Requests

support
Showing: limited to 100 requests
Correct spectrum selection for non-redundant library
Juan C. Rojas E. 2020-09-30

Dear support,

Attached you can find a few slides displaying my issue.

In multiple instances I have observed that the spectrum selected for the non-redundant library is suboptimal compared to some other spectra available (Slide 1 and 2). For DDA files the problem is easily circumvented by working with the redundant library, but for library generation for DIA, MRM, and/or PRM methods could lead to comparisons to suboptimal representative library spectrum.

The .mzXML files (exported from PEAKS) for data acquired in resolution mode are kept in resolution mode format when exported (Slide 3). Maybe this is the reason for the mismatch due to better random matching (i.e. in mass tolerance consideration) to some of the "split" peaks of some spectra compared to others even if the absolute abundance is lower.

Is it possible to manually exchange the best representative spectrum for the non-redundant library?

If not, could a peak picking step be included in the library building procedure? Or should I just perform it with MSConvert externally?

As always, thank you for your time and support.
Sincerely,
JC

 Library_msms_spectrum_selection.pptx 
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Broken compatibility with Shimadzu LCMS 8050 after replacing Shimadzu DataReader with IoModule for MRM files
Pawel 2020-09-30
Hi Skyline Team!

Thanks for your continuous work on the software. Just wanted to make you aware that the most recent update 20.1.9.268 broke the compatibility with Shimadzu LCMS 8050. The following error appears:

"Failed importing results file 'C:\LabSolutions\Data\Chalani\20200929_QC(100x)_021.lcd'.
[ShimadzuReader::getSpectrum] GetMSSpectrumByScan: E_INVALIDARG"

Old version of Skyline still works but I am unable to use Skyline for importing Shimadzu data on any computer on which I updated the software to the above version. Would you mind having a look into this problem?

Regards,
Pawel
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Skyline CMD help
(3 responses) smanda 2020-08-13

Hi Brendon/Kaipo,

I am trying to build a spectral library all using command line as we are trying to automate some of the steps at our lab. To this, I am unable to figure out, how can I choose peptides standards for RT calibration (after I import the library). My current steps are:

  1. SkylineCmd.exe --in=Empty.sky --out=New_library.sky --import-search-file=searchresults.mzid --import-fasta=20190717_Uniprot_concat_decoys.fasta
    This step creates a nr library and adds the peptides as targets.
    I do not specify RT peptides here.
    I then import a report format

  2. SkylineCmd.exe" --report-add=skyline.skyr

  3. SkylineCmd.exe --in=New_library.sky --report-name=OpenSWATH --report-file=test_report.tsv --report-format=tsv

I see that the exported reported as several NAs in the iRT column. We have around 40 endogenous peptides that are added as iRT standards in all experiments, can you please let me know, how to I add them at RT peptides and export the report with iRT values.

Regards,
Srikanth

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Retention time recognized in sec format
Khang Huynh 2020-09-29

Hi Brendan and Team,

I have just updated to the latest version of Skyline-daily today. When I imported the LC-MS/MS data files, the retention times on the chromatograms were in "second" format instead of in "minute" as usual (photo attached). I have tried to look into the Settings but could not figure out how to change it back to the "minute" format. Could you please help?

Operating system: Windows 10.
Small molecules for Quantification.
Raw data files: Shimadzu .lcd

Thanks,
Khang Huynh

 Screenshot 2020-09-29 172658.jpg 
view request
Broad mass range for peak extraction
(4 responses) harald schoeny 2020-09-18

Dear Skyline Team,

I have lipid data measured on an HILIC- Orbitrap QExactive HF with an all ion fragmentation (AIF) approach. I figured it out to extract single masses with the help of the forum but I would like to extract a broader mass range (m/z 530-650 ) withing one peak. It should be the sum of all lipid species within one lipid class. Unfortunately, I couldn't find a way to do that in Skyline. I have tried to set the MS/MS filtering to Isolation scheme/Use results data isolation targets/ Isolation width/fixed/120 mz but nothing really happened. Is there another workaround for that?

Thank you in advance.
Best, Harald

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Problem in building of spectral library
(1 response) arvindverma0078 2020-09-29

Dear Skyline team,

Recently I used Skyline to build a spectral library from .mgf files. However, I want to convert .mgf file to blib format, because skyline do not accept .mgf file. If a direct conversion is not possible, perhaps there is a way to convert the library to an intermediate format that you are aware of?
Moreover, it is very helpful to me. If you assist me to building of spectral library from my data generated from Thermo Scientific Orbitrap Fusion MS platform.

Best regards,
Arivnd

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Reduction in the number of true peptide identification as the number of DIA raw files increases in the spectral library search
Chinmaya k 2020-09-28

Hello,

I have carried out a Spectral Library search for one of our DIA data generated with a 25m/z isolation window from 400-1000 m/z range. The spectral library search of single DIA.Raw file is able to identify 8000+ peptides with Q-Value < 0.01 from mProphet model. However, when multiple replicates of the same sample (Which includes the DIA file used for the first search) were searched against the same spectral library keeping all other parameters as the same is resulting in a drastically less number of peptides (<5000) with Q-value < 0.01 from mProphet model. And this goes on as the number of files increases.

What might be the reason for this? Why the number of true peptide identifications is decreasing as the number of DIA files are increased?

Prior to applying the mProphet model, the peptides RT was calibrated using Pierce iRT standard spiked in the sample. The screenshots of parameters used for RT prediction and calculation are attached below for your references.

Please let me whether any parameters have to be changed and if so, why? How does it will effect the DIA Spectral library search and statistical validation?

--
Chinmaya

 Peptide_Setting.PNG  Edit_iRT_Calculator.PNG  Edit_Retention_Time_Predictor.PNG 
view request
Missing light product ions in SureQuant experiment
(3 responses) carmen.gonzaleztejedo 2020-09-28
Dear Team, We performed a SureQuant experiment in an Orbitrap Exploris 480. When we load the data into the Skyline, we can see the XIC for the light and heavy precursors and the XICs for the heavy fragments. However, we cannot see the fragments of the light precursors (see attached). I have checked all the peptide and transition settings but I cannot figure out what the issue is. I would really appreciate if you can help me with that. Thanks a lot in advance. Regards, Carmen.
 SkylineSupport_OE480_SureQuant.pptx 
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Custom iRT peptides
(3 responses) benoit fatou 2020-09-24

Dear Skyline Team,

I was wondering if it is possible to create custom iRT peptides for RT adjustment in MRM experiments. In fact, I am working on plasma samples and I would like to use some endogenous peptides from the serum albumin in my samples.
Thanks for your help,
Best,
Benoit

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Crosslink search from Mascot to create library for Skyline
(6 responses) sandberg 2020-08-05

Hi,
Is it possible to use crosslinking results from the Mascot search engine to create a library to search the files for crosslinked peptides in Skyline?

Best,
Magdalena

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DIA 3-plex SILAC
(1 response) trinidad martincampos 2020-09-27

Hello all,

First of all, thank you very much for the effort you put into Skyline for all of us ;)

I took a look at the post with "DIA SILAC" but my problem is still not solved... I have a 3-plex SILAC (light, medium and heavy) experiment that I am going to analyse with DIA in an orbitrap fusion.
Any hint about how could I do that? What happens with a peptide whose light and medium (for example)tags are in different windows? Any possible workflow to do this?

Thank you very much!!!
Trini

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How did skyline read the OpenSWATH results?
(6 responses) andyzcq 2020-09-19

I followed the tutorial, and found there is no merged.osw file in OpenSWATH results.

I have used OpenSWATH (V. 2.5) to analyze my DIA data.

So how did Skyline read OpenSWATH (V. 2.5) results?

view request
Oxonium ions for MRM glycan analysis
(3 responses) giulia lambiase 2020-09-24

Hello,
I am writing an MRM method for monitoring glycans in mAbs. I need to add oxonium ions in my transition list but cannot work out how to do it in Skyline. Could you please support me with this?
Thanks,
Julia

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Skyline-daily Program Won't Open
(2 responses) ehubb004 2020-09-22

Yesterday, for no clear reason, the skyline-daily program (the most current version) stopped opening on my computer. When I try to open it, my computer briefly registers that it's loading, but nothing actually happens, and task manager never registers Skyline activity. I've tried restarting my computer, as well as completely reinstalling skyline-daily and even redownloading the installer program, but nothing has affected this issue.

view request
Retention time selection
(6 responses) ojulien 2020-09-17

Hi,

We imported 4 .raw files into skyline, and used a bibliospec file to import all the sequences into skyline.

We then created an inclusion list for all the peptides of interest (~700) for PRM.

However, the RT in the inclusion list are all off if I select "average RT", since the peptides selected were not found in all datasets. The other option is to use a single dataset out of 4, but then the RT will be off for the peptides that were not found in that specific dataset.

The best way would be to use the RT from the dataset that matches the spectral library. Is there a way to do this, am I missing something? If not, is it possible to add this as an option in future release?

This would be great, as of right now, we are adjusting the RT window manually for each peptides in each datasets...

OJ

view request
Waters Method Export Failure (Masslynx 4.2 SCN986)
(1 response) Will Thompson 2020-09-23

Hi Brian et al

We have recently upgraded two of our Waters TQ-S systems to Windows 10, and purchased licenses for Masslynx 4.2 SCN986 to do this upgrade. When trying to export *.exp methods for the instrument, we are now getting errors that the method cannot be exported. I am pasting in the long text of the method below. Do you have any hints from this text on whether or not it is a masslynx version issue, or something else? We have confirmed the method export is functional on MassLynx 4.2 SCN1007, which runs on the TQ-XS. I have asked Waters if there is a more recent upgrade for the TQ-S, or if we can run SCN1007 on the TQ-S. Maybe there are other questions you guys can ask your Waters contacts?

