Deep Structural Characterization of Protein-Bound Lipids via Native MS and Ultraviolet Photodissociation

Protein-lipid interactions are critical for maintaining membrane protein structure and regulating diverse protein functions. Native mass spectrometry (MS) has emerged as a powerful technique for the direct observation and characterization of protein-lipid complexes. However, intact mass measurements alone cannot resolve important structural details such as the identity of lipid acyl chains and their modifications. To fully characterize protein-bound lipids, we present a multistage native MS method that leverages ultraviolet photodissociation to elucidate the precise molecular composition of heterogeneous protein-lipid assemblies. We demonstrate the utility of this approach for both soluble and membrane proteins. First, we comprehensively define the endogenous lipids bound to the bacterial transporter MlaC, distinguishing between unsaturated and cyclopropane lipids, and localizing acyl chains and their modifications. Next, we characterize and quantify phospholipids associated with the bacterial membrane protein AqpZ and show that the approach can be extended to more complex cardiolipins containing four lipid chains. Together, our workflow provides detailed structural insights into protein-lipid interactions and offers a path toward uncovering protein-specific metabolic regulation that is not accessible through classical lipidomics workflows.