Ion-chromatogram libraries assembly in DIA proteomic analysis of post-exercise skeletal muscle in prediabetic subjects

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. Due to its molecular composition proteomic analysis of skeletal muscle is challenging. To identify optimum approach, 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. GPF-fractionated, hybrid DDA/DIA libraries yielded the best overall balance between the speed of preparation, data collection and protein identification. 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 selection of appropriate approach in the analysis of challenging clinical samples and reveals the potential explanation for deficiency of muscle function in the prediabetic state.