This would mean that any Waters TQ-S users who upgrade to Windows 10 will not be able to export methods from Skyline any more... :(

Cheers,

Will

Error text below


System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

Command-line: Method\Waters\BuildWatersMethod -w 2 -s -m "C:\VerifyETemplate.exp"
Working directory: D:\5633
Standard input:
D:\5633~SKEB17.tmp
D:\5633\5633_TQS_v1.exp
protein.name,peptide.seq,precursor.mz,precursor.retT,product.m_z,collision_energy,cone_voltage,peptide_unmod.seq,ion_name,library_rank
1,C0.[M+],162.1,0.56,85.1,18,32,C0,Ion [85.100549/85.100549],-1
2,d3-C0.[M+],165.2,0.56,85.1,18,32,d3-C0,Ion [85.100549/85.100549],-1
3,C2.[M+],204.1,0.9,85.1,18,26,C2,Ion [85.100549/85.100549],-1
4,d3-C2.[M+],207.1,0.9,85.1,18,26,d3-C2,Ion [85.100549/85.100549],-1
13,C3.[M+],218.1,1.73,85.1,18,28,C3,Ion [85.100549/85.100549],-1
14,d3-C3.[M+],221.1,1.73,85.1,18,28,d3-C3,Ion [85.100549/85.100549],-1
15,iC4.[M+],232.1,2.19,85.1,18,28,iC4,Ion [85.100549/85.100549],-1
16,C4.[M+],232.1,2.29,85.1,18,28,C4,Ion [85.100549/85.100549],-1
12,d3-C4.[M+],235.2,2.27,85.1,18,28,d3-C4,Ion [85.100549/85.100549],-1
21,C5:1.[M+],244.1,2.66,85.1,20,32,C5:1,Ion [85.100549/85.100549],-1
22,C4:1-M.[M+],244.1,2.71,85.1,20,32,C4:1-M,Ion [85.100549/85.100549],-1
23,C5-P.[M+],246.2,2.69,85.1,18,30,C5-P,Ion [85.100549/85.100549],-1
24,C4-2M.[M+],246.2,2.81,85.1,18,30,C4-2M,Ion [85.100549/85.100549],-1
25,iC5.[M+],246.2,2.89,85.1,18,30,iC5,Ion [85.100549/85.100549],-1
26,C5.[M+],246.2,3.01,85.1,18,30,C5,Ion [85.100549/85.100549],-1
5,C3-DC.[M+],248.1,0.8,85.1,21,30,C3-DC,Ion [85.100549/85.100549],-1
6,C4-OH.[M+],248.1,1.25,85.1,21,30,C4-OH,Ion [85.100549/85.100549],-1
19,d9-C5.[M+],255.2,2.86,85.1,18,30,d9-C5,Ion [85.100549/85.100549],-1
20,C6.[M+],260.2,3.56,85.1,18,30,C6,Ion [85.100549/85.100549],-1
7,C4-DC.[M+],262.2,1.2,85.1,21,32,C4-DC,Ion [85.100549/85.100549],-1
8,C3-DC-M.[M+],262.2,1.5,85.1,21,32,C3-DC-M,Ion [85.100549/85.100549],-1
9,C5OH-I.[M+],262.2,1.78,85.1,21,32,C5OH-I,Ion [85.100549/85.100549],-1
29,d3-C6.[M+],263.2,3.55,85.1,18,30,d3-C6,Ion [85.100549/85.100549],-1
10,C5-DC.[M+],276.1,1.71,85.1,20,32,C5-DC,Ion [85.100549/85.100549],-1
11,C6-OH.[M+],276.1,2.53,85.1,20,32,C6-OH,Ion [85.100549/85.100549],-1
31,C8:1.[M+],286.2,4.16,85.1,22,34,C8:1,Ion [85.100549/85.100549],-1
32,C8.[M+],288.2,4.28,85.1,22,34,C8,Ion [85.100549/85.100549],-1
17,C6-DC.[M+],290.2,2.01,85.1,20,34,C6-DC,Ion [85.100549/85.100549],-1
18,C5-M-DC.[M+],290.2,2.08,85.1,20,34,C5-M-DC,Ion [85.100549/85.100549],-1
33,d3-C8.[M+],291.2,4.27,85.1,22,34,d3-C8,Ion [85.100549/85.100549],-1
28,C10:1.[M+],314.2,4.76,85.1,22,37,C10:1,Ion [85.100549/85.100549],-1
34,C10.[M+],316.2,4.86,85.1,22,38,C10,Ion [85.100549/85.100549],-1
27,C8-DC.[M+],318.2,2.94,85.1,22,35,C8-DC,Ion [85.100549/85.100549],-1
35,d3-C10.[M+],318.9,4.85,85.1,22,38,d3-C10,Ion [85.100549/85.100549],-1
37,C12:1.[M+],342.3,5.29,85.1,24,40,C12:1,Ion [85.100549/85.100549],-1
39,C12.[M+],344.2,5.37,85.1,22,38,C12,Ion [85.100549/85.100549],-1
30,C10-DC.[M+],346.2,3.62,85.1,22,36,C10-DC,Ion [85.100549/85.100549],-1
40,d3-C12.[M+],347.2,5.37,85.1,22,38,d3-C12,Ion [85.100549/85.100549],-1
36,C12-OH.[M+],360.3,4.87,85.1,23,38,C12-OH,Ion [85.100549/85.100549],-1
38,C14:2.[M+],368.3,5.33,85.1,23,39,C14:2,Ion [85.100549/85.100549],-1
42,C14:1.[M+],370.2,5.76,85.1,23,39,C14:1,Ion [85.100549/85.100549],-1
43,C14.[M+],372.2,5.85,85.1,24,40,C14,Ion [85.100549/85.100549],-1
44,d3-C14.[M+],375,5.85,85.1,24,40,d3-C14,Ion [85.100549/85.100549],-1
49,cis-9-C16:1.[M+],398.3,5.98,85.1,25,42,cis-9-C16:1,Ion [85.100549/85.100549],-1
50,trans-2-C16:1.[M+],398.3,6.2,85.1,25,42,trans-2-C16:1,Ion [85.100549/85.100549],-1
51,C16.[M+],400.4,6.27,85.1,24,44,C16,Ion [85.100549/85.100549],-1
52,d3-C16.[M+],403.4,6.27,85.1,24,44,d3-C16,Ion [85.100549/85.100549],-1
41,C16:1-OH.[M+],414.3,5.51,85.1,26,44,C16:1-OH,Ion [85.100549/85.100549],-1
45,C16-OH.[M+],416.3,5.85,85.1,26,44,C16-OH,Ion [85.100549/85.100549],-1
48,C18:2.[M+],424.4,6.09,85.1,26,45,C18:2,Ion [85.100549/85.100549],-1
54,C18:1.[M+],426.4,6.34,85.1,27,45,C18:1,Ion [85.100549/85.100549],-1
55,C18.[M+],428.4,6.62,85.1,28,50,C18,Ion [85.100549/85.100549],-1
56,d3-C18.[M+],431.4,6.61,85.1,28,50,d3-C18,Ion [85.100549/85.100549],-1
46,C18:1-OH.[M+],442.4,5.97,85.1,28,46,C18:1-OH,Ion [85.100549/85.100549],-1
53,C18-OH.[M+],444.3,6.27,85.1,28,46,C18-OH,Ion [85.100549/85.100549],-1
47,C20:4.[M+],448.3,6.08,85.1,28,48,C20:4,Ion [85.100549/85.100549],-1
---> System.IO.IOException: ERROR: Method not found: 'Boolean CompoundDatabaseClassLibrary.CompoundDatabase.InsertNewIonDetails(Int32, Int32, TransMode, Int32, Int32, Int32, Int32, Int32, Int32)'.

Command-line: Method\Waters\BuildWatersMethod -w 2 -s -m "C:\VerifyETemplate.exp"
Working directory: D:\5633
Standard input:
D:\5633~SKEB17.tmp
D:\5633\5633_TQS_v1.exp
protein.name,peptide.seq,precursor.mz,precursor.retT,product.m_z,collision_energy,cone_voltage,peptide_unmod.seq,ion_name,library_rank
1,C0.[M+],162.1,0.56,85.1,18,32,C0,Ion [85.100549/85.100549],-1
2,d3-C0.[M+],165.2,0.56,85.1,18,32,d3-C0,Ion [85.100549/85.100549],-1
3,C2.[M+],204.1,0.9,85.1,18,26,C2,Ion [85.100549/85.100549],-1
4,d3-C2.[M+],207.1,0.9,85.1,18,26,d3-C2,Ion [85.100549/85.100549],-1
13,C3.[M+],218.1,1.73,85.1,18,28,C3,Ion [85.100549/85.100549],-1
14,d3-C3.[M+],221.1,1.73,85.1,18,28,d3-C3,Ion [85.100549/85.100549],-1
15,iC4.[M+],232.1,2.19,85.1,18,28,iC4,Ion [85.100549/85.100549],-1
16,C4.[M+],232.1,2.29,85.1,18,28,C4,Ion [85.100549/85.100549],-1
12,d3-C4.[M+],235.2,2.27,85.1,18,28,d3-C4,Ion [85.100549/85.100549],-1
21,C5:1.[M+],244.1,2.66,85.1,20,32,C5:1,Ion [85.100549/85.100549],-1
22,C4:1-M.[M+],244.1,2.71,85.1,20,32,C4:1-M,Ion [85.100549/85.100549],-1
23,C5-P.[M+],246.2,2.69,85.1,18,30,C5-P,Ion [85.100549/85.100549],-1
24,C4-2M.[M+],246.2,2.81,85.1,18,30,C4-2M,Ion [85.100549/85.100549],-1
25,iC5.[M+],246.2,2.89,85.1,18,30,iC5,Ion [85.100549/85.100549],-1
26,C5.[M+],246.2,3.01,85.1,18,30,C5,Ion [85.100549/85.100549],-1
5,C3-DC.[M+],248.1,0.8,85.1,21,30,C3-DC,Ion [85.100549/85.100549],-1
6,C4-OH.[M+],248.1,1.25,85.1,21,30,C4-OH,Ion [85.100549/85.100549],-1
19,d9-C5.[M+],255.2,2.86,85.1,18,30,d9-C5,Ion [85.100549/85.100549],-1
20,C6.[M+],260.2,3.56,85.1,18,30,C6,Ion [85.100549/85.100549],-1
7,C4-DC.[M+],262.2,1.2,85.1,21,32,C4-DC,Ion [85.100549/85.100549],-1
8,C3-DC-M.[M+],262.2,1.5,85.1,21,32,C3-DC-M,Ion [85.100549/85.100549],-1
9,C5OH-I.[M+],262.2,1.78,85.1,21,32,C5OH-I,Ion [85.100549/85.100549],-1
29,d3-C6.[M+],263.2,3.55,85.1,18,30,d3-C6,Ion [85.100549/85.100549],-1
10,C5-DC.[M+],276.1,1.71,85.1,20,32,C5-DC,Ion [85.100549/85.100549],-1
11,C6-OH.[M+],276.1,2.53,85.1,20,32,C6-OH,Ion [85.100549/85.100549],-1
31,C8:1.[M+],286.2,4.16,85.1,22,34,C8:1,Ion [85.100549/85.100549],-1
32,C8.[M+],288.2,4.28,85.1,22,34,C8,Ion [85.100549/85.100549],-1
17,C6-DC.[M+],290.2,2.01,85.1,20,34,C6-DC,Ion [85.100549/85.100549],-1
18,C5-M-DC.[M+],290.2,2.08,85.1,20,34,C5-M-DC,Ion [85.100549/85.100549],-1
33,d3-C8.[M+],291.2,4.27,85.1,22,34,d3-C8,Ion [85.100549/85.100549],-1
28,C10:1.[M+],314.2,4.76,85.1,22,37,C10:1,Ion [85.100549/85.100549],-1
34,C10.[M+],316.2,4.86,85.1,22,38,C10,Ion [85.100549/85.100549],-1
27,C8-DC.[M+],318.2,2.94,85.1,22,35,C8-DC,Ion [85.100549/85.100549],-1
35,d3-C10.[M+],318.9,4.85,85.1,22,38,d3-C10,Ion [85.100549/85.100549],-1
37,C12:1.[M+],342.3,5.29,85.1,24,40,C12:1,Ion [85.100549/85.100549],-1
39,C12.[M+],344.2,5.37,85.1,22,38,C12,Ion [85.100549/85.100549],-1
30,C10-DC.[M+],346.2,3.62,85.1,22,36,C10-DC,Ion [85.100549/85.100549],-1
40,d3-C12.[M+],347.2,5.37,85.1,22,38,d3-C12,Ion [85.100549/85.100549],-1
36,C12-OH.[M+],360.3,4.87,85.1,23,38,C12-OH,Ion [85.100549/85.100549],-1
38,C14:2.[M+],368.3,5.33,85.1,23,39,C14:2,Ion [85.100549/85.100549],-1
42,C14:1.[M+],370.2,5.76,85.1,23,39,C14:1,Ion [85.100549/85.100549],-1
43,C14.[M+],372.2,5.85,85.1,24,40,C14,Ion [85.100549/85.100549],-1
44,d3-C14.[M+],375,5.85,85.1,24,40,d3-C14,Ion [85.100549/85.100549],-1
49,cis-9-C16:1.[M+],398.3,5.98,85.1,25,42,cis-9-C16:1,Ion [85.100549/85.100549],-1
50,trans-2-C16:1.[M+],398.3,6.2,85.1,25,42,trans-2-C16:1,Ion [85.100549/85.100549],-1
51,C16.[M+],400.4,6.27,85.1,24,44,C16,Ion [85.100549/85.100549],-1
52,d3-C16.[M+],403.4,6.27,85.1,24,44,d3-C16,Ion [85.100549/85.100549],-1
41,C16:1-OH.[M+],414.3,5.51,85.1,26,44,C16:1-OH,Ion [85.100549/85.100549],-1
45,C16-OH.[M+],416.3,5.85,85.1,26,44,C16-OH,Ion [85.100549/85.100549],-1
48,C18:2.[M+],424.4,6.09,85.1,26,45,C18:2,Ion [85.100549/85.100549],-1
54,C18:1.[M+],426.4,6.34,85.1,27,45,C18:1,Ion [85.100549/85.100549],-1
55,C18.[M+],428.4,6.62,85.1,28,50,C18,Ion [85.100549/85.100549],-1
56,d3-C18.[M+],431.4,6.61,85.1,28,50,d3-C18,Ion [85.100549/85.100549],-1
46,C18:1-OH.[M+],442.4,5.97,85.1,28,46,C18:1-OH,Ion [85.100549/85.100549],-1
53,C18-OH.[M+],444.3,6.27,85.1,28,46,C18-OH,Ion [85.100549/85.100549],-1
47,C20:4.[M+],448.3,6.08,85.1,28,48,C20:4,Ion [85.100549/85.100549],-1

