Combining native mass spectrometry and lipidomics to uncover specific membrane protein–lipid interactions from natural lipid sources

Abstract

While it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative...

Native mass spectrometry of proteoliposomes containing integral and peripheral membrane proteins

“…Proteoliposomes containing POPE and TOCDL were selected because previous work has shown PE enhances AmtB–CDL interactions. 53,56 AmtB could readily be ejected from the proteoliposomes containing different lipids in the presence of m -NBA (Fig. 4).…”

“…This observation is consistent with previous work showing that PE enhances AmtB–CDL interactions. 53,56 Moreover, the intensities of AmtB-lipid bound species is complex and the irregularity indicates these may likely represent mixed lipid bound states. TOCDL is about twice the mass of POPE, and the broadness of the adduct peaks makes it difficult to assign with confidence.…”

“…We have recently developed an approach that combines native MS and lipidomic approaches to identify specific protein–lipids interactions from natural sources, such as a brain polar lipid extract 56 (Fig. S9 † ).…”

“…This result is consistent with previous findings that PE enhances AmtB–CDL interactions. 48,56 TRAAK reconstituted in 10% POPA ejected bound to POPA and POPC, and in combination with copper( ii ). The observation of POPA bound, despite being only 10% in composition, is consistent with results obtained in detergent, where POPA binds TRAAK with about four-fold higher affinity over POPC.…”

Advancing Kir4.2 Channel Ligand Identification through Collision-Induced Affinity Selection Mass Spectrometry

“…Investigation of membrane proteins poses challenges owing to their amphipathic nature, low natural abundance, and difficulties in overexpression and purification . Native mass spectrometry (native MS) has emerged as a robust platform for investigating membrane proteins, using collisional activation to liberate membrane proteins from detergent micelles. − Native MS has proven valuable in characterizing protein–lipid interactions. − Within the context of Kir channels, lipids binding to Kir3.2 and Kir3.4 have been reported. − The selectivity of Kir3.2 toward various phospholipids was investigated, revealing that Kir3.2 exhibits a preference for phosphatidylinositide (PIP) isoforms, particularly for the phosphatidylinositol 4,5-bisphosphate (PI­(4,5)­P 2 ) headgroup, compared to other phospholipids. , Kir3.4 also displayed differences in lipid binding selectivity, with weaker interactions with PIPs compared to that of Kir3.2 . The binding thermodynamics between Kir3.2 and lipids showed that the interaction between Kir3.2 and specific PIPs is highly influenced by entropy …”