using .wiff files or .mzml files converted from .wiff files, but as soon as I try to import a .wiff2 file or .mzml file generated from the .wiff2, I don’t see any chromatogram information. The problem only seems to occur for my MS/MS transitions of interest, as the MS1 trace is visible irrespective of the file-type.

Do you have any idea how this could happen and how to circumvent this issue? Unfortunately, I need to use the .wiff2 file (or .mzml file generated from the .wiff2) for my data analysis. Using SeeMS I can clearly see that MS2 spectra are present.
I use Skyline 64 bit 23.1.0.268 with Windows Enterprise and try to analyze data generated on a zenoTOF 7600 from SCIEX.

I would highly appreciate your input and look forward to hearing from you.
Best,
Lisa

Hello,

I quantify small molecules on Skyline. I wanted to use metabolite B as an internal standard for metabolite A, so I defined marked metabolite B as a standard surrogate in the tree structure. Unfortunately, in the Normalization Method column of the Document Grid, the name of the metabolite is replaced by its InChIKey, which makes it very difficult to read (see attached file).

How can we make the "Ratio to surrogate..." option show the metabolite name (as defined in the tree structure) and not an identifier?

Thanks
Amandine

Very, very new to Skyline, so please be gentle:

We are working on our Quality Assurance plan, and I am looking into the possibility of importing endpoint Auto QC data into our existing quality control software and want to better understand Auto QC's output. Is there an output file other than the monthly zip file that could be saved externally? And if so, what is the format or file type? Any help would be appreciated!

Thank you,

Lindsay :)

Sorry to bother u, my laptop is Mac m1 pro(ventura 13.5.2), i tried to run the download skyline exe on my parallel, but it was failed, can u help me?

Good day,

I am trying to use TurnoveR tool in Skyline. I have a question regarding "Set Reference". I am not sure What can we use for Reference? is it like DDA of control sample (unlabeled sample)? can you please explain what Set Reference and why need to use?

Thank you,
G

Hi all,
newbie in skyline and I'm trying (unsuccessfully to import some data). I read that skyline looks in .d files for .tdf files. I do have .tdf files however skyline doesn't recognize it.

Any ideas/tips/tricks/help?

Hi All,
Is there a way to populate Skyline target list with the library generated by MSFragger/EasyPQP (library.tsv)?

Tomas

We are trying to upload MSFragger results from a search on DIA GPF, following the instruction (https://fragpipe.nesvilab.org/docs/tutorial_skyline.html) using both DDA and DIA search results workflow in Skyline. But in both cases we get error that the spectra cannot be found. We have both interact-xx.pep.xml and the mzML files in the same directory, so I am not sure what is going on.
Apparently there has been previously similar issue with this when .mgf file were used.
https://skyline.ms/announcements/home/support/thread.view?rowId=57930

Thanks for any advice.

Tomas

Hey,

thanks for the great software.
I analyzed a larger DDA dataset with MS1filtering. Unfortunately, the external disk with the RAW files crashed. I still have the skyline file on my computer and a backup of the maxquant search plus raw files of the corresponding analysis. However, Skyline expects the original location of the raw files on the broken external memory.

1. Is it possible to reimport the data in the skyline files from a new folder?
   or 2. is there another way to reconnect skyline analysis and raw data (plus maxquant search)?

Thanks in advance!

Ingo

Would like the ability to save item from Targets list to iRT database. This would allow user to work in a cycle of exploration / update targets library.

Maybe right click?

Hi Skyline developers,

I have been using Proteome Discoverer 3.0.0.757 (Sequest node) with Skyline 21.2.0.425 for building spectra libraries. This combination has worked fine for several projects this year till now. The PD result file, generated .blib and Skyline document seem to give a reasonable number of proteins and peptides, but library details are somehow not showing (screencap attached). Nothing in our workflow has changed between the previous projects and this current one except for the number of files in library run dataset: 80 files compared to 20-30 from before. I have also tried re-do of PD search and importing of library under different filename and get the same problem.

Thanks for your help.

Dear,

I encountered an error when trying to import a transition list (which I exported from another Skyline project); it is not extracting the isotopic peaks. I tried looking into the transition settings, but I couldn't find any restrictions there for the isotopic peaks. I added the Skyline file and transition list in the attachment. Could you help me with this?

Thank you!

Laura

Hello Skyline Team,

I have set up my skyline document to look for both heavy and light peptides. Is there a way, once i've imported my MS data, to have skyline show me the intensities and/or peak areas of the precursors and fragments for both the heavy and light peptides? Currently it only shows me the light peptides. Thanks!

I no longer have access to the raw data files which were imported into a skyline document as results to establish retention times. Is there a way to transfer these imported results from one skyline document to a new one without having access to the original files?

Good afternoon,
I have been trying to implement a Prosit Spectral Library in Skyline which I have done lots before with no trouble, but this morning a new error appeared. Any ideas what this could be caused by?

Best wishes,
Colleen