RESUMEN
Mass photometry (MP) is a rapidly growing optical technique for label-free mass measurement of single biomolecules in solution. The underlying measurement principle provides numerous advantages over ensemble-based methods but has been limited to low analyte concentrations due to the need to uniquely and accurately quantify the binding of individual molecules to the measurement surface, which results in diffraction-limited spots. Here, we combine nanoparticle lithography with surface PEGylation to substantially lower surface binding, resulting in a 2 orders of magnitude improvement in the upper concentration limit associated with mass photometry. We demonstrate the facile tunability of degree of passivation, enabling measurements at increased analyte concentrations. These advances provide access to protein-protein interactions in the high nanomolar to low micromolar range, substantially expanding the application space of mass photometry.
Asunto(s)
Fotometría , Polietilenglicoles , Polietilenglicoles/química , Fotometría/métodos , Propiedades de Superficie , Nanopartículas/química , Proteínas/química , Proteínas/análisisRESUMEN
Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (â¼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of â¼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Unión Proteica , Espectrometría de Masas , PolisacáridosRESUMEN
RNA molecules have emerged as increasingly attractive biomaterials with important applications such as RNA interference (RNAi) for cancer treatment and mRNA vaccines against infectious diseases. However, it remains challenging to engineer RNA biomaterials with sophisticated functions such as non-covalent light-switching ability. Herein, light-responsive RNA-protein nanowires are engineered to have such functions. It first demonstrates that the high affinity of RNA aptamer enables the formation of long RNA-protein nanowires through designing a dimeric RNA aptamer and an engineered green fluorescence protein (GFP) that contains two TAT-derived peptides at N- and C- termini. GFP is then replaced with an optogenetic protein pair system, LOV2 (light-oxygen-voltage) protein and its binding partner ZDK (Z subunit of protein A), to confer blue light-controlled photo-switching ability. The light-responsive nanowires are long (>500 nm) in the dark, but small (20-30 nm) when exposed to light. Importantly, the co-assembly of this RNA-protein hybrid biomaterial does not rely on the photochemistry commonly used for light-responsive biomaterials, such as bond formation, cleavage, and isomerization, and is thus reversible. These RNA-protein structures can serve as a new class of light-controlled biocompatible frameworks for incorporating versatile elements such as RNA, DNA, and enzymes.
Asunto(s)
Aptámeros de Nucleótidos , Nanocables , ARN/química , Aptámeros de Nucleótidos/química , Interferencia de ARN , Péptidos , Proteínas Fluorescentes VerdesRESUMEN
Glycosaminoglycans (GAGs) are heterogeneous acidic polysaccharides involved in a range of biological functions. They have a significant influence on the regulation of cellular processes and the development of various diseases and infections. To fully understand the functional roles that GAGs play in mammalian systems, including disease processes, it is essential to understand their structural features. Despite having a linear structure and a repetitive disaccharide backbone, their structural analysis is challenging and requires elaborate preparative and analytical techniques. In particular, the extent to which GAGs are sulfated, as well as variation in sulfate position across the entire oligosaccharide or on individual monosaccharides, represents a major obstacle. Here, we summarize the current state-of-the-art methodologies used for GAG sample preparation and analysis, discussing in detail liquid chromatograpy and mass spectrometry-based approaches, including advanced ion activation methods, ion mobility separations and infrared action spectroscopy of mass-selected species.
Asunto(s)
Disacáridos , Glicosaminoglicanos , Animales , Glicosaminoglicanos/análisis , Glicosaminoglicanos/química , Mamíferos , Espectrometría de Masas/métodos , Monosacáridos , Oligosacáridos , Polisacáridos , Sulfatos/análisisRESUMEN
Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Lípidos/química , Lípidos/farmacología , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Multimerización de Proteína/efectos de los fármacos , Sitios de Unión/genética , Cardiolipinas/química , Cardiolipinas/metabolismo , Cardiolipinas/farmacología , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Moritella/química , Estabilidad Proteica/efectos de los fármacos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Termodinámica , Thermus thermophilus/químicaRESUMEN
Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.
