Abstract
Physical exercise of even a moderate intensity is beneficial in both the prevention of prediabetes and management of Type 2 diabetes mellitus, as skeletal muscle is a primary tissue responsible for glucose uptake. Exercise-evoked proteomic alterations in muscle of subjects with prediabetes are of great importance for the study of relationships between insulin resistance and exercise. Although data-dependent (DDA) proteomic analysis is a cornerstone of deep proteome profiling employed in the elucidation of skeletal muscle biology, data-independent (DIA) approaches gain popularity in the studies focused on data reproducibility and throughput. We compared various ion-chromatogram libraries assembled with the use of off-line high-pH fractionation (HpH), gas-phase fractionation (GPF) and libraryless DirectDIA in LC/MS/HRMS DIA proteomic analysis of muscle from normoglycemic (NGT) and prediabetic (IGT) subjects after 3 months of supervised, mixed-mode exercise. In our hands, GPF-fractionated, hybrid DDA/DIA libraries yielded the best overall balance between the speed of preparation, data collection and protein identification among tested approaches. Analysis revealed, that despite 3-month exercise intervention skeletal muscle from IGT subjects displayed significant alterations in pathways and molecules relevant to muscle contraction, extracellular matrix composition and protein synthesis as compared to NGT counterparts. In conclusion, our study underlines the importance of the ion library assembly in the DIA analysis of clinical samples and confirms at molecular level changes connected with deficiency of muscle function in the prediabetic state.