at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_20_2_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.Skyline.Util.Extensions.UtilProcess.RunProcess(ProcessStartInfo psi, String stdin, String messagePrefix, IProgressMonitor progress, IProgressStatus& status) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Extensions\UtilProcess.cs:line 45
at pwiz.Skyline.Model.MethodExporter.ExportMethod(String exeName, List1 argv, String fileName, String templateName, Dictionary2 dictTranLists, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 4162
at pwiz.Skyline.Model.WatersMethodExporter.ExportMethod(String fileName, String templateName, IProgressMonitor progressMonitor) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Export.cs:line 3959
at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254 --- End of inner exception stack trace --- at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1942 at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 204
at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140 at pwiz.Skyline.FileUI.ExportDlgProperties.PerformLongExport(Action1 performExport) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\ExportMethodDlg.cs:line 2095

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Export method to MassLynx 4.2 version
(6 responses) danielacgranato 2018-11-27

Dear,

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

Thank you very much. Best, Daniela

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Cannot Roundtrip Small Molecule Transition List
(3 responses) damien ready 2020-09-17

Greetings,
We are looking into using Skyline for some of our small molecule processing, and I ran into a rough edge I thought could be improved around managing transition lists. If I graphically build a small molecule transition list in Skyline and export it, Skyline fails to import the list into a new document.

If you follow the below, I was able to manipulate the exported document into a format that Skyline would import, but the workflow is cumbersome for something that feels like it should be natively handled by the GUI.

If you do intend to investigate the issue, I should be clear that my actual interest is in automating the creation of Skyline documents + transition lists through the Skyline Commandline runner. This work through the GUI was just to establish POC.

Skyline Daily 20.1.9.234
To reproduce:

  • Start Skyline
  • Select Small Molecule workflow
  • Edit->Insert-Transition List
    • Molecule List Name: aspirin
    • Precursor Name: aspirin
    • Precursor Formula: C9H8O4
    • Precursor Adduct: [M+H]
    • Precursor m/z: 181.0495348
    • Precursor Charge: 1
  • Insert molecule
  • File->Export->Transition List
  • File->New
  • File->Import->Transition List
    Results in following error message:
System.IO.InvalidDataException: Failed to find peptide column., line 1. ---> System.IO.InvalidDataException: Failed to find peptide column., line 1. ---> pwiz.Skyline.Model.LineColNumberedIoException: Failed to find peptide column., line 1.
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, ColumnIndices indices, IDictionary`2 dictNameSeq, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 485
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 423
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.MassListImporter.Import(IProgressMonitor progressMonitor, String sourceFile, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Import.cs:line 427
   at pwiz.Skyline.Model.SrmDocument.ImportMassList(MassListInputs inputs, IProgressMonitor progressMonitor, IdentityPath to, IdentityPath& firstAdded, List`1& irtPeptides, List`1& librarySpectra, List`1& errorList, List`1& peptideGroups) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\SrmDocument.cs:line 1454
   at pwiz.Skyline.SkylineWindow.<>c__DisplayClass1079_0.<ImportMassList>b__2(IProgressMonitor longWaitBroker) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1925
   at pwiz.Skyline.Controls.LongWaitDlg.RunWork(Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 254
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Util.Helpers.WrapAndThrowException(Exception x) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Util\Util.cs:line 1940
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 202
   at pwiz.Skyline.Controls.LongWaitDlg.PerformWork(Control parent, Int32 delayMillis, Action`1 performWork) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Controls\LongWaitDlg.cs:line 140
   at pwiz.Skyline.SkylineWindow.ImportMassList(MassListInputs inputs, String description, Boolean assayLibrary) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1923
   at pwiz.Skyline.SkylineWindow.ImportMassList(String fileName) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1902

Consulting the Help on the "Transition List" window, I attempted to hand-edit the exported CSV to include the columns (MoleculeGroup, PrecursorName, ProductName, PrecursorFormula, ProductFormula, PrecursorMz, ProductMz, PrecursorCharge, ProductCharge, PrecursorAdduct, ProductAdduct, PrecursorRT, PrecursorRTWindow, PrecursorCE, PrecursorDT, HighEnergyDTOffset, PrecursorIM, HighEnergyIMOffset, IMUnits, PrecursorCCS, SLens, ConeVoltage, CompensationVoltage, DeclusteringPotential, Note, LabelType, InChiKey, CAS, HMDB, InChi, SMILES, KEGG, ProductNeutralLoss), added the required data, but this too failed to import. Lastly, I added the suggested columns, filled in the extra data, and deleted the originally exported columns - this worked.

Attached:

  • small_molecule_ts_export.csv - failed to import - Skyline small molecule built and exported transition list
  • small_molecule_ts_export_extracolumns.csv - failed to import - Skyline small molecule export + manually added suggested columns
  • small_molecule_ts_export_extracolumns_only.csv - successful import - Skyline small molecule export + manually added suggested columns only

Thanks for your attention. Happy to give any more details that you require.

 small_molecule_ts_export.csv  small_molecule_ts_export_extracolumns.csv  small_molecule_ts_export_extracolumns_only.csv 
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Comments need to be deleted
(5 responses) mattkarasu 2020-09-11

When exporting an MS Method for our Waters Xevo TQ from Skyline it seems to be creating an .exp file with comments added to every line of the MRM table. Oddly, there seem to be two different comments though? One mentioning Quanpedia and the other mentioning VerifyESkylinLibrary.

It is also worth pointing out that the output .exp file has these comments present even when opened as a raw text file, so I know it is not something our MS File Editor is adding or misinterpreting.

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Extracting MS1 peak area for peptides of known mass from DDA/bbCID experiments
(3 responses) Mus 2020-09-14
Hi folks, I have 6 datafiles from DDA runs on a Bruker QTOF. I collected MS/MS fragmentation data in bbCID mode (similar to all ion fragmentation), - only to assign MS1 peaks confidently. Each of the 6 samples are varying but defined mixtures of the following, in a sea of trypsin digested BSA: - 35 synthetically made LIGHT peptides (with K or R C-terminus to mimic a trypsin digest) - 35 synthetically made HEAVY (deuterium labelled) peptides (with K or R C-terminus to mimic a trypsin digest) The peptide sequences are known and I have precurser masses for +2/+3 charge states of all the 70 peptides. The samples contain varying ratio's of LIGHT:HEAVY peptides and I am trying to determine these ratio's by extracting peak area for MS1 peaks for each of 70 peptides. What's the best way to do this in Skyline? I have tried MS1 filtering workflow without success. Thanks in advanvce.
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support in Shimadzu LabSOlutions LCMS 5.99 SP2
(4 responses) Stephane MOREAU 2020-08-28

Hi,

it seems that Skyline cannot read data coming from the latest version of SHimadzu LabSolutions LCMS 5.99 SP2 ?

sincerely

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Prosit Server Unavailibility
(3 responses) Chinmaya k 2020-09-14

Hello,

I am not able to connect to the Prosit server (131.159.152.7:8500) through Skyline. What is the issue?

--
Chinmaya

 Skyline_Prosit_Server_Unavailability.PNG 
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error building Prosit library
(1 response) bin fang 2020-08-28
pwiz.Skyline.Model.Prosit.PrositException: Status(StatusCode=Unknown, Detail="CUDNN_STATUS_BAD_PARAM
in external/org_tensorflow/tensorflow/stream_executor/cuda/cuda_dnn.cc(1249): 'cudnnSetTensorNdDescriptor( tensor_desc.get(), data_type, sizeof(dims) / sizeof(dims[0]), dims, strides)'
     [[{{node encoder1/CudnnRNN}} = CudnnRNN[T=DT_FLOAT, direction="unidirectional", dropout=0, input_mode="linear_input", is_training=true, rnn_mode="gru", seed=87654321, seed2=0, _device="/job:localhost/replica:0/task:0/device:GPU:0"](encoder1/transpose, encoder1/ExpandDims_1, encoder1/Const, encoder1/concat)]]
     [[{{node out/Reshape/_55}} = _Recv[client_terminated=false, recv_device="/job:localhost/replica:0/task:0/device:CPU:0", send_device="/job:localhost/replica:0/task:0/device:GPU:0", send_device_incarnation=1, tensor_name="edge_605_out/Reshape", tensor_type=DT_FLOAT, _device="/job:localhost/replica:0/task:0/device:CPU:0"]()]]") ---> Grpc.Core.RpcException: Status(StatusCode=Unknown, Detail="CUDNN_STATUS_BAD_PARAM
in external/org_tensorflow/tensorflow/stream_executor/cuda/cuda_dnn.cc(1249): 'cudnnSetTensorNdDescriptor( tensor_desc.get(), data_type, sizeof(dims) / sizeof(dims[0]), dims, strides)'
     [[{{node encoder1/CudnnRNN}} = CudnnRNN[T=DT_FLOAT, direction="unidirectional", dropout=0, input_mode="linear_input", is_training=true, rnn_mode="gru", seed=87654321, seed2=0, _device="/job:localhost/replica:0/task:0/device:GPU:0"](encoder1/transpose, encoder1/ExpandDims_1, encoder1/Const, encoder1/concat)]]
     [[{{node out/Reshape/_55}} = _Recv[client_terminated=false, recv_device="/job:localhost/replica:0/task:0/device:CPU:0", send_device="/job:localhost/replica:0/task:0/device:GPU:0", send_device_incarnation=1, tensor_name="edge_605_out/Reshape", tensor_type=DT_FLOAT, _device="/job:localhost/replica:0/task:0/device:CPU:0"]()]]")
   at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   at System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   at Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   at Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   at Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, CallOptions options) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 96
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 86
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 230
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 234
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.PredictBatches(PredictionServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, IList`1 inputs, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 314
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibraryOrThrow(IProgressMonitor progress, IProgressStatus& progressStatus) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 113
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 69
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Cannot fill in Protein Description field in 'Edit>Insert>Peptides'
(2 responses) shaoen 2020-09-09

Hi,

I would like to fill all three fields (peptide sequence, protein name, protein description) when adding a list of peptides but it seems I can't enter anything in the protein description field. Data in the first two columns work fine. What am I missing?

