Authors: Frank Antony | A. K. Bhattacharya | Hiroyuki Hiroyuki | Rupinder Singh Jandu | Abdualrahman Mohammed Abdualkader | Rami Al Batran | Mohan Babu | Franck Duong
Source: bioRxiv (Cold Spring Harbor Laboratory) (preprint, 2025-08-29)
Field: Chemistry — Spectroscopy

Comparative Evaluation of Solid-Phase and Membrane Mimetic Strategies in Membrane Proteome Coverage and Disease-State Analysis Abstract 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. Highlights Systematic comparison of seven workflows for membrane proteomics Solid-phase methods enrich soluble proteins; mimetics enrich membrane proteins SMALP displays intermediate performance between other workflows Peptidisc captures the most dysregulated membrane proteins in diseased liver Peptidisc most accurate in detecting membrane protein dysregulation In Brief Statement This 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. Abstract Figure Proteome ; Membrane ; Phase (matter) ; Chemistry ; Disease ; Membrane protein ; Computational biology ; State (computer science) ; Computer science ; Biology ; Biochemistry ; Medicine ; Internal medicine ; Algorithm Advanced Proteomics Techniques and Applications ; Bacteriophages and microbial interactions ; SARS-CoV-2 detection and testing