Non-targeted Proteomics of Muscle Biopsy

Non‐targeted proteomics was performed on muscle biopsy samples taken pre‐ and post‐intervention. Samples preparation is described in Supplementary Information 6. Samples were analyzed using liquid chromatography‐coupled mass spectrometry. Peptides were separated on an EASY‐Spray column ES803 and analyzed on a Dionex UltiMateTM 3000 UHPLC system and an Orbitrap FusionTM LumosTM platform (both Thermo Fisher Scientific). (Davis et al., 2017 (link)) Raw data were imported into Progenesis QI (Waters, UK) using default parameters. Tandem mass spectrometry data were searched using Mascot (v.2.5, Matrix Science) against the Universal Periodic Review human database. Mass tolerances were set to 10 ppm for precursor and 0.5 Da for fragment masses. Peptide‐level false discovery rate was adjusted to 1%. Peptides with a score of <20 were discarded. Data were cantered and normalized in Progenesis QI (Waters), before being extracted for further data processing in Perseus (Max‐Plank Institute of Biochemistry) (Tyanova & Cox, 2018 (link)).

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Dearlove D.J., Soto Mota A., Hauton D., Pinnick K., Evans R., Miller J., Fischer R., Mccullagh J.S., Hodson L., Clarke K, & Cox P.J. (2022). The effects of endogenously‐ and exogenously‐induced hyperketonemia on exercise performance and adaptation. Physiological Reports, 10(10), e15309.

Publication 2022

Dionex UltiMateTM 3000 UHPLC system Progenesis QI Mascot

Biopsy Human Liquid chromatography Mass spectrometry Muscle Peptides Tandem mass spectrometry Tolerances

Corresponding Organization : University of Oxford

Other organizations : Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital

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Variable analysis

independent variables

Intervention

dependent variables

Protein expression

control variables

Muscle biopsy samples
Liquid chromatography-coupled mass spectrometry
Peptide separation on EASY-Spray column ES803
Analysis on Dionex UltiMate 3000 UHPLC system and Orbitrap Fusion Lumos platform
Progenesis QI software for data import and processing
Mascot v.2.5 for tandem mass spectrometry data search against the Universal Periodic Review human database
Peptide-level false discovery rate adjusted to 1%
Peptides with a score of <20 discarded
Data centered and normalized in Progenesis QI

controls

No positive or negative controls were explicitly mentioned in the provided information.

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