Skyline version: Skyline-Daily 20.1.9.234

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System.IO.IOException: ERROR: Error parsing modifications file: modifications.xml(line 5662): Missing required attribute 'composition'.
(2 responses) m kuppusamy 2020-09-09

I am trying to load some DDA data on skyline using the import DDA peptide search option. I get an error "System.IO.IOException: ERROR: Error parsing modifications file: modifications.xml(line 5662): Missing required attribute 'composition'." when I try to load the msms.txt file from the Maxquant search. I already have modifications.xml file from maxquant in the same directory as well. Could you please tell me what could be the problem here?

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issue when importing peptide search from a MaxQuant msms.txt file - ignored modifications
(2 responses) marie locard-paulet21061 2020-09-08

Hello,

I would like to use Skyline to generate a spectral library compatible with a library-based DIA search in DIA-NN.
My issue is that when I import an output from MaxQuant (1.6.14) using the "import peptide search" pipeline (msms.txt file in the same folder as the raw files, the mqpar.xml and the modifications.xml containing the list of variable modifications -I added the Ammonium and Ammonia loss in the bottom-), the only modifications that I find in the output are oxidation (M) and carbamydomethylation (C). So several modifications are missing.
I am pretty sure that I miss something but I cannot figure out what.

The idea is to then export a custom report from Skyline that would be in a format that I can make compatible with a DIA-NN input library. This bit works.

I work with Skyline (64-bit) 20.1.0.155 (a0e7323e3).
I have uploaded the MaxQuant outputs I work with on the skyline upload site, with the name "Ticket_MLocardPaulet.zip".
Please let me know if you need more information.
Thank you for your help.
Kind regards,
Marie.

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Bibliospec for spectral library searching
(2 responses) david morgenstern 2020-09-06

Hi,

I'm considering moving to using spectral library searching for glycoprotoemics due to the prohibitive search times (especially for large datasets). apart from the old manuscript - is there any tutorial I can use for it? there are multiple questions I've got regarding the use of the software:

  1. how does FDR performed after the search?
  2. how does it deal with very small database (50 entries) for FDR calculations?
  3. how does the scoring algorithm (as well as the annotator) deal with c/z B/Y spectra from glycopeptides and EThcD/sHCD?
  4. can it look for modifications on top of what comes out of the library?
  5. Can you convert the .blib libraries to .msp?
  6. can it deal with PRM data for library preparation? PRM with separate MS1?

I'd appreciate any resource you can point out to me.

Thanks!
David.

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bibliospec error
(4 responses) dkueltz 2020-09-05

Hi Nick, Brendan,
I have used the integrated bibliospec for many years and never had a problem but now for some files the error below pops up when trying to build a new spectral library. I am using pep.xml + corresponding mzxml files generated in PEAKS Xplus for building libraries. DDA data from two consecutive runs that look similar in terms of peptide and protein coverage and file size give different results. One file works fine for library generation but the other throws this error. I tried this with Skyline v19, v20, and the latest daily. I also tried windows admin and regular user accounts - same result. There is no disk space issue either. I can upload an example pep.xml + mzxml file pair that works and another one that does not work if that would help troubleshoot the issue - let me know.
Thanks,
Dietmar
System.IO.IOException: Error occurred running process.

Command-line: C:\Users\dkueltz\AppData\Local\Apps\2.0\CHO9N7RC.HQC\PCZXTA8K.NOQ\skyl..tion_e4141a2a22107248_0013.0001_e50c2c68eab478d7\BlibBuild -s -A -H -o -c 0.95 -i t -S "C:\Users\dkueltz\AppData\Local\Temp\tmpA47.tmp" "D:\Spectral-Libraries\t.redundant.blib"
Working directory: D:\LR0013_Oremo-Kidney\PEAKS_mzID-mzXml_Export\PEAKS10P__LR0013_Oremo-Kidney_gradual2weeks_PEAKS PTM_42
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 62
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String[]& ambiguous) in C:\proj\skyline_19_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 186
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_19_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 144

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Peaks not found
(2 responses) jessica medina 2020-08-27

Dear Skyline team,

I am trying to process my lipidomics data in positive mode, but some peaks are not found (mostly the ammonium adducts).
I have tried many options but nothing works yet.

I have processed already the same samples from negative mode and everything worked well.

I am using the paramenters and files in the attachment.

Thank you very much for your help

 OPtPOSSCIEX.wiff  OPtPOSSCIEX.wiff.scan  positive.sky  Lipidomics data positive mode.pptx 
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Question about calculating standard errors of mean and CVs for group comparison
(2 responses) roman sakson 2020-09-06

Dear Skyline team,

I did an experiment where I monitored four peptides for a particular protein and afterwards decided to include all transitions for my first peptide but only one quantitative transition for the other three peptides, respectively, when performing the group comparison. I noticed that the peptide with several transitions had a higher significance in the volcano plot compared to others (see volcano_plot.jpg) and I investigated further. When I had a look at the respective CVs for all transitions for that peptide across the two conditions, CV values were terrible (around 90 %, see CVs_Transitions.jpg), which did not agree with the significance in the volcano plot nor with my expectations based on "okayish" to good XICs. However, once I summarized transitions from "all" to "total", overall CVs dropped below 10 %, which would correspond to the volcano plot (see CVs_All.jpg). For the other peptides, where there are no transitions to sum up, I do not see this pattern. This effect is present in the latest daily version as well as in 20.1. I assume that CVs for individual transitions are somehow off. Do you have an idea what the reason might be?

Thank you,

Roman

 Volcano_Plot.jpg  CVs_Transitions.jpg  CVs_All.jpg  200906_Support_Case.sky.zip 
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Array dimensions exceeded supported range error on DIA .raw file import - ELIB from EncyclopeDIA
(1 response) cabarnescabarnes 2020-09-05

Hi all,

I have been doing the gas phase fractionation workflow with EncyclopeDIA to generate an ELIB from the "Save Quant Reports" in EnclyclopeDIA. Then, I import the ELIB generated after that step into Skyline and usually can import .raw DIA files cleanly and look at all the data in Skyline. I'm working on an experiment now, however, which has a lot of transitions (238,774 transitions / 58,099 peptides / 6310 proteins) and I'm importing 36 injections. I've tried the import multiple times with this library and it fails with the below error. However, if I delete all of the peptides down to a single peptide, I can get it to import just fine. When it fails, it looks like it gets all the way to the end and then fails after the visual import status graph looks finished. Is this a memory problem? Or something else?

At 11:47 AM:
Failed importing results file 'C:\TPP\data\smORF_discovery_Experiements_BAT_scWAT_huWAT_C2C12\2020_Beige_BAT_WAT_secretome_proteome\Proteome\20200731_LUM1_CPBA_EASY04_085_30_SA_90mingrad_80B_DIA_400_1000_8mzol_15k_20IIT_4e5agc_1850_02_01.raw'.
Array dimensions exceeded supported range.

Thank you so much!!

Best,

chris

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MS/MS Spectrum screen
(1 response) jfarrera 2020-09-04

Dear Skyline team,

I'm new on Skyline, I have started working with Skyline recently doing peptide sequencing. Till now I have created some MRM methods with Skyline (v. 20.1.0.155, a0e7323e3) which I could run in a triple quadrupole LCMS8040 (LabSolutions, Shimadzu). I was able to open and process the acquired MRM data in the Skyline platform, but I was not able to open a MS/MS spectrum with Skyline. Is it possible to open in Skyline a MS/MS spectrum from a triple quadrupole LCMS8040 (Shimadzu)? When I open the View menu, the MS/MS Spectra option is not available (is not in the menu). Any idea?

Thank you very much for your support!! Best regards,

Josep

 Skyline screen.odp 
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Spectral library export
(3 responses) Juergen 2020-09-02

Dear Skyline team,

recently I used Skyline to build a spectral library from mascot search results (.dat files). However, I want to use the very same library also in spectraST later. Is there any way to convert the blib format to splib? I remember, that some years (~10) ago it was possible to import these libraraies into spectraST directly. But it seems that this support was stopped in the meantime. If a direct conversion is not possible, perhaps there is a way to convert the library to an intermediate format that you are aware of?

Best regards,
Juergen

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Wrong position of amino acids?
(3 responses) fcsigloch 2020-01-13

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

 skyline_aa_numbering.JPG 
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Small molecules Bracketed calibration and data consolidation
(1 response) anders honore 2020-09-02

Hi,

Hoping you could point me to webinars/tutorials to assist me in solving the following questions

  • How to (conveniently) import concentration data for calibration for multiple components?

  • How to perform bracketed calibration , i.e. applying the repeated inj. of calibration solutions surrounding a given set of unknowns in sequence?

Finally:

  • How to consolidate technical replicates after calculations, i.e such calculated amt/conc in unknowns replicates A, B, C are reported as either mean or median and stdev?

Fingers crossed that your advise will help me cut some corners in a complex method implementation

BR,
Anders

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Spectrum missing
(7 responses) cunain1gx 2020-08-31

I am using Mascot DAT file to build a library in skyline. I can find a MS/MS spectrum for one peptide in mascot search result, but cant find this peptide spectrum in the skyline library. As I am trying to extract transition for this peptide from skyline, now it just not showed in the library. Should I change any parameters or anything? Because the spectrum can be observed with mascot search result. Thank you

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Small molecule (non-global) standard concentrations
(2 responses) jrenders 2020-09-01

Hi there,
Thanks so much for adding the ion ratio features to skyline - we are currently rebuilding all of our small molecule methods in skyline and have unfortunately hit a snag. I think you previously made mention of (in the future) adding the ability to assign calibration curve regression and weighting on a per-analyte basis. Right now, via the "Document grid --> Reports --> Replicates" report we can assign analyte concentrations, but only globally. However, we have methods where our calibrators do not have all analytes at the same concentration. Is there plans to allow for a per-analyte concentration assignment as well in the future? Or perhaps there is already a way to handle this via the document grid or pivot table? Thanks for any guidance you can suggest.

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Isotope labels on some residue
(2 responses) Vincent Lacasse 2020-08-31

Hello Skyline team,

I'm currently preparing an MRM assay using an internal calibration curve with two internal standards spiked-in a different concentrations. Although I was able to add my different labels to my skyline file, some of the residue of the same amino acid are labelled and not others. For instance:

SQPGQDCR
The first "Q" is not labelled, but the second "Q" is labelled. I have 28 different peptides and there are multiple instances where this problem shows up.

