Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics

Abstract

Vendor-independent software tools for quantification of small molecules and metabolites are lacking, especially for targeted analysis workflows. Skyline is a freely available, open-source software tool for targeted quantitative mass spectrometry method development and data processing with a ten-year history supporting 6 major instrument vendors. Designed initially for proteomic analysis, we describe the expansion of Skyline to data for small molecule analysis, including selected reaction monitoring (SRM), high-resolution mass spectrometry (HRMS), and calibrated quantification. This fundamental expansion of Skyline from a peptide-sequence centric tool to a molecule-centric tool makes it agnostic to the source of the molecule while retaining Skyline features critical for workflows in both peptide and more general biomolecular research. The data visualization and interrogation features already available in Skyline -such as peak picking, chromatographic alignment, and transition selection -have been adapted to support small molecule data, including metabolomics. Herein, we explain the conceptual workflow for small molecule analysis using Skyline, demonstrate Skyline performance benchmarked against a comparable *

Chasing the Major Sphingolipids on Earth: Automated Annotation of Plant Glycosyl Inositol Phospho Ceramides by Glycolipidomics

“…Even though lactosyl ceramide (d18:1/16:0) may be present in plants, we could not detect it in our samples, thus, making it suitable as the internal standard in our workflow. Normalization by the internal standard (area ratio) and dry weight was performed for MS1-based relative quantification by Skyline [ 25 ] ( Figure 3 A–C). Estimated concentrations in the nmol to µmol range per gram dry weight were observed, which is consistent with the literature [ 12 , 18 ].…”

“…The correct GIPC annotation was ensured by a manual inspection of the results. MS1-based relative quantification of annotated GIPCs was performed with Skyline [ 25 ]. Total areas were divided by the corresponding calculated dry weights and areas of the IS, resulting in normalized ratios per g dry weight, of which the average was taken based on the number of replicates (3 for salad and spinach, 4 for strawberries and raspberries).…”

A New Approach to Developing Long-Acting Injectable Formulations of Anti-HIV Drugs: Poly(Ethylene Phosphoric Acid) Block Copolymers Increase the Efficiency of Tenofovir against HIV-1 in MT-4 Cells

“…MS1 spectra were recorded in positive mode with 70K resolution, 150–1500 scan range, 3e6 automatic gain control (AGC) target value, 50 ms maxIT. Peak areas were analyzed in Skyline (MacCoss Lab, Seattle, WA, USA) [ 69 ]. TFD, tenofovir monoester, and tenofovir base were detected, respectively, with m/z values of 520.1903, 404.1330, and 288.0856.…”

Benchmarking non-targeted metabolomics using yeast derived libraries

“…www.ebi.ac.uk/metabolights/MTBLS1782. Skyline (version 20.1.0) was used to create MS1 area based quality control charts 29…”

“…The higher match of our internal Pichia pastoris yeast extract database with the HMDB compared to YMDB can be explained by a more frequent comprehensive database curation of the HMDB (e.g: additional "status" ontology in HMDB including Thus, the obtained biological repeatability of the fully controlled fermentations together with the storage stability of the dried extraction aliquots over three years, makes the yeast benchmark fit for purpose for a whole range of metabolomics measurement platforms. Orbitrap mass analyzers (HF 1, HF 2) using Skyline for visualization 29 .…”