Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.Key words: Lipid-protein interactions, Structural biology, Structural lipidomics, Protein mass spectrometry Lipid-Protein Interactions P roteins and lipids, both integral for the function of all biological systems, are chemically distinct. A protein's three-dimensional structure and ultimately its function are defined by peptide-bonded amino acids and subsequent posttranslational modifications. In contrast, lipids are smaller molecules than proteins, but have a larger array of building blocks and linkage chemistries. Because of these inherent chemical differences, the optimal tools for de novo structural characterization differ between proteins and lipids.Lipid-protein interactions are critical for maintaining biological function in the membrane bilayer, the cytosol, and extracellular space. In the membrane, the chemical structures of lipid solvent molecules are important for the correct folding, insertion, structure, and function of membrane proteins. The interactions between enzymes and their respective lipid substrates are critical for correct substrate recognition and catalysis (e.g., lipoxygenase [1]). Additionally, lipid-protein interactions are increasingly being realized as important in maintaining cellular structure (e.g., cytoskeletal anchoring [2]), scaffolding and localization of multi-subunit protein complexes (e.g., associated kinase anchoring protein complexes [3]), and as second messengers in signal transduction (e.g., protein kinase C [4]).