How can I tell Skyline to label only some of the amino acids? I've also tried to put it as a variable modification on the protein, however it does the same thing, it labels both amino acids.

Thank you and have a nice day!

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Error when copy + pasting a peptide list into Skyline daily
(5 responses) roberthardt 2020-08-28

Dear Skyline-Team,

if I want to insert peptides via "Edit - Insert - Peptides" in Skyline Daily (20.1.9.234, 77dodc4a4) the software always freezes. In contrast, if I do exactly the same in the current public Skyline release (20.1.0.155, a0e7323e3) it works without a glitch.

Regards

Robert

 pep_import_freeze_daily.pptx 
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Stable isotope tracing experiments: Is is possible to change the order of isotopologues in the peak area replicate comparison?
(2 responses) Michi 2020-08-27

Dear Skyline team,

Thank you so much for all your effort, I don't know what I would do without Skyline anymore. I recently discovered that you also implemented new plots for the peak area replicate comparison that allows you to directly see the labeling patterns when doing stable isotope tracing experiments in the format I'm used to. It works very well for small metabolites. However, when looking at compounds with more than 10 carbons, the ordering of isotopologues is not by mass, but alphanumeric?, i.e. it shows the M-H, then the M10C13-H, M11C13-H,...., and only at the end the M1C13-H, M2C13-H, etc.

Is there an option to sort these differently? I need to export the data to do natural isotope abundance correction and plot the different isotopologues as fractions afterwards anyway, but it would be very helpful to see them in the right order in Skyline already. It is currently confusing if you don't look very closely and realize how they are ordered.

I attached an example.

Thanks,
Michi

 Skyline_isotopologue_order.PNG  Isotopologues_order_example.sky 
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peak integration
(6 responses) monasharar 2020-08-26

Good evening,

I could see that for samples if they have 4 min peak width for example, some of them all the peak appears
while for others there is a cut, so first min appears then the line stops in the middle.
Manual peak integration cannot do anything other than adjusting boards, while if I view TIC for the run I cannot do manual integration.
Can you please advice ?

Thank you

 Skyline.png 
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Problems with import of peak boundaries in smallmolecule mode for .wiff data
(2 responses) Max 2020-08-24

Hi,

i am trying to import peak boundaries for a number of data sets based on .wiff data. We have set up some small molecule transitions for small molecules and peptides (due to qunatifiaction porpuses). I have used the allready discussed workarounds for small molecules (see https://skyline.ms/issues/home/issues/details.view?issueId=671) which is perfectly working for data from e.g. waters were a single filne represents a single injection. However, in the case of AbSciex 5500 the data is stored in a single .wiff file. I added therefore the sample name (alias Sample identifier) column to the peak boundaries list as stated in the FAQ for importing peak boundaries (see here: https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/ImportingIntegrationBoundaries-2_6.pdf). Nevertheless, skyline gives me an error message back stating that the sample names are not fitting to the sample names in the raw file . We have not changed the names of the single sample upon importing in Skyline thus i am looking for help to resolve this. I know i am missing something but i can not figure it out....

Looking forward to hear from you guys!

Max

PS: I have example files ready but can only send them confidentally.

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DDA Spectral Library building from Thermo Lumos ,
(1 response) sray 2020-08-24

Hello All,

I'm building a spectral library from DDA peptide search results generated on Thermo Lumos.

In "Configure MS1 Full-Scan Settings" section, under "precursor mass analyzer" , which option is better, "Centroided" or "Orbitrap" (with corresponding resolution settings) ?

Thanks

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DIA overlap with margin demultiplex in MSConvert
raghuram 2020-08-24

Dear Skyline team,

I had issues demultiplexing overlap with margin in MS convert. Is there any other open-source code that I can use to demultiplex overlap with margin files. And also is there a way to control demultiplexing by providing the isolation scheme?

cheers,
Bharath

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IMS Predictor questions
(2 responses) Juan C. Rojas E. 2020-08-21

Dear Skyline team,

I have a few questions I hope you can help me clarifying.

  1. How does Skyline determine the CCS area from WATERS HD files? What equation is implemented?

I checked at some point if the CCS areas reported in Skyline matched the ones calculated with Driftscope and I noticed slight differences. Maybe due to small differences in the application of the calibration equation stored in the files?

  1. When the IMS predictor is run automatically with "Use Results" it will attempt to only use the areas inside of the RT boundaries?

I import my DIA data with small retention time windows to limit false integrations and then run the predictor. However, after running the predictor I noticed that the EICs show the full chromatogram and not only the small retention windows imported in beginning.

  1. Is there a bias for optimizing the IMS window of the fragments (i.e. high energy scan) above the one for the precursors (i.e. low energy scan)? Or is it some sort of weighed contribution?

I have tried using the automatic predictor and very often its determining incorrect offset estimations. In the image attached you can see that when automatic is ran it usually ends up with lots of positive offsets which is the complete opposite of what is expected from associated fragment ions in the HDMSE high energy scans. This happened because it chose same fragment ions that came from other co-eluting or closely eluting molecules.

Of course, with my manual corrections I still had some mistakes due to badly curated peaks, but at least the bias is towards negative offsets for most of them. I should mention that I use a low resolving power of 15 since is the value the encompasses all area for most of my molecules with my current instrument settings and analytes relative abundance.

Sorry if these were too many questions for one post, but I thought they were all related.

Sincerely,
JC

 Offset estimations.png 
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Export of Transition List and Method for Sciex 6500+ broken
(13 responses) Will Thompson 2020-08-19

Hi Brendan et al

Export of transition lists and of methods for Sciex 6500+ is not currently working in Skyline -daily or 20.x build, meaning method development is not possible on this instrument. Specifically this is the case for negative ion small molecule data. Some problems may be specific to that use case, but others seem generic. Not sure how long this has been going on, we have not done any routine method development on this system in a while. There are a number of issues. Recognized so far:

  1. Skyline will not allow a negative collision energy or declustering potential. These are required for negative ion acquisition for Sciex. When you paste in a negative CE or DxP using the import or Ctrl+V method, Skyline does not give an error, but it just puts nothing in the appropriate fields.
  2. Even if you change the polarity of the CE in Skyline to (the incorrect) positive value, then you export an acquisition method, the method contains a random negative number instead of the CE that was programmed.
  3. Even if you change the polarity of the CE in Skyline to (the incorrect) positive value, then you export a transition list, the TL is the incorrect format and Analyst cannot import it.

I hope you can reproduce these errors with the attached documents but if not we can set up a Zoom call for me to walk through in more detail. Please let me know if you need more information.

Cheers

Will

 forSkylineSupport.sky.zip  forSkylineSupport.xlsx 
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How can I select endogenous peptides not spiked-in iRT peptides for RT nomalization in Skyline?
(1 response) andyzcq 2020-07-23

I have acquired diaPASEF data, but I forgot to spike iRT peptide into the samples.

So how can I select endogenous peptides for RT normalization?

CiRT peptides in the CiRT calculator are not applicable.

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Applying manual integration windows when adding new transitions/fragments for an existing compound in Targets
(6 responses) Emmanuel 2020-07-27

Dear Skyline team,

I've manually adjusted the integration of a targeted small molecule across a dozen of samples and I'd like to add few more transitions/fragments for that compound (additional fragments found in another database than the first ones).

Is there a simple way to apply the manual integration boundaries to the new fragments without using the workaround of exporting/re-importing the peak boundaries as a report (as suggested by Tobi some years ago)?

When re-importing the results, it seems that the new transitions are integrated according to the default integration settings without taking into account the manual integration from the previous transitions. Therefore, I need to manually re-integrate all the samples ....

Thanks in advance for your help.

Emmanuel

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MS Amanda DDA search
sstoychev 2020-08-21

Hi Skyline team,

I'm trying to do a DDA search but a following error pops up during method set-up:

Skyline version: 20.1.9.233-4850f5574 (64-bit)
Installation ID: ed56f5a5-04ae-49bc-bfc9-021aa0ce9225
Exception type: Exception
Error message: Obo files (psi-ms.obo and unimod.obo) not found


System.Exception: Obo files (psi-ms.obo and unimod.obo) not found
at pwiz.Skyline.Model.DdaSearch.MSAmandaSearchWrapper..ctor() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\DdaSearch\MSAmandaSearchWrapper.cs:line 88
at pwiz.Skyline.FileUI.PeptideSearch.ImportPeptideSearchDlg.NextPage() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\FileUI\PeptideSearch\ImportPeptideSearchDlg.cs:line 565
at System.Windows.Forms.Control.OnClick(EventArgs e)
at System.Windows.Forms.Button.OnClick(EventArgs e)
at System.Windows.Forms.Button.OnMouseUp(MouseEventArgs mevent)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.ButtonBase.WndProc(Message& m)
at System.Windows.Forms.Button.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
Exception caught at:
at System.Windows.Forms.Application.ThreadContext.OnThreadException(Exception t)
at System.Windows.Forms.Control.WndProcException(Exception e)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Form.ShowDialog(IWin32Window owner)
at pwiz.Skyline.SkylineWindow.ShowImportPeptideSearchDlg(Nullable`1 workflowType) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 3006
at pwiz.Skyline.SkylineWindow.importPeptideSearchMenuItem_Click(Object sender, EventArgs e) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 2988
at System.Windows.Forms.ToolStripItem.RaiseEvent(Object key, EventArgs e)
at System.Windows.Forms.ToolStripMenuItem.OnClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleClick(EventArgs e)
at System.Windows.Forms.ToolStripItem.HandleMouseUp(MouseEventArgs e)
at System.Windows.Forms.ToolStrip.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.ToolStripDropDown.OnMouseUp(MouseEventArgs mea)
at System.Windows.Forms.Control.WmMouseUp(Message& m, MouseButtons button, Int32 clicks)
at System.Windows.Forms.Control.WndProc(Message& m)
at System.Windows.Forms.ToolStrip.WndProc(Message& m)
at System.Windows.Forms.ToolStripDropDown.WndProc(Message& m)
at System.Windows.Forms.NativeWindow.Callback(IntPtr hWnd, Int32 msg, IntPtr wparam, IntPtr lparam)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.UnsafeNativeMethods.DispatchMessageW(MSG& msg)
at System.Windows.Forms.Application.ComponentManager.System.Windows.Forms.UnsafeNativeMethods.IMsoComponentManager.FPushMessageLoop(IntPtr dwComponentID, Int32 reason, Int32 pvLoopData)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoopInner(Int32 reason, ApplicationContext context)
at System.Windows.Forms.Application.ThreadContext.RunMessageLoop(Int32 reason, ApplicationContext context)
at pwiz.Skyline.Program.Main(String[] args) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Program.cs:line 306

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Skyline and QTRA6500-CE and DP optimization method export
(1 response) qin fu 2020-08-19

Dear Skyline team,
when I tried to export CE or DP optimization method using QTRAP6500 setting, I have encountered the error messages, enclosed here. My guess is the choice of the instrument in the transition setting, I must have gotten some details wrong in the setting. What did I do wrong?
Thanks
Best
Qin Fu

 6500QTRAP_CV optimizationwitherrormessage.pptx 
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Retention time shift
(15 responses) yulun 2017-04-28
Hi all,
I run some standards (metabolite) in different runs and would like to analyze by Skyline. Please see the attachment. The most right panel is the correct peak identified (~13.5 min) by the Skyline. However, the most left (1.5 min) and the middle (22.8 min) ones return the wrong retention time. I know the Skyline did the good job to pick the highest peak. My question is: Is there any possible way from setting to get rid of these 2 peaks identification since it is out of rt windows (0.2 min for rt window)?
Thank you
Yulun
 RT_shift.png 
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DIA TIC does not match DDA
(4 responses) Mark Athanason 2020-08-14

Hi again Brendan,

I know this is not a skyline question, but since I followed your tutorial for setting up the DDA and DIA methods in Xcalibur I figured I would ask here. In the past this worked perfectly well with a 2 hour gradient, but now I'm running the two methods as a one hour gradient and something funky is going on with the DIA acquisition. The DIA and DDA chromatography parameters are exactly the same, and the DIA instrument method is as shown in your tutorial. I have attached the two method files along with a screen shot of the TICs to demonstrate what I am talking about. If you can see something I'm obviously doing wrong, I would be very appreciative. This is being run on a nanoAquity LC with a picochip spray source coupled to a Thermo QE HF mass spec.

