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Full Scan Data

Hi Brendan, I have been using skyline in full scan mode to qc our ion traps (works great so far). One small problem I am having is that it is really hard to keep track of all the data files in skyline after a while. Is there a way to organize or annotate the data when I say change a column or qc vial. Maybe a panel that has a list view instead of a tab view, where I can add custom columns for example....

How to analyze Shimadzu .lcd files?

I outsourced LCMS running and received .lcd files for targeted metabolites. Files contain (samples with replicates, blanks , standard curv / dilution series ). I don’t have access to shimadzu software to process targeted LCQQQ .lcd files. This was not possible with MSDIAL, I heaerd the best open source software for processing targeted LCQQQ data. How can I analyze .lcd files from targeted metabolics?...

SRM - Force Skyline to integrate scans with different/multiple headers

Hi, I'm currently working on optimization in MRM/SRM using a quantiva. Let's say I didn't want to miss any transition and I asked skyline to produce a,b,y transition list which I imported into a quantiva. After analysis, skyline integrates only a few ions which seemed surprising in the first place as i knew that some ions would be produced by CID but were missing from Skyline integration. After...

Dilution Factor

I am wondering what the purpose of the dilution factor column in the Results grid / document view / Replicate table is. This would be a very useful column since it can be populated manually but shouldn't a difference in dilution factors among samples be reflected in the quantitative comparisons? I do not see any change in quantitative values when populating this column with highly different dilution...

Problem with Peak picking

Hi, I like to do label free quantitation from some MSe runs with PLGS (identity E) outputs. Build of library worked with the final fragment csv output from PLGS, also import of raw files. However, when I compare the RT from the PLGS output for a peptide and the peak RT he piked for in skyline it is off. For example in the output I find the peptide with RT 20.7 and skyline labeled a peak at 27.3....

5 - Jaffe.pptx

Discovery to Targets for a Phosphoproteomic Signature Assay: One-stop shopping in Skyline Jake Jaffe Skyline Users Meeting June 2013 Proteomics and Biomarker Discovery Idea: Phosphosignaling is coordinated! Phosphosignaling is likely coordinated Kinase-substrate relationships tend to be 1-to-many A priori expectation of coordinate regulation of sites Don’t need to monitor every phosphosite...

Data Normalization with Heavy Peptide along with Global Standard

Hi Skyline Support Team, I'm a new user learning targeted proteomics data analysis with Skyline and have some questions about normalization approaches for my experiment. My current setup includes: Around 2-3 peptides per protein Around 2-3 transitions per peptide One transition for each labeled peptide (for chromatography and instrument variation normalization) BSA protein as a global standard...

Dealing with huge sanple set in Skyline

Dear Skyline Support Team, We have just finished a PRM experiment comparing different treatments and time points, in total, 396 samples. I have already uploaded all the files in Skyline and now, as I usually do, I would like to confirm the peak picking by manually checking all the peaks (correct peaks at expected RT and peak boundaries). However, as I have 90 target peptides and 396 samples that...

2014-ASMS-Amodei.pdf

A Multi-Instrument, Skyline-Based Comparison of DIA Peptide Detection and Statistical Confidence Tools Dario Amodei1, Don Marsh2, Hannes Rost3, Lucia Espona Pernas3, George Rosenberger3, Reudi Aebersold3, Parag Mallick1, Michael J Maccoss2, Brendan MacLean2 1 Canary Center for Early Cancer Detection, Stanford University 2 Department of Genome Sciences, University of Washington 3 ETH Zurich,...

Peak picking: (Qex) creating data fro PRM experiment from preliminary DIA experiment

Dear Skyline support team and Brendan, I had a course in targeted proteomics in Zurich last jun. Now I am trying to apply it to my experiment. I am working with phosphorylated proteomics. I have 4 biological replicates (each replicate with 2 fractions) comparing Jurkat T-cells conntrol vs cisplatin treated. After LC-MS/MS run, I performed search on Mascot using the default (0.05 Da and 10 ppm)...

Small molecule import Peak Boundaries trouble

Hi all, I am unable to import a peak boundaries file for small molecules since "FullPeptideName" is a required column, and for small molecules, this is always #N/A. If I put #N/A in for this column, skyline won't import it. Is there a way to do this for small molecules? Thanks! jay Hi Jay, There are still a lot of places in Skyline where we say "peptide" when we really mean "molecule"....

6 - Amodei.pdf

Multi-Instrument, Skyline-based Comparison of DIA Peptide Detection and Statistical Confidence Tools Dario Amodei Postdoctoral Scholar, Skyline Developer Mallick Lab Stanford University 6/15/2014 Acquisition Methods Targeted Discovery Data Dependent Acquisition (DDA) Many proteins Poor reproducibility Poor sensitivity Data Independent Acquisition (DIA) Selected Reaction Monitoring...

Export of Transition List and Method for Sciex 6500+ broken

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...

Q & A

Dear Skyline Users, We had some good questions during our sixth Skyline Tutorial Webinar so we have provided the answers! Q: Can you describe how you normalize data in Skyline? Ans:1. Ratio to Global Standards and 2. Ratio to heavy (or any other internal standard isotope label type). It is important to note that for targeted proteomics is it not a valid assumption that you can normalize to the...

Event Information

REGISTRATION OPEN - 2025 Skyline User Group Meeting in Baltimore, June 1, 2025 Welcome The Skyline Team is pleased to announce this year's big event, the Annual Skyline User Group Meeting, which will be held in Baltimore, MD on Sunday afternoon before ASMS. We love seeing you all in-person -- so be sure to register and attend! We would like to thank the event sponsors (see below) for their generosity...

Thanks!

Thanks to everyone who joined us in Baltimore for the Skyline User Group Meeting at ASMS 2025. It was great to meet with all of you passionate Skyline users and share the opportunity to experience varied presentations about recent work enabled by (and for) Skyline software! Below are links to the speakers' pages with options to view their slides or watch their presentations as [video]s. At the...

Failed to build cache error

Hi, I am experiencing the following error: "failed to build cache: 'file location and name'." Array dimensions exceeded supported range. Skyline ran out of memory. I have seen this with latest version of Skyline and Skyline-Daily. The system is 64-bit Win7 with 64 GB RAM. Skyline is using about 12 GB when the error occurs. I think the problem is I have too many transitions in my document,...

Thanks!

Thanks to everyone who joined us in Houston for the Skyline User Group Meeting before ASMS 2023. It was great to meet with all of you passion Skyline users and share the tremendous opportunity to hear terrific and varied presentations about recent work enabled by Skyline software! Below are links to the speakers' pages with options to view their slides or watch their presentations. At the bottom...

Specific-modified peptides don't appear

Hi! I am writing to ask you help about some trouble I'm having in seeing in Skyline some specific modified peptides; it's probably something easy to solve, but after trying many things, I still couldn't get to solve it. I have MS/MS data of a digested pure protein, acquired by a universal method in an Orbitrap fusion. I have a control condition and two conditions were the enzyme was treated with...

S1-5_Krieger_v2_05312025.pdf

Improved targeted quantitation with diagonal-PASEF using Skyline Stephanie Kaspar-Schönefeld Applications Development Proteomics Jonathan Krieger Head of Research at Bruker ProteoScape || ||© 2025 Bruker For Research Use Only. Not for use in clinical diagnostic procedures. Outline 2 June 2025Innovation with Integrity 2  Targeted quantitation using timsTOF instruments  Introduction...