Thanks for helping make this year's online Skyline User Group Meeting very much a success!

With coronavirus still a concern, we reluctantly opted to take our annual face-to-face event online again over two days -- Oct 27 and 28, 2021 - hopefully for the very last time! Without missing a beat, Skyline users rallied once again to hear 11 speakers on a diverse spectrum of mass spectrometry topics plus Skyline Principal Developer Brendan MacLean deliver his always-entertaining annual state-of-the-project update on the past and future of Skyline. Thanks again for the -- as always -- spirited Q&A sessions after each presentation.

Session recordings: including the Q&A sessions are available from the Speaker's page under each speaker's bio below.

Q&A recap: Either written response or time stamp location of live answer in video: Day 1, Day 2

We would like to thank our speakers who never wavered in their commitment to sharing their work. They volunteered their time to create and deliver informative presentations despite myriad timezones, practice sessions, and technical challenges. Thanks too for the ongoing support from instrument vendors who help fund our efforts and lastly, to our users who continually inspire us with the new and innovative applications for Skyline.

But let's not wait until the next ASMS to keep the discussion going: got frustrations? suggestions? praise? or any other issues, let's talk here ... now ...:

We look forward to seeing and conversing with you in person! at ASMS Minneapolis in 2022!

-- Mike MacCoss, Brendan MacLean and Event Organizers



Michael J. MacCoss, Ph.D. (University of Washington): Introduction and event host

Brendan MacLean (MacCoss Lab, University of Washington): Status of the Skyline Open-source Software Project 13 Years after its Inception
The Skyline project started just after ASMS 2008 as a 2-year effort to bring better SRM/MRM software tools to the NCI-CPTAC Verification Working Group that could support the variety of mass spectrometers in use in participating laboratories. Nearly 13 years later, the Skyline project is a thriving multi-omics community open-source collaboration supporting 6 mass spec instrument vendors integrated with a wide variety of external software, with many thousands of users worldwide and over ten thousand instances started each week. (More info...)

Chris Ashwood, Ph.D., (Glycomics Core, BIDMC): High-throughput Glycan Composition Profiling Enabled by MALDISkyLink and the Skyline Ecosystem
Protein glycosylation is the most frequent and varied type of co- and post-translational modification seen in eukaryotic cells. Using our newly developed software, MALDISkyLink, individual spectra are converted into a format compatible with the Skyline ecosystem, which is typically limited to LC- or CE- MS data. This enables the use of Skyline Batch for MALDI-based glycan analysis by extracting precursor information for over 500 pre-defined glycan compositions, refining for only high-quality precursors, and exporting the relative intensities of each high-quality glycan composition detection. (More info...)

Natan Basisty, Ph.D., (NIH): Analysis of Protein Turnover Rates in Skyline with the TurnoveR External Tool
While the measurement of protein turnover relevant in many biological settings, the study of in vivo protein turnover in whole organisms remain computationally difficult for most scientists due to lack of tools on widely accessible and user-friendly platforms. Here we will describe and demonstrate a new Skyline external tool, 'TurnoveR', for the accurate calculation, visualization, and statistical analysis of protein turnover rates based on mass spectrometry analysis of metabolic labeling experiments in whole animals, and demonstrate the application of TurnoveR in mouse studies of aging and muscle atrophy. (More info...)

Michelle Kennedy, (Cristea Lab, Princeton University): Leveraging Skyline to Develop and Analyze Data from a Targeted Mass Spectrometry Assay for Pan-herpesvirus Protein Detection
By taking advantage of the targeted mass spectrometry tools available in the Skyline interface, we have designed and experimentally validated a targeted mass spectrometry assay for monitoring human viruses representing the three Herpesviridae subfamilies. Our study provides a reproducible framework for monitoring the progression of herpesvirus infections under different conditions and it benefitted heavily from the accessible environment afforded by Skyline for data analysis and integration.(More info...)

Bini Ramachandran, Ph.D., (FARRP, University of Nebraska-Lincoln): Matrix-independent Calibration: A Consensus Strategy to Quantify an Analyte from Different Types of Matrices.
The novel method appeared to be sensitive, accurate, and precise in the detection of milk allergens in five different test matrices evaluated, containing known amounts of allergen.
In this study, we are demonstrating a matrix-independent strategy for targeted detection of milk allergens from multiple food matrices. An inert carrier protein was incorporated as a background matrix for both the calibrants and test samples from the sample preparation stages. This matrix-independent strategy could improve the adoption and applicability of targeted mass spectrometry approaches for samples that are otherwise challenging for immunochemistry approaches in routine testing labs. (More info...)

