Al Batran, R. | bioRxiv Science

bioRxiv2025

Comparative Evaluation of Solid-Phase and Membrane Mimetic Strategies in Membrane Proteome Coverage and Disease-State Analysis

Membrane proteins (MPs) are vital to cellular signaling, metabolism, and disease pathology, yet remain underrepresented in proteomics. To address this, several independent workflows have been developed to enable the profiling of the membrane proteome, however the relative advantages and limitations of each method remain poorly defined. Here, we systematically compare four classical solid-phase membrane proteomic workflows (SP3, SP4, FASP, S-Trap) and three membrane mimetic strategies (Peptidisc, nanodisc, and SMALP copolymer) for mass spectrometry-based membrane proteome profiling, using healthy (LFD) and obese (HFD) mouse liver tissue. We found that the solid-phase methods yield higher total protein identifications, while the membrane mimetic systems enrich MPs. SMALP copolymer displays intermediate characteristics between the solid-phase and membrane mimetic workflows. Peptidisc and nanodisc stand out for their enrichment of MPs, although Peptidisc shows better enrichment of plasma membrane integral MPs, particularly those with 11+ transmembrane segments. In the context of HFD-induced liver proteome remodeling, the Peptidisc workflow outperformed the other six methods by capturing the highest number of differentially expressed MPs and demonstrating the greatest accuracy in detecting MP-level dysregulation. Collectively, this comparative analysis highlights the trade-offs between depth of proteome coverage and MP enrichment across workflows, underscoring the importance of method selection based on total protein counts, MP enrichment, and the accurate detection of MP-level dysregulation. HighlightsO_LISystematic comparison of seven workflows for membrane proteomics C_LIO_LISolid-phase methods enrich soluble proteins; mimetics enrich membrane proteins C_LIO_LISMALP displays intermediate performance between other workflows C_LIO_LIPeptidisc captures the most dysregulated membrane proteins in diseased liver C_LIO_LIPeptidisc most accurate in detecting membrane protein dysregulation C_LI In Brief StatementThis study presents a systematic comparison of seven proteomic workflows for membrane protein profiling. Solid-phase methods yield higher total protein identifications, whereas membrane mimetics enrich more membrane proteins. Among tested methods on the diseased mouse liver, Peptidisc captures more differentially expressed membrane proteins and demonstrates superior accuracy in detecting membrane protein-level dysregulation. These findings provide a practical framework for selecting proteomic strategies tailored to membrane protein enrichment and biological insight. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/672181v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1217dc7org.highwire.dtl.DTLVardef@1c8c188org.highwire.dtl.DTLVardef@119db19org.highwire.dtl.DTLVardef@7f5642_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Loss of Skeletal Muscle Pyruvate Dehydrogenase Induces Lactic Acidosis and Adaptive Anaplerotic Compensation via Pyruvate-Alanine Cycling and Glutaminolysis

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation that links glycolysis-derived pyruvate with the TCA cycle. Although skeletal muscle is a significant site for glucose oxidation and is closely linked with metabolic flexibility, the importance of muscle PDH during rest and exercise has yet to be fully elucidated. Here, we demonstrate that mice with muscle-specific deletion of PDH exhibit rapid weight loss and suffer from severe lactic acidosis, ultimately leading to early mortality under low-fat diet provision. Furthermore, loss of muscle PDH induces adaptive anaplerotic compensation by increasing pyruvate-alanine cycling and glutaminolysis. Interestingly, high-fat diet supplementation effectively abolishes the early mortality and rescues the overt metabolic phenotype induced by muscle PDH deficiency. Despite increased reliance on fatty acid oxidation during high-fat diet provision, loss of muscle PDH worsens exercise performance and induces lactic acidosis. These observations illustrate the importance of muscle PDH in maintaining metabolic flexibility and preventing the development of metabolic disorders. HighlightsO_LISkeletal Muscle PDH is essential for survival C_LIO_LILoss of muscle PDH induces lactic acidosis and premature death C_LIO_LILoss of muscle PDH enhances pyruvate transformations and glutaminolysis C_LIO_LIHigh-fat diet supplementation abolishes early mortality and overt phenotype induced by muscle PDH loss C_LI

cell biology↗