Dear Skyline Users,
The Skyline Team is excited to release Skyline 24.1, the product of another 10 months of effort in response to your feedback and use of Skyline.

Since our last release, you have started 15,000 instances each week (on average) of Skyline or Skyline-daily, and downloaded the software 1600 times per month. There have been 1291 posts to the support board.

Improvements in Skyline 24.1 include:

• New! File > Search submenu putting all our search pipeline options in a place you can find.
• New! Complete support for FragPipe and DIA-NN integration.
• New! Complete EncyclopeDIA pipeline with narrow and wide window searches and staggered window demultiplexing.
• New! File > Import > Features Detection - MS1 peptide feature finding with Hardklor/Bulseye.
• New! MS/MS spectrum and RT prediction with Koina (Prosit model available).
• New! Peak Areas > Relative Abundance plot.
• Added right-click Auto Arrange Labels option on the Group Comparison - Volcano and Peak Areas - Relative Abundance plots for publishable figures.
• Added matching small molecule versions of all standard peptide reports in Document Grid.
• Added gene level parsimony option in protein association dialog.
• Added File > Open from Panorama with anonymous account access to Panorama, e.g. Panorama Public.
• Added "Peptide Spectrum Match Count" to the PeptideResult in the Document Grid.
• Added support for Proteome Discoverer 3.1 in spectral library builder.
• Added modification and special fragment ions for TMTpro.
• Added support for precursor isotopes and reporter ions to transition list import.
• Added support in Spectral Library Explorer for contains search by using preceding asterisk, e.g. *IDE.
• Added tooltips to the Peak Areas - Replicate Comparison plot.
• Added command-line arguments for File > Export > Spectral Library.
• Added command-line arguments for File > Export > mProphet Features.
• Added command-line arguments for peptide digestion settings.
• Added command-line arguments for peptide filter settings.
• Added command-line arguments for File > Export > Annotations.
• Added command-line argument --import-pep-list.
• Added command-line argument --associate-proteins-fasta for associating existing document peptides with a FASTA.
• Added command-line arguments --pep-add-mod*, --pep-clear-mods, and --integrate-all.
• Added command-line argument —pep-add-mod-variable=<true|false> for explicit setting of peptide modifications to “variable” or “fixed”.
• Added command-line arguments to add annotations.
• Added command-line argument --verbose-errors to help troubleshoot unexpected errors.
• Added support for reading chemical formulas with Unicode numeric subscript text.
• Added "Surrogate External Standard" that can be set on Peptides and Molecules in the Document Grid which enable using a different molecule's calibration curve for quantification.
• Added ion mobility columns for library building SSL format.
• Added support for Agilent MassHunter 12 method export.
• Added support for Thermo Astral and Orbitrap GC instruments.
• Added method export support for the Thermo Stellar instrument.
• Added support for SCIEX OS software (exporting acquisition methods and quantitation methods) for QQQ/QTRAP and QTOF platforms.
• Added support to treat WIFF1 and WIFF2 as separate types (e.g. for purposes of import results form)
• Added File > Export > Transition List for Agilent MH 12.1.
• Added Dot Product value to Full-Scan view property grid that compares the spectrum in the plot with the expected distribution.
• Added "Replicate Name" to the things that can be set using Result File Rules.
• Added warning to the immediate window when a CCS<=>IM conversion fails.
• Added warning when adding decoys or training an mProphet model if document uses explicit peak boundaries from spectral libraries.
• Added red text and warning tooltip on Transition Settings - Full Scan tab if retention time filtering is selected with "PRM" or "SureQuant" acquisition methods.
• Added isotope distribution matching to pick best ion mobility value in ion mobility library "Use Results".
• Added support for MSFragger pepXML/mzML pairs for Bruker timsTOF data with plain integer scan numbers in the spectrumNativeID attribute.
• Update to MassLynxSDK 4.11.0.
• Updated Shimadzu data reader DLLs.
• Updated SCIEX method export DLLs.
• Updated to MSFragger 3.8.
• Updated available modifications to current UniMod, Mascot naming, and ProteinPilot abbreviations.
• Improved peak quality indicators in the Targets view for the colorblind.
• Improved performance of .sky file reading.
• Improved performance of "Aligning Retention Times".
• Improved performance displaying protein tooltips (and Library Match window) when Max Neutral Losses is high.
• Improved CE Optimization method and transition list export for Agilent to stop adding 0.01 to the Q3 m/z values.
• Improved Associate Proteins form to keep unmapped peptides in an "Unmapped Peptides" list, and hide min peptides per protein option when called from the Refine menu.
• Improved sort order for adducts so that molecule precursor ions are ordered by mass, as with peptides.
• Improved "Apply Peak" to work with multiple-selection in the Targets tree.