Asunto(s)
Gránulos Citoplasmáticos/enzimología , Glicopéptidos/metabolismo , Neutrófilos/enzimología , Peroxidasa/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Glicopéptidos/química , Glicosilación , HumanosRESUMEN
Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.
Asunto(s)
ADN/química , Espectrometría de Masas/métodos , Fotometría/métodos , Imagen Individual de Molécula/métodos , ADN/análisisRESUMEN
Glycoproteins are inherently heterogeneous and therefore resolving structures in their entirety remains a major challenge in structural biology. Native mass spectrometry has transformed our ability to study glycoproteins, and despite advances in high-resolution instrumentation, there are comparatively a few studies demonstrating its potential with data largely limited to an overall measure of monosaccharide composition for all glycans across glycosylation sites for a given protein. Clearly, these readouts lack glycan topology information, namely, monosaccharide linkage and glycan branching. To address this deficiency, we developed a new approach that joins native mass spectrometry with glycan exoglycosidase sequencing, the combination of which provides remarkable glycoprotein structural details. We show how N-glycan branching, terminal fucosylation, LacNAc extensions, and N- and O-glycan occupancy (i.e., total number of glycans) can be directly characterized on intact glycoproteins with minimal sample preparation. Taken together, native exoglycosidase sequencing mass spectrometry (NES-MS) notably improves our ability to characterize protein glycosylation, addressing a significant need in structural biology that will enable new routes to understand glycoprotein function.
Asunto(s)
Glicómica , Glicoproteínas , Glicoproteínas/metabolismo , Glicosilación , Espectrometría de Masas , PolisacáridosRESUMEN
Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2â3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.
Asunto(s)
Glicoproteínas/química , Espectrometría de Movilidad Iónica/métodos , Polisacáridos/análisis , Polisacáridos/química , Acetilgalactosamina/química , Glicoproteínas/genética , Células HEK293 , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , Humanos , Nitrógeno/química , Multimerización de Proteína , Proteínas Recombinantes/genética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Structural determination of N-glycans by mass spectrometry is ideally performed by negative ion collision-induced dissociation because the spectra are dominated by cross-ring fragments leading to ions that reveal structural details not available by many other methods. Most glycans form [M - H]- or [M + adduct]- ions but larger ones (above approx. m/z 2000) typically form doubly charged ions. Differences have been reported between the fragmentation of singly and doubly charged ions but a detailed comparison does not appear to have been reported. In addition to [M + adduct]- ions (this paper uses phosphate as the adduct) other doubly, triply, and quadruply charged ions of composition [Mn + (H2PO4)n]n- have been observed in mixtures of N-glycans released from viral and other glycoproteins. This paper explores the formation and fragmentation of these different types of multiply charged ions with particular reference to the presence of diagnostic fragments in the CID spectra and comments on how these ions can be used to characterize these glycans.
Asunto(s)
Glicoproteínas/química , Polisacáridos/análisis , Polisacáridos/química , Espectrometría de Movilidad Iónica/métodos , Iones , Espectrometría de Masa por Ionización de Electrospray/métodos , ortoaminobenzoatos/químicaRESUMEN
Protein-protein interactions are involved in the regulation and function of the majority of cellular processes. As a result, much effort has been aimed at the development of methodologies capable of quantifying protein-protein interactions, with label-free methods being of particular interest due to the associated simplified workflows and minimisation of label-induced perturbations. Here, we review recent advances in optical technologies providing label-free in vitro measurements of affinities and kinetics. We provide an overview and comparison of existing techniques and their principles, discussing advantages, limitations, and recent applications.
Asunto(s)
Proteínas/química , Cinética , Fenómenos Ópticos , Unión Proteica , Proteínas/metabolismoRESUMEN
Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein-protein, or protein-small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin-hemoglobin (Hp-Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp-Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein-drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.