Best
Mark

 ms_DDAvsDIA.PNG  DIA_400to1260by20_60min_trapping.meth  DDA_top20_60min_trapping.meth 
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Calibration curve X axis Analyte Concentration/Light:Heavy con ratio
(3 responses) Zac 2020-08-14

I have calibartion curve data with heavy labelled peptides for normalization. For most peptides the x-axis of the calibration curve is Analyte Concentration but for one peptide this changes to Light:Heavy concentration ratio. Not sure how to standardize all peptides to show Analyte concentration, the X-axis values are also different.

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Skyline not recognizing in house Labkey server as 'Panorama server'
(5 responses) mlane 2020-08-13

Hi,
We're using Skyline-daily 20.1.1.175 and just set up new Community edition of Labkey so not sure if this is Labkey or Skyline issue. I can directly connect to Labkey server through browser, log-in fine and create Panorama folders, etc. However, when I try to add server to be recognized in Skyline, an error comes back on the url that it is not recognized as Panorama server.
See attached for url and error.

Thanks!
Monica

 Labkey_error_08132020.pdf 
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An error occurred attempting to install Skyline
(1 response) shobhabr20 2020-08-12

Hi Skyline team,

I get an error when trying to install skyline daily and I've included the install.log file.

Skyline-daily 20.1.1.213 (https://skyline.ms/project/home/software/Skyline/daily/begin.view?)

I am installing skyline daily onto our lab server. If the error is stemming from our end, please let me know so I can tell my IT person.

Thanks,
Shobha

 Installation_Error.png  install.log 
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Feature detection capability for small molecules
(1 response) FC 2020-08-13

Hello Skyline team,

I am new to Skyline and would like to know if Skyline has the capability to detect features/peaks from DIA-MS/MS data for small molecules without first going through a compound identification process. I am interested in using these features/peaks to build a spectral library. Thank you!

Kind regards,
FC

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Peaks integration with different boundaries between light and heavy
(3 responses) Anne Incamps 2013-11-06
Hi Brendan

I don't manage to integrate manually light and corresponding heavy peptides with different boundaries.
If I clic on the light peptide and integrate the right peak, it automatically shift the same boundaries for the heavy one. I cannot set different boundaries for light and heavy. How can I sort this out ?

Thanks for your answer,

Anne
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Exclude counting of decoys from prot, pept, precurs, transitions numbers in target list
(2 responses) dkueltz 2020-08-06

Hi Brendan et al.,
When constructing DIA assay libraries from raw spectral libraries by sequentially applying QC filter criteria my lab is routinely recording the numbers of proteins, peptides, precursors, and transitions remaining after each filter step. We get these numbers from the display on the lower right hand corner of the skyline app. However, these numbers currently include decoys and we have to delete the decoys and then paste them back tp the target list after every filter step. It would be nice to not have to do that. So I am asking if you could program Skyline such that it excludes decoys automatically from the display in the lower right hand corner of the screen.
Thanks,
Dietmar

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(Loss of) resolving power when importing mzXML files from HRAM QTOF
(8 responses) anders honore 2020-08-04

Hi
Working with small molcules in MS1 from Bruker QTOF instrument. Data is stored as centroided and converted to mzXML (by Bruker converter) prior to upload. Resolution is typically +40,000.
After import resolving power is reduced to <10,000 and otherwise obvious peaks when reviewed e.g. in MZmine2 becomes severely distorted. Appears as though resolution is reduced to <<10,000
Transition Settings: precursor as monotopic, full scan count, peaks 3, precursor mass analyzer TOF, resolving power 50,000
Include all matching scans

Probably error-40 by a novice user - but please advise...

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PTM annotation
(2 responses) rajirathore3 2020-08-06

Hi Skyline Team

I am working on Glycation (a PTMs) of selected peptides of Albumin protein where I look for different types of glycation modifications of each peptide but when I add them in workflow it sums up the modifications shown in attachment. I have tried first with one of the representative peptide KVPQVSTPTLVEVSR where I specified 3 different glycation modifications on first lysine site. I added them separately in modification list and I was expecting it to be annotated separately but it has summed up two different lysine modifications and which does not meet my experimental workflow.
I want it to be annotated like K(58)VPQVSTPTLVEVSR and K(72)VPQVSTPTLVEVSR not K(130)VPQVSTPTLVEVSR. Please suggest me how this can be performed.

Thanks
Rajeshwari

 modification issue_SKY.pptx 
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Assigning peak colors for small molescules
(3 responses) sarah williams 2020-08-06

Hello,

I am working on a project with tmt labeled small molecules. I would like to assign colors for the MS1 peaks I have assigned for each of the small molecules and their tag. I noticed there is a tutorial for this for peptides, but I can not seem to make it work for my small molecules.

 Colors Peaks skyline support.JPG 
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Prosit - error
Ihor 2020-08-06
Hi,

1) I am trying to build a Prosit library in Skyline v.20.1.0.155 and I get the error message (copied below). This error message also pops up on diff. computer systems and Skyline files. Please note that when I click on Options-->Prosit it does mention 'Server Unavailable' (proteomicsdb.org:8500) - is this related to the error?
2) Prosit is able to predict optimal (N)CE based on synthetic peptide library - is Skyline able to use that information from Prosit and suggest the best NCE for each peptide?

---------------------------
Skyline
---------------------------
Status(StatusCode=Unavailable, Detail="failed to connect to all addresses")
---------------------------
OK More Info
---------------------------
pwiz.Skyline.Model.Prosit.PrositException: Status(StatusCode=Unavailable, Detail="failed to connect to all addresses") ---> Grpc.Core.RpcException: Status(StatusCode=Unavailable, Detail="failed to connect to all addresses")
   at System.Runtime.ExceptionServices.ExceptionDispatchInfo.Throw()
   at System.Runtime.CompilerServices.TaskAwaiter.HandleNonSuccessAndDebuggerNotification(Task task)
   at Grpc.Core.Internal.AsyncCall`2.UnaryCall(TRequest msg)
   at Grpc.Core.DefaultCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.<BlockingUnaryCall>b__3_0[TRequest,TResponse](TRequest req, ClientInterceptorContext`2 ctx)
   at Grpc.Core.ClientBase.ClientBaseConfiguration.ClientBaseConfigurationInterceptor.BlockingUnaryCall[TRequest,TResponse](TRequest request, ClientInterceptorContext`2 context, BlockingUnaryCallContinuation`2 continuation)
   at Grpc.Core.Interceptors.InterceptingCallInvoker.BlockingUnaryCall[TRequest,TResponse](Method`2 method, String host, CallOptions options, TRequest request)
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, CallOptions options) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 96
   at Tensorflow.Serving.PredictionService.PredictionServiceClient.Predict(PredictRequest request, Metadata headers, Nullable`1 deadline, CancellationToken cancellationToken) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\ProtocolBuffers\GeneratedCode\PredictionServiceGrpc.cs:line 86
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 230
   --- End of inner exception stack trace ---
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.Predict(PredictionServiceClient predictionClient, TPrositIn inputData, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 234
   at pwiz.Skyline.Model.Prosit.Models.PrositModel`6.PredictBatches(PredictionServiceClient predictionClient, IProgressMonitor progressMonitor, IProgressStatus& progressStatus, SrmSettings settings, IList`1 inputs, CancellationToken token) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\Models\PrositModel.cs:line 314
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibraryOrThrow(IProgressMonitor progress, IProgressStatus& progressStatus) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 113
   at pwiz.Skyline.Model.Prosit.PrositLibraryBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Prosit\PrositLibraryBuilder.cs:line 69
---------------------------

Thank you
Ihor
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Software crashing while importing result files
(3 responses) alaine garrett 2020-08-05

Hi,

I am currently working through the tutorials and have had the software crash during the import of results steps in both the Targeted Method Refinement and Processing Grouped Study Data tutorials. I am able to open the raw data files in Qual Browser so I believe that it is not a function of the raw data files being corrupted. We did have a difficult time getting the software installed on our off line system.

Any assistance would be greatly appreciated.

Thank you

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Max LOQ bias calculation
(2 responses) Zac 2020-08-06

Is there any documentation on how the Max LOQ bias is determined, or can you it explain it briefly? Thanks

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Manuscript on Biotin Acceptor Peptide
akulyyasov 2020-08-06

Dear Skyline/Panorama team and users,
I have uploaded the graphical abstract of the manuscript which I prepared during lockdown time.
In this paper, I summarized the application of Biotin Acceptor Peptide 1070 which is used in proximity utilizing biotinylation method.
The paper contains the description of its design, construction, and quantitation by Skyline software on the example of DNA dependent interaction of Sox2 and Oct4.
If someone would like to read the text of the article, please send a message to my e-mail kulyyasov@biocenter.kz
I will be very grateful for any help in improving the text, remarks, and comments on this manuscript.

With best regards,
Arman

 JP_BAP_graphical_abstract.png 
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Issue downloading skyline daily
(1 response) wlstutts 2020-08-05

Hi,

I receive an error message when trying to download Skyline Daily. Any suggestions? I have tried two different computers. Thanks!

 skyline.PNG 
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Ion Mobility Window Type settings with Bruker timsTOF data
(1 response) Sebastian Winter 2020-08-05

Hi,

I have experienced trouble with the latest Skyline Daily version 20.1.1.213 when exporting a method for Bruker timsTOF. Regardless of the parameters entered for the "Window Type" under "Ion Mobility" in the Transitions Settings, the exported targets are always isolated with a 1/K0 window of 0.4. Setting different values for "Fixed Width" or"Resolving Power" have no effect on the exported window size.
Does anyone have an idea what could be wrong?

Best,

Sebastian

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Waters HDMSE Raw Data Visualization
(14 responses) brad williams 2020-04-13

I have been testing the latest Skyline builds (20.1) and Skyline Daily (20.1.1.83) and they both do not allow the raw data to be visualized for MS1 precursors. The raw data plot shows up blank and the MS/MS information is there. Has this been reported before? I have attached a screen capture to show an example. The datafile type is HDMSE.

 MS1 Spectrum Viewer_041320.pptx 
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Time - course analysis: data export
(1 response) david morgenstern 2020-08-04

Hi,

I'm running a time course analysis of a certain compound - and i have a quantification trace for that compound through time.
how can export the intensity value (i.e. height) for each sampled time point? is it possible to generate a smoothed trend line and export those values?