Juan Rojas,, (University of Leipzig - Hoffmann Lab): Skyline for the Parallel Analysis of LC-TWIMS-MS/MS DDA and DIA Data
Recently, a solution to inherent poor ion beam sampling of time-of-flight (TOF) mass analyzers has been proposed by restricting detection to ions expected at specific arrival times after being separated in a travelling wave ion mobility spectrometry (TWIMS) cell. Here, we show that this approach can be applied to data independent acquisition (DIA) using a so-called signal enhancement MSE (seMSE) experiment, which was compared to two different TWIMS-DDA and three TWIMS-MSE different methods with respect to the spectral quality of peptide identifications and, more importantly, fragment ion sensitivity. Although seMSE provided limited peptide identifications due to isobaric fragment ion interferences, the sensitivity of fragment ions was much improved. Thus, seMSE provided up to tenfold higher signal intensities than the other tested quantitative methods at the fragment level, which can be favorably supplemented with high quality TWIMS-DDA spectral libraries. (More info...)

Lightning Talks

Robert Ahrends, Ph.D., (University of Vienna): Targeting the Lipid Metabolism with LipidCreator and STAMPS to Investigate Fat Cell Differentiation of Mesenchymal Stem Cells
We recently introduce tools working natively with Skyline. Here we would like to present two which we use in our daily lab routine to reduce time and effort in the development of targeted omics workflows such as we need for the investigation of fat cell differentiation. STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150. LipidCreator, is a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. Both tools are fully computationally and analytically validated and prove that there are capable to generate concise targeted experiments to analyze and to dissect the lipid metabolism and signaling. (More info...)

Elena Barletta, (University of Zurich): Mass Spectrometry-based Identification of Allergen Proteins Involved in Seafood-related Allergic Reactions
We are presenting our bottom-up proteomics approach using LC-MS/MS coupled with Parallel Reaction Monitoring (PRM) technique to target alergen pepties that cause food allergies from shellfish -- a major cause of food-induced anaphylaxis. (More info...)

Muluneh Fashe, Ph.D., (University of North Carolina - Lee Lab): Using Skyline to Quantify Drug Metabolizing Enzyme and Transport Protein Concentrations in Sandwich-Cultured Primary Human Hepatocytes
We used a targeted quantitative proteomic isotope dilution nanoLC-MS/MS method to quantify absolute concentrations of over 70 phase I and II drug-metabolizing enzyme (DME) and transport proteins in membrane fractions isolated from sandwich-cultured primary human hepatocytes in the absence or presence of inducers. We analyzed the quantitative proteomics data using the Skyline software and then using MultiQuant (SCIEX) software and compared the concentrations between the platforms using correlation and Bland-Altman plot analyses. This presentation highlights the applicability of Skyline for processing and evaluating quantitative targeted absolute proteomics data for multiple DMEs and transporter proteins in the membrane fractions of human primary hepatocytes. (More info...)

MaKayla Foster, (North Carolina State University) Utilizing Skyline for the Evaluation and Quantitation of Per- and Polyfluoroalkyl Substances
Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants characterized by extensive fluorination along extended aliphatic chains, rendering them chemically inert, thermally stable and hydrophobic. These physicochemical properties make PFAS uniquely suited for many household and industrial applications, but also cause them to bioaccumulate and become toxic as they increase in concentration in both the environment and biological systems. To characterize PFAS in various sample types, we utilized a simultaneous liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) platform to evaluate 100 PFAS standards and create a Skyline library containing LC retention times, IMS collision cross sections (CCSs) and m/z values. This PFAS library was then applied to water, tissue and blood samples to determine PFAS content where numerous biomolecules were also present. (More info...)

Virag Sagi-Kiss, Ph.D., (Imperial College London): Rapid Sample Preprocessing of a Large Number of Targeted Metabolites with Skyline
We are developing and applying targeted profiling methods for discovering molecular pathways underlying healthy aging. We use a workflow in Skyline that allows us to speed up the data pre-processing steps: adjust retention time windows, filter contamination present in blanks, monitor retention time drifts, evaluate stability of Quality Control (QC) samples, evaluate the linearity of analytes via dilution curve of QC and aid extracting data for close eluting isobaric compounds.(More info...)

Nikunj Tanna, (Waters Corporation): MassLynx Skyline Interface - Enabling automated MRM method development for targeted proteomics and peptide bioanalysis workflows
Protein therapeutics and biomarkers are increasingly becoming commonplace in laboratories traditionally performing small molecule LC-MS assays. Developing MRM methods for peptides v/s small molecules has some fundamental differences and often requires retraining of personnel. Skyline has been the informatics platform of choice within the proteomics world, and lends itself perfectly to aid and guide MRM method development for TQ assays. MassLynx Skyline interface is an informatics tool which integrates, simplifies and automates the TQ MRM method development process, ensuring systematic, consistent and robust method development across all user levels. (More info...)