• Improved Spectral Library Explorer to suggest adding "common" modifications instead of less common modifications when unknown modifications are found in library.
• Improved Spectral Library Explorer for a better experience with larger small molecule libraries.
• Improved error handling when attempting to parse formulas with errors in mass offsets, e.g. "C12H5H3[+3.2/3x3]"
• Improved TMT support by excluding reporter ions when calculating library dot products and detecting peaks.
• Improved tolerance for ion mobility data with bad CCS calibrations.
• Improved error message display interacting with Panorama.
• Improved peptide Unimod modification defaults for "variable" and "fixed" to make only alkylation, isobaric tag labels, and loss-only modifications "fixed" by default.
• Expanded DIA isolation scheme detection to 1000 spectra cycles for gas phase fractionation (GPF) and Astral.
• Reduced the minimum number of values required for a dot product to be calculated from 3 to 2.
• Changed so that the "Do you want to add decoys" message only gets shown for DIA when importing results if the document has at least 20 peptides in it.
• Changed to reset standards in File > New to decrease confusion over documents with unexpected "light" as standard instead of "heavy".
• Changed values such as "Normalized Area", "Calculated Concentration" to have a value even when some peaks are missing or truncated.
  • Warning messages are displayed in Document Grid.
  • Old behavior is available with "Normalized Area Strict" sub-property in Document Grid.
• Fixed error that sometimes happens choosing "Kernel Density Estimation" on Retention Times run to run regression graph.
• Fixed error that could happen when clicking on an ID line on the chromatogram graph.
• Fixed calculation of bogus isolation window offsets when WIFF2 file returns 0 for lower/upper window bounds.
• Fixed Associate Proteins form to use final document counts for proteins and peptides in the "prot, pep, prec, tran" string (so it includes decoys, iRT, and the unmapped peptide group in the count).
• Fixed MSAmanda to write PeptideEvidence with isDecoy attribute.
• Fixed IdentData mzIdentML parser to be case-insensitive on isDecoy attribute.
• Fixed case where View > Transitions > QC shows blank menu item instead of list of available QC graphs.
• Fixed Import Transition List / Assay Library to allow users to deal with errors after click on OK if errors were previously reviewed.
• Fixed "Chromatogram Information Unavailable" in small molecule document with spectrum filters.
• Fixed centroiding for Waters non-IMS data to be done spectrum by spectrum instead of all at once.
• Fixed repeated neutral loss labels on crosslinked peptide transitions in Library Match view.
• Fixed unexpected error copying and pasting protein groups.
• Fixed unexpected error hovering over Protein in Targets tree if .skyd file cannot be read.
• Fixed incorrect TIC area in documents with QC traces and transition full scan retention time filtering.
• Fixed unexpected error in small molecule transition list reader.
• Fixed edge case in detection of Waters lock mass channel.
• Fixed "times and intensities disagree in point count" error applying a Spectrum Filter to an MS1 Transition.
• Fixed reading compensation voltage values from mzML files.
• Fixed unexpected error extracting chromatograms from raw file when there are pressure traces but no MS1 spectra.
• Fixed command-line output of transition full-scan settings changes.
• Fixed unexpected error attempting to modify base molecule in targets tree with an invalid chemical formula.
• Fixed WIFF SIM/SRM chromatogram extraction to operate on the entire time range instead of within the scheduled limits.
  • Works around a bug(?) with Sciex WIFF where it records the wrong scheduled limits but the data is there if you tell it to ignore the limits.
• Fixed Bruker TSF reader crashing when enumerating chromatograms when the file has empty spectra.
• Fixed library build from pepXML to check for spectrum files in the base_name's parent path (if present).
• Fixed small molecule chromatogram extraction to limit time range based on retention time prediction.
• Fixed to update Document Grid when annotations are removed from the document.
• Fixed DIA-NN speclib N-terminus mods to be moved/merged to the N-terminal AA.
• Fixed spurious Skyline Batch error about empty directory when directory contained .d folders.
• Fixed error displaying dot product line on Peak Area Replicate Comparison graph when currently selected peptide has no transitions of the type (precursor or product).
• Changed to display a warning message in the Immediate Window when Skyline discards chromatograms because the Explicit Retention Time is outside the retention time range over which the chromatogram was extracted.
• Fixed BiblioSpec to use the spectrumNativeID attribute in pepXML when reading MSFragger pepXML.
• Fixed unexpected error ("Attempt to add integration information for missing file") when doing a rescore with multiple injections if it fails because of missing iRT standards.
• Fixed transitions getting incorrectly added/removed from siblings when changing children of a precursor with a Spectrum Filter.
• Fixed unexpected error in Edit Spectrum Filter form.
• Fixed unexpected error when selected QC trace is not present in a particular replicate.
• Fixed truncation of report text copied to the clipboard when report columns include "CleavageAa".
• Fixed command-line arguments --tran-product-*-special-ion not being processed if they are the only filter arguments passed.
• Fixed Skyline detection of Waters RAW folders to be case-insensitive.
• Fixed error that could happen if DIA-NN chosen peak was outside of extracted chromatogram range.
• Fixed unexpected error in Retention Times Replicate Comparison graph when aligning retention times and there are missing results.
• Fixed unexpected error in the Detections plot.
• Fixed unexpected error importing peak boundaries with malformed peptide modification.
• Fixed unexpected error when spectrum from an extracted chromatogram cannot be found in the raw file.
• Fixed incorrect isotope dot product for newly imported small molecule precursor transition.
• Fixed MS1 chromatogram extraction when doing PRM CE Optimization.
• Fixed adducts like [M+H-H2O] on molecules described as mass-only.
• Fixed unexpected error in Peak Areas > Replicate Comparison graph when a peptide has missing results for one replicate.
• Fixed unexpected error bringing up Full-Scan spectrum view on some datasets.
• Fixed unexpected error displaying TIC chromatogram when not available in some datasets.
• Fixed parsing spectrum IDs from MSFragger pepXML files with MGF spectrum files.
• Fixed unresponsive long wait when doing "Equalize Medians" in huge documents.
• Fixed unexpected error using "Apply Peak to All" when one replicate has missing chromatograms.
• Fixed unexpected error importing a small molecule transition list.
• Fixed unexpected error after Modify Molecule form to change charge state in a way that makes no sense with the current adduct.
• Fixed unexpected error after Modify Transition form to change the precursor adduct in a way that makes no sense with parent molecule, or vice versa.
• Fixed unexpected error exporting a report definition to a .skyr file that cannot be written to.
• Fixed to output a warning message to the Immediate Window if a single transition's chromatogram is being discarded because of the Explicit Retention Time.
• Fixed unexpected error using "Edit Modifications" form.
• Fixed volcano plot formatting when Match Expression includes both fold change and p-value.
• Fixed loss annotations in MS/MS spectra to work for mass-only losses.
• Fixed unexpected error in Spectral Library Explorer when amino acid 'J' had a modification on it.
• Fixed unexpected error showing Detections graph when document has no results.
• Fixed preventing multiple spectrum filters from being added to heavy precursor.
• Fixed optimization step incorrect for some Agilent collision optimization data.
• Fixed "Matrix must be positive definite" error that sometimes happened with bilinear fit calibration curves.
• Fixed handling explicit adduct charge declarations (e.g. the trailing "+" in "[M+2CH3+Cl]+").
• Fixed Panorama error "Documents with same GUID should have same first audit log entry" when audit log restarted.
• Fixed unexpected error showing full scan graph when transitions differ only be neutral loss.
• Fixed unexpected error doing "Apply Peak to All" when retention times have been aligned.
• Fixed unexpected error searching for missing peak scores when peptide is missing results for one replicate.
• Fixed chromatogram weirdness when renamed molecule used to have the same name as another molecule in the document.
• Fixed disk error that would sometimes happen at end of chromatogram extraction.
• Fixed Edit > Find (Ctrl-F) form to set focus to the text box for immediate typing.
• Fixed several command-line operations that were not being recorded in the Audit Log.
  • Including "--import-annotations", "--import-peak-boundaries", "--reintegrate", "--add-decoys", and "--import-file".
• Fixed graphs sometimes blank when displaying more than 100 precursors at the same time.
• Fixed error adding decoys to a document with high resolution MS1 and sulfur containing amino acids.
• Fixed exporting negative RT values in Agilent instrument methods.
• Fixed using too much memory outputting report with "Normalized Area" column when document has Peptide Quantification regression method set to something other than "None".
• Fixed reading CE from WIFF file spectra.
• Fixed search errors from Search control to be more specific.
• Fixed ion mobility values from spectral libraries (with no .imsdb file) not appearing in reports.
• Fixed to gracefully handle network error during chromatogram loading.
• Fixed potential hang extracting chromatograms when some protein groups have proteins with nonstandard accession numbers.

This release will require a manual upgrade. We hope you'll take the time to visit the Skyline installation page:

https://skyline.ms/skyline64.url

And install this latest release. It should be worth the effort.

Thanks for your continued use, feedback, and support of the Skyline Project.

Brendan MacLean
Skyline Principal Developer