Cheers,
D.

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Skyline truncating small molecule peaks that appear to have more data...
(8 responses) jrenders 2020-07-17

Hi There,
I am running a small molecule calibration curve using skyline. On my instrument, I am acquiring data via an inclusion list, so MS1 data matching to my list triggers an MS2 event for that mass. This inclusion list is also scheduled but my RT's are set correctly with 1.5 min of buffer on each side of the peak (on a 10 min method - so a pretty wide window).

When I import the data into skyline I see all expected peaks but often times the leading edge of the peak is highly truncated. This truncation often throws an error which can prevent proper integration and calibration ("MS1.PNG") - "The selected replicate has missing or truncated transitions". I am able to "rescue" these peaks using a trick Brendan mentioned in a similar thread (by bumping the integration boundary inward), however, this truncation is an error as far as I can tell because when I interrogate the data in a vendor spectral viewer I can see that there is data that doesn't seem to make it into skyline. I have attached an image from the vendor software ("Truncated_Peak.PNG") which shows that this mass appears (within the mass tolerance) in the MS1 dimension at a RT that is not properly tracked by skyline in the MS1 dimension. I am thinking I may have some setting set too strictly which is causing the peak to not be picked up until part way through it's elution?

I have attached the skyline document .zip here. In this document I am quanting using both MS1 and MS2 data. Most of the truncation occurs in the MS1 dimension (see for example the 1000 cal point which throws an error). I also see some strange integration in the MS2 dimension ("MS2.PNG") where it appears skyline is trying to "connect" data points that are separated by a long stretch of time (see for example the 500 cal point) and therefore not properly allowing the peak to discontinue or return to baseline. This also appears to me to be related to peak truncation and an improper parsing of data that I believe is actually present in the raw file and which would make these peaks look more normal...

Thanks for any help you can provide!

 20200717_Skyline_Truncated_Peaks_Error.sky.zip  MS1.PNG  Truncated_Peak.PNG  MS2.PNG 
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SMILES InChi in Reports
(14 responses) Christina lucas 2020-07-27

Hi there,

maybe I am to lost in all the report possibilities...:)

In SM Mode, you can enter SMILES and Inchi/InchiKey, HMDB and all this...

If I want to add it to my Report, where can I find it in the Report Editor?
I had several guesses, thing what it might be in "Proteinlanguage" , like Proteinname for Molecule Name e.g., but Proteinsequence and so on did not work...

I would be very grateful for a tip :)
Best, Christina

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LC-MS/MS data file input for Skyline 20.1.0.155
(3 responses) avaldiviezo 2020-07-29
I've been using Skyline for a few months to analyze data files generated by Ion Mobility - Mass Spectrometry and the software works very well. Recently, I have been trying to use Skyline to analyze data generated by Liquid Chromatography - Tandem Mass Spec, but I cannot see any peaks when I upload the data files after importing my transition list. Is there a particular way I need to set up the molecule/transition settings or transition list settings in order to analyze LC-MS/MS data?

Any help with this issue is greatly appreciated!

Thanks,
Alan
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Skyline Access Error
(2 responses) diana saggese 2020-07-29

I cannot access Skyline documents made using a previous version. Could I please get access to these files?

Thanks!

 Skyline error.PNG 
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ERROR: illegal character _ found in sequence [xxxxxxxx].... (line xxx)
(2 responses) siljebt 2020-07-26

I´m experiencing a problem using MaxQuant v1.6.14 and Skyline v20.1.0.155, trying to build a library in Skyline using msms.txt files from MQ. The message I get states "ERROR: illegal character _ found in sequence [ ].... (line xxx)". I see that the message refers to a "_", but I cannot find it.

Skyline import /was/ working nicely with the most conventional PTMs (oxidation, carboxylation, etc), but when I built on with additional PTMs in my MQ search the problem arose. It at first seemed like the problem arises when deamidation and amidation occur in the same peptide, but after manually deleting the lines that are causing problems - one by one, it seems like my initial assumtions about the cause for the error is not the case.

 Annotation 2020-07-26 184546.png 
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histone quantification
(8 responses) mariette matondo 2020-07-22

Hi,
I like to do some quantification of modified peptides with the Skyline software.
For identification I use the MaxQuant software v1.6
To quantify my data, I import the msms.txt from the MaxQuant search into Skyline I also paste the modification.local.xml file from MQ.
But I cannot import my peptides
in attached files the msms files and modification.local. xml file

Could you please help me

Many thanks,
Mariette

 msms.txt  modifications.local.xml 
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Intermittent Error: Could not connect to Skyline
(1 response) mmarx 2020-07-22

I am running the Agilent Automation tool and intermittently I am getting the error message when I click 'Create Project':
Error: Could not connect to Skyline.
Make sure you have a valid Skyline installation.

It usually occurs after Skyline and the Automation tool have been idle for a period of time.
Sometimes, if I just try again it will work. Other times, the error continues to occur.

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Proteomics data processing into sample vs protein intensity matrices
(3 responses) saharak 2020-07-19

Hi,

I am interested in downloading and processing proteomics data into sample vs protein intensity matrices for various disease types that include healthy control samples.

For the datasets that have been processed with skyline, I found that there is a viewer for windows, however, I wasn’t able to find any library to parse them. How do you recommend that I process them into sample vs protein intensity matrices?

I also found "MSstats" package::SkylinetoMSstatsFormat and lipidr::read_skyline, however both require sky csv files and I am only able to download .sky file. Where can I find the csv format and if I can't directly download it, how do I process the .sky files into csv files?

Also, where would I be able to obtain information on each sample, e.g. whether diseased or normal?

Thank you

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AutoQC upload on Panorama issue
(2 responses) Cedric 2020-07-21

Hi,

I am using AutoQC for uploading my Waters file on Panorama (local installation). For most of my analysis it is working well, but for some project, AutoQC cannot update the results in Panorama. I got the log error message attached. What should I do to fix this?

Thanks in advance for your help. Kind regards

Cédric

 p174_SST_Odin_Followup_2020-07-21_14-15-07.sky_2020-07-21_14-15-08.log 
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small feature improvement
(1 response) Tobi 2020-07-13

Hi Skyline Team,

hope you had a good start into the week.

The M-1 chromatogram feature is so valuable but heavily underutilized as the user needs to click several times for each single precursor to enable it. Also, its quite a hidden feature.

Can you please add the option on having it enabled for all Precursors, for example by writing "M-1" or sth. else in the window next to p, y, b under Transition filter settings?

With best wishes,
tobi

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failed attempting to check for an update
(4 responses) andyzcq 2020-07-21

The details about the failure are shown below.

System.Deployment.Application.DeploymentDownloadException: 下载 file:///C:/Users/zcq/Downloads/Skyline-64_20_1_0_76/Skyline.application 未成功。 ---> System.Net.WebException: 未能找到路径“C:\Users\zcq\Downloads\Skyline-64_20_1_0_76\Skyline.application”的一部分。 ---> System.Net.WebException: 未能找到路径“C:\Users\zcq\Downloads\Skyline-64_20_1_0_76\Skyline.application”的一部分。 ---> System.IO.DirectoryNotFoundException: 未能找到路径“C:\Users\zcq\Downloads\Skyline-64_20_1_0_76\Skyline.application”的一部分。
at System.IO.__Error.WinIOError(Int32 errorCode, String maybeFullPath)
at System.IO.FileStream.Init(String path, FileMode mode, FileAccess access, Int32 rights, Boolean useRights, FileShare share, Int32 bufferSize, FileOptions options, SECURITY_ATTRIBUTES secAttrs, String msgPath, Boolean bFromProxy, Boolean useLongPath, Boolean checkHost)
at System.IO.FileStream..ctor(String path, FileMode mode, FileAccess access, FileShare share, Int32 bufferSize, FileOptions options, String msgPath, Boolean bFromProxy)
at System.Net.FileWebStream..ctor(FileWebRequest request, String path, FileMode mode, FileAccess access, FileShare sharing, Int32 length, Boolean async)
at System.Net.FileWebResponse..ctor(FileWebRequest request, Uri uri, FileAccess access, Boolean asyncHint)
--- End of inner exception stack trace ---
at System.Net.FileWebResponse..ctor(FileWebRequest request, Uri uri, FileAccess access, Boolean asyncHint)
at System.Net.FileWebRequest.GetResponseCallback(Object state)
--- End of inner exception stack trace ---
at System.Net.FileWebRequest.EndGetResponse(IAsyncResult asyncResult)
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
--- End of inner exception stack trace ---
at System.Deployment.Application.SystemNetDownloader.DownloadSingleFile(DownloadQueueItem next)
at System.Deployment.Application.SystemNetDownloader.DownloadAllFiles()
at System.Deployment.Application.FileDownloader.Download(SubscriptionState subState, X509Certificate2 clientCertificate)
at System.Deployment.Application.DownloadManager.DownloadManifestAsRawFile(Uri& sourceUri, String targetPath, IDownloadNotification notification, DownloadOptions options, ServerInformation& serverInformation)
at System.Deployment.Application.DownloadManager.DownloadDeploymentManifestDirect(SubscriptionStore subStore, Uri& sourceUri, TempFile& tempFile, IDownloadNotification notification, DownloadOptions options, ServerInformation& serverInformation)
at System.Deployment.Application.DownloadManager.DownloadDeploymentManifest(SubscriptionStore subStore, Uri& sourceUri, TempFile& tempFile, IDownloadNotification notification, DownloadOptions options)
at System.Deployment.Application.DeploymentManager.BindCore(Boolean blocking, TempFile& tempDeploy, TempDirectory& tempAppDir, FileStream& refTransaction, String& productName)
at System.Deployment.Application.DeploymentManager.Bind()
at System.Deployment.Application.ApplicationDeployment.CheckForDetailedUpdate(Boolean persistUpdateCheckResult)
at pwiz.Skyline.UpgradeManager.AppDeploymentWrapper.CheckForDetailedUpdate() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\UpgradeManager.cs:line 339
at pwiz.Skyline.UpgradeManager.updateCheck_DoWork(Object sender, DoWorkEventArgs e) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\UpgradeManager.cs:line 103

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Isotope ([M+1], [M+2])
(2 responses) dbergmann 2020-07-22

What could be the issue for precursor isotopes ([M+1], [M+2]) not to show up at all? Filter is set up okay as well as full scan in my opinion; could there be any other setting that might be missing or a missing column whenever transition list is uploaded?

 Test1.sky 
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Avoidance of missed cleavages, methionine, cysteine
(2 responses) ddickerson 2020-07-20

I understand that proteases with high numbers of missed cleavages are to be avoided if using Skyline. Also, peptides which contain methionines or cysteines, which can be post-translationally modified, are also to be avoided. I was asked to work on a project which involved trypsin+Glu-C digested peptides (with high numbers of missed cleavages), and also many peptides which contained methionine and/or cysteine. A lab mate had already done some work on samples of such peptides using DDA, and he asked me to do the PRM. The samples were in quadruplicate. My results gave similar results to his, and many of the peptides detected by PRM had coefficients of variation <.05 for the 4 repeats, and the differential expression volcano plots showed many peptides had significant p-values. I used 9 maximum missed cleavages for digestion and included a background proteome with 9 max missed as well. The results seem plausible, and the corresponding trypsin-only digests gave far fewer results. Can you please confirm whether I need to throw away this data.

