Electrospray ionization (ESI) is essential for application of mass spectrometry in biological systems, as it prevents the analyte being split into fragments. However, due to lack of a clear understanding of the mechanism of ESI, the interpretation of mass spectra is often ambiguous. This is a particular challenge for complex biological systems. Here, we focus on systems that include nanodiscs as membrane environment, which are essential for membrane proteins. We performed microsecond atomistic molecular dynamics simulations to study the release of nanodiscs from highly charged nano-droplets into the gas phase, the late stage of ESI. We observed two distinct major scenarios, highlighting the diversity of morphologies of gaseous product ions. Our simulations are in reasonable agreement with experimental results. Our work provides a detailed atomistic view of the ESI process of a heterogeneous system (lipid nanodisc), which may give insights into the interpretation of mass spectra of all lipid-protein systems.

Electrospray ionization (ESI) prevents the analyte being split into fragments and is widely used to analyze biomolecules. Nanodiscs provide a native-like environment for membrane proteins, while making them accessible in aqueous solution for analysis. We performed microsecond molecular dynamics simulations at different temperatures, to provide atomistic insight in the release of intact nanodiscs from charged nano-droplets, the late stage of the ESI process. Two distinct main scenarios, at-center and off-center, were observed. The at-center scenario follows the charged residue model, and in the process the nanodisc stays well in the droplet interior, with the membrane scaffold protein (MSP) keeping its annular geometry. As solvent evaporates, