Mass spectrometry profiling of proteins and their ubiquitin modifications in ageing skeletal muscle

Lord, Samuel Oliver (2025). Mass spectrometry profiling of proteins and their ubiquitin modifications in ageing skeletal muscle. University of Birmingham. Ph.D.

Lord2025PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (30MB)

Abstract

Skeletal muscle mass and function progressively decline with age. This decline is exacerbated in later life, contributing to the development of sarcopenia which is prevalent in many chronic diseases and a predictor of mortality. The molecular mechanisms driving this decline are not fully understood, slowing the development of pharmaceutical interventions. This thesis uses mass spectrometry to profile age-related changes in proteins and their ubiquitin modifications in mouse skeletal muscle to elucidate potential driving mechanisms of sarcopenia. We begin by reviewing the application of proteomics to study ubiquitylated proteins – termed ubiquitylomics – highlighting its application in skeletal muscle research. We developed a workflow designed to address challenges associated with skeletal muscle proteome profiling, later modifying the protocol to allow for the detection of ubiquitylated sites. Our workflow was applied on skeletal muscle obtained from young (6 month) and old (21-22 month) C57BL/6 male and female mice. Bioinformatics analysis of the proteomics dataset highlighted profound changes to extracellular matrix, mitochondria, energy metabolism, proteostasis, sarcomere and spliceosome proteins. Of note, we show that the unfolded protein response in the endoplasmic reticulum was a male-specific trait in our aged cohort. The ubiquitylomics dataset revealed age-related changes on sites from mitochondrial proteins, histones, ribosomal subunits and UPS-associated proteins. There was no clear relationship between muscle protein abundance and their ubiquitylation status, indicating a more complex mechanism than ubiquitin-mediated degradation. Ubiquitylation of histone H2B – increased during ageing of simpler organisms – was consistently downregulated in both males and females, occurring on an isoform not previously mentioned in the ageing field. Altogether, this thesis improves our mechanistic understanding of ageing skeletal muscle preceding sarcopenia, offering some potential targets for future research.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):