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Base Peak not showing in 20.1.0.155 but visible in 4.1.0.11714
(2 responses) H.Schmidt 2020-07-20

Dear Skyline community,

I have FullScan-PRM data from a QExactive.
Importing those to Skyline 20.1.0.155 let me see the TIC but give me back a "No base peak chromatogram found" when selecting Transitions-->Base Peak.
However, Skyline version 4.1.0.11714 shows me the Base Peak of the very same data with Transitions-->Base Peak.
MS1 filtering is setup for both Skyline files in the same way.

I would like to know how I receive the Base Peak in the actual Skyline version.

Best regards.

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Peak area of molecular contaminant during specified time window
(1 response) lbergluecke 2020-07-17

Hello,

Is is possible to look at peak areas (in the replicate comparison view) for a specified time window ? I am interested to see what a molecular contaminant's peak area is during the gradient not the wash.

Let me know if my question does not make sense and I will try to explain better.

Thanks,
Linda

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Skyline document with cross-linked peptides
(9 responses) danielacgranato 2020-06-27

Sorry, I forgot to attach the documents in the prior message (pLINK output and prox.xml converted). Thank you very much. Best, Daniela

 plink_12_maio_2020.zip  proxl.xml_convert.zip 
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Transition export for small molecule CE optimization not working
(2 responses) stolltho 2020-07-14

Hi sky team.

I followed the CE opt tutorial, but the transition export does not seem to work for me. It's only exporting transitions for one (the lowest) CE.
Could you please check?

Cheers,
Thomas

PS.: It would be nice if you could also adjust MRM and sMRM transition export templates for Agilent QQQ, this would save time re-formatting each export
(Agilent QQQ export template attached)

 200715_Prostaglandin_shared.sky.zip  PGD2 tran.csv  Agilent QQQ export template.xlsx 
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Export Peak Areas light and heavy separate
(1 response) camiel aarents 2020-07-15

Dear operators,

For my experiment i spiked heavy labelled isotope peptides in all my experimental samples to quantify my peptide of interest.

i found that via export-> report -> Peptide Ratio Results i get the ratio between sum of heavy and light transitions in my sample which is very usefull. However i would also like to have the sum of light and heavy transitions separate (thus not a calculated ratio). Is this also available in an export file?

Best, Camiel

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Top 5 quantitation
(1 response) swg42 2020-07-15

I'm new to using skyline and was hoping to get some help on how best to output the top 5 intensity peptides for label-free quantitation? Any suggestions?

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Identifying proteins
(1 response) p pereira79 2020-07-14

Dear ones,

I am using Skyline to identify proteins from a TOF analysis.
Well, I'm using a UniProt FASTA file as a library (75,000 proteins, and countless peptides), and the upload of RAW files takes too long time (it's taking more than 24 h just one file of size 1.32 Gb!).
Then, I would like to know:

  • Does Skyline work without internet?
  • Why is the upload taking so long?
  • Is it possible for the skyline to connect with a library (like Uniprot) online?

Best regard.

 kkkk.png 
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Error MSConvert demultiplexing
(12 responses) wendy white 2020-07-09

Hi Skyline team!

My goal is to acquire staggered DIA using a Thermo Exploris 480 ( Garcia Lab Histone methods on QE or HF). Unfortunately, the Exploris is missing features that allow it to upload an inclusion list from Skyline. The method I made on Exploris looks similar but I can't get MSConvert to demulitplex. Could you please take a look at error and let me know if there is a problem with the method, or can I change the MSConvert settings to process my data?

Thank you!

C:\Skyline Documents\2020_0708_histone_std_combinedList_01.raw

Starting...
Opening file "C:\Skyline Documents\2020_0708_histone_std_combinedList_01.raw" for read...
Calculating SHA1 checksum...
Processing...
Writing "C:\Skyline Documents\output\2020_0708_histone_std_combinedList_01.mzML"...
Failed - System.Exception: SpectrumToIndices() Number of demultiplexing windows changed. Minimum window size or window boundary tolerance may be set too low.
at pwiz.CLI.msdata.MSDataFile.write(MSData msd, String filename, WriteConfig config, IterationListenerRegistry iterationListenerRegistry)
at MSConvertGUI.MainLogic.processFile(String filename, Config config, ReaderList readers, Map2 usedOutputFilenames) at MSConvertGUI.MainLogic.Go(Config config, Map2 usedOutputFilenames)

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Maintaining Integration Windows When Re-importing Data
(8 responses) meyermr 2014-02-28
Hi Skyline Team,

I'd like to know if Skyline can maintain integration windows manually set by the user when re-importing a data file? For example, when I manually set the integration window for a given peak, add additional fragment ions to the peptide tree, and then re-import the data...Skyline will shift the integration windows slightly. Is there a way to override this so I don't have to manually go through and reset the integration windows?

Thanks for the help,
Matt
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iRT regression based failure to import DIA runs
(3 responses) kguehrs 2020-07-09

Dear Skyline team,

I know that similar issues were already discussed but I am somewhat frustrated and not sure how ti further proceed.

I did 6 DIA runs for 2 preparation schemes of 3 samples similar to 3 biological replicates for 2 conditions. I added the Biognosys 1 peptides iRT reference to all sample and also used the Biognosys 11 calculator in Skyline in the peptide settings.

Four of the six runs were imported without problems. The remaining two run were not imported with the failure message shown in one of the screenshots in the attached zip archive. I added 6 additional figures that show the regression line for each of the samples together with the extracted peaks for one of the Biognosys 11 reference. We use for some special reason rather complex gradients that are optimized for the main samples that are often measured and that might be one possible explanation for the deviations of the retention times of some of the iRT peptides, but this behaviour is shared wth all samples.

I have tried to use different iRT calculated based on the Biognosys 11 with several samples peptides added but without success. I have also tried to use a Biognosys reference with only 9 or 10 peptides but this did not result in successful import. I also reduced the transitions to 3 product ions only without success of reimport.

I have asked a colleague to use the dataset with Biognosys software and he managed to import the sampled without any problem. Therefore, I assume that the problem is in Skyline and you might think one more time about the idea of r*r>=0.99 for the iRT regression being the most appropriate setting for import of DIA data that are more noisy and not that simple as PRM chromatograms.

Another suggestion relates to the import procedure. As far as I understood from previous discussions of the import issue the r*r>=0.99 for the iRT regression is calculated at the beginning of the import process but the error message is only shown after finalizing data import. In my trials I always had to wait some 10 minutes (rather old computer) until I had to realize the failure. If my assumption of the process timing is correct it should be possible to give an early message and possibility that the user can cancel the process to change settings and retry more early.

Perhaps, you can supply some more ideas how to progress as I am running out of ideas for further steps.

Thank you for your good work to offer Skyline for the community.

 Skyline_failure_200709.zip 
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Making libraries for synthetic peptides and packaging them into single file in skyline
(4 responses) SRS 2020-07-07

Hi There,

I have been trying to make a spectra library of about few thousands synthetic peptides from a DDA run. I was able to import the data using "mziden" and "mzxml" files from the search and mass spectrometry data, respectively. But I have two questions here, 1) how can I export all the spectra libraries into a single file in skyline?

  1. How could I do the peak picking for over thousand peptides, as they are so many, it is not possible for me to open them to make sure the RT is aligned.

Do I need to make sure the alignment of the RT is correct then package them?

I can send you the skyline file if that help,

Thank you, looking forward to hearing from you.

Regards,
SRS

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Quantifying on multiple fragment ion isotopic peaks
(3 responses) johan gobom 2020-07-08

Dear Skyline Team,

I’m setting up a PRM method for large peptides, around 4.5 kDa, +4-charged, on an Orbitrap. For many of the fragments, the monoisotopic peak is not the strongest by far, so I would like to quantify on up to four isotopic peaks for each fragment. Is this possible to do in Skyline?

All the best,

Johan

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Bug in document grid reporting
(4 responses) jmeyer 2020-07-08

Dear Skyline team,

I believe I found a bug in skyline reporting identifications in runs where the peptide was in fact not identified.

Please see the two attached screenshots where there is clearly no identification line in the chromatogram, but the document grid reports "TRUE" for an identification in that run (top line in the document grid). These are two separate peptides. When I manually drag the chromatogram to re-integrate anywhere in that run, even the same area, it goes to FALSE.

Example 1 is R.SEQEDEVLLVSSSR.Y [27, 40] ++

Example 2 is R.YPDQWIVPGGGMEPEEEPGGAAVR.E [41, 64] +++

I also attached a minimal skyline share with these two peptides, and the other peptides in the same protein.

Any help fixing this or providing a workaround would be greatly appreciated.

Best regards,
Jesse

 Inkedwrong ID 1 skyline_LI.jpg  wrong ID 2 skyline.JPG  wrong_IDs_example_small.sky.zip 
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Failed to export method for CE optimization
(10 responses) benoit fatou 2020-06-08

Dear Skyline Team,
I tried to export a MRM method to perform CE optimization and I have got this error message (see attached file).
Could you please help me solve this issue?
Thanks,
Best,
Benoit

 ErrorSkyline.PNG 
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TMT0 Labeled Peptide as Internal Standard for TMT10 Targets
drothenberg 2020-07-07

Instead of using a heavy labeled synthetic peptide as an internal standard, I'm interested in using a TMTzero labeled peptide to compare against TMT-10plex labeled targets. I know ratios can be calculated using the Surrogate Standard option, but is it possible to have synchronized zooming and transition matching similar to heavy-labeled peptides? This would be similar to the TOMAHAQ approach, but without MS3. Thanks.

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QuaSAR Error
msj0506 2020-07-06

Dear Skyline Team,

I have a problem using QuaSAR tool in Skyline.

Now, my desktop system is window 10 and QuaSAR tool is connected R 4.0.0 ver in Skyline.

But, When i used QuaSAR for calculating LOD and LOQ, the error occurred continuously. i attach the related file.

Reinstallation does not solve the problem, and the same problem occurs in my laptop. Can you tell me how to solve this?

 Error(1).PNG  Error(2).PNG 
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Calibration curve calculations problems.
(2 responses) monika lysakowska 2020-07-06

I have a problem calculating the calibration curve. The calculations Skyline does are different from my calculations in Excel.​I have entered all the appropriate values for settings and grids
and I got a good calibration curve. R square and intercept is identical to the calculations in the excel spreadsheet, slope differs by two decimal places (I think it is related to the Y-axis conversion). However, the calculated values by Skyline Quan differ slightly from heavy normalized to light values from the excel worksheet.

The data should match the normalized skyline. Currently they are about a percentage point off, which is substantial. It’s about 20% difference between the two. What can explain this?​

Regards,
Monika Lysakowska

 1.png  2.png  3.png 
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Install issue
(3 responses) Brett Phinney 2018-01-30
Hey everyone, not sure if this is just my messed up win10 computer but I tried to install

https://skyline.ms/software/Skyline-release-64_4_1/setup.exe

And win10 would not let me install it even as the admin.

This seemed to fix it

https://superuser.com/questions/1252575/unable-to-install-clickonce-application-due-to-security-settings-windows-10


just an FYI if someone else has this issue

Cheers

Brett
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