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. 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