Asunto(s)
Glucanos/química , Haptoglobinas/química , Modelos Químicos , Orosomucoide/química , Warfarina/química , Glucanos/metabolismo , Haptoglobinas/metabolismo , Humanos , Orosomucoide/metabolismoRESUMEN
In the version of this article initially published, authors Sarah E. Wilkins, Charlotte D. Eaton, Martine I. Abboud and Maximiliano J. Katz were incorrectly included in the equal contributions footnote in the affiliations list. Footnote number seven linking to the equal contributions statement should be present only for Suzana Markolovic and Qinqin Zhuang, and the statement should read "These authors contributed equally: Suzana Markolovic, Qinqin Zhuang." The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(II)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.
Asunto(s)
Biocatálisis , GTP Fosfohidrolasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , GTP Fosfohidrolasas/química , Humanos , Hidroxilación , Histona Demetilasas con Dominio de Jumonji/química , Modelos MolecularesRESUMEN
Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Fiebre Hemorrágica Americana/inmunología , Pruebas de Neutralización , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Glicoproteínas/química , Células HEK293 , Humanos , Sistema Inmunológico , Virus Junin , Unión Proteica , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/químicaRESUMEN
The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Herein, we report the development and application of a mass-spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Coreâ 1 and 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the DC-SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites.
Asunto(s)
Moléculas de Adhesión Celular/química , Lectinas Tipo C/química , Polisacáridos/química , Receptores de Superficie Celular/química , Moléculas de Adhesión Celular/análisis , Glicosilación , Células HEK293 , Humanos , Espectrometría de Movilidad Iónica/métodos , Lectinas Tipo C/análisis , Espectrometría de Masas/métodos , Polisacáridos/análisis , Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Estabilidad Proteica , Receptores de Superficie Celular/análisisRESUMEN
Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single-molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.
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Proteínas/química , Espectrofotometría Ultravioleta/métodos , CinéticaRESUMEN
The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to design guidelines that improve the reporting and reproducibility of glycoanalytical methods. Previously, the MIRAGE Commission has published guidelines for describing sample preparation methods and the reporting of glycan array and mass spectrometry techniques and data collections. Here, we present the first version of guidelines that aim to improve the quality of the reporting of liquid chromatography (LC) glycan data in the scientific literature. These guidelines cover all aspects of instrument setup and modality of data handling and manipulation and is cross-linked with other MIRAGE recommendations. The most recent version of the MIRAGE-LC guidelines is freely available at the MIRAGE project website doi:10.3762/mirage.4.
Asunto(s)
Glicómica , Polisacáridos/análisis , Cromatografía Liquida , HumanosRESUMEN
Glycosylation is one of the most important post-translational modifications essential for modulating biological functions on cellular surfaces and within cells. Glycan structures are not predictable from the genome since their biosynthesis is nontemplate driven and subject to multiple sequential and competitive glycosyltransferases/glycosidases. From a structural viewpoint, their analysis presents a particular challenge in terms of sensitivity and structural characterization. Porous graphitized carbon liquid chromatography coupled mass spectrometry (PGCLC-MS) is arguably the gold-standard for the structural characterization of glycoconjugates, especially complex mixtures typical in biological samples. This high performance is due in large part to chromatographic separation of isomers and the information delivered by collision induced fragmentation of each glycan in the mass spectrometer. More recently, ion mobility mass spectrometry (IM-MS) has emerged as an effective tool for gas-phase separation of isomeric oligosaccharides that has been demonstrated with small oligosaccharides and N-glycans. Here, we present a direct comparison of the IM- and LC-separation of O-glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by PGCLC and/or IM, validating the combination of the two methods. Taken together, the incorporation of both techniques into a single platform would be powerful and undoubtedly valuable for determining the full glycome of unknown samples.
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Mucinas Gástricas/química , Mucosa Bucal/química , Polisacáridos/aislamiento & purificación , Animales , Cromatografía Liquida , Humanos , Espectrometría de Movilidad Iónica , Espectrometría de Masas , Tamaño de la Partícula , Polisacáridos/química , Porosidad , Propiedades de Superficie , PorcinosRESUMEN
Small molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry-based method for analyzing intact membrane protein-ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.