Deuterium oxide labeling methods for bulk and single muscle protein synthesis measures, and measures of integrated muscle protein synthesis and breakdown rates

ABSTRACT: This methodological pilot study explored the feasibility of an integrated tracer approach for simultaneous measurement of human skeletal muscle protein synthesis and breakdown during prolonged deuterium oxide (²H₂O) labelling. Four healthy men completed a five week protocol combining daily oral ²H₂O intake with selected stable isotope amino acid tracers to assess bulk fractional synthesis rates, fractional breakdown rates, and single protein turnover. The study specifically addressed the methodological question whether bulk or individual protein synthesis measurements remain consistent over a prolonged 2H₂O labelling period. We demonstrate that measurements of mixed-protein fractions in muscle are vulnerable to error due to the rise-to-plateau kinetics associated with longer-duration labelling. Mean mixed-protein synthesis rates can be underestimated when labelling exceeds approximately four weeks. Increasing precursor enrichment mitigated this effect but highlights the need for controlled enrichment strategies in long term studies. In contrast, dynamic proteome profiling (DPP) provided more robust single protein turnover estimates across the full period because the different isotopic plateaus of individual peptides could be modelled. The study also tested the feasibility of combining ²H₂O labelling with ¹⁵N alanine–based breakdown measurements and evaluated potential interference from multiple tracers on DPP outputs. Together, these findings outline practical considerations, limitations, and feasibility of an integrated multi tracer framework for studying human muscle protein turnover over extended periods.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skeletal Muscle

SUBMITTER: Jatin Burniston  

LAB HEAD: Prof Jatin G Burniston

PROVIDER: PXD078552 | Pride | 2026-09-07

REPOSITORIES: Pride