Simple and rapid TLC blotting-based methodology for screening membrane protein-specific lipids

Abstract

We present a novel methodology for the rapid and simplified screening of membrane protein (MP)-specific lipids, utilizing thin-layer chromatography (TLC) to separate lipid mixtures, followed by colorimetric detection using our recently developed MP-immobilized gold nanoparticles (AuNPs). After separation, the lipids on the TLC plate were transferred to a polyvinylidene difluoride (PVDF) membrane and visualized colorimetrically with MP-functionalized AuNPs. Lipid species on the TLC or PVDF membrane were further annotated via mass spectrometry. To enhance the visualization of specifically interacting lipids and minimize non-specific binding on the PVDF membrane, several factors, including AuNP size, blocking with bovine serum albumin, and detergent washing, were optimized using bacteriorhodopsin (bR)-immobilized AuNPs. Consequently, S-TGD-1, a known bR-specific lipid, was strongly detected, confirming the effectiveness of this method. We then applied this approach to the potassium channel KcsA and demonstrated that cardiolipin band on the PVDF was most strongly stained by KcsA-immobilized AuNPs. This is consistent with previous reports identifying cardiolipin as a KcsA-specific lipid. To more quantitatively identify MP-specific lipids, we also introduced the Binding Index (BI), which reproduced that S-TGD-1 and cardiolipin are specific to bR and KcsA, respectively. Thus, the utility of this methodology was demonstrated through studies with bR and KcsA. This technique offers a broadly applicable approach for identifying MP-binding lipids without requiring extensive lipid purification or sophisticated instrumentation, providing a versatile tool for studying MP–lipid interactions.

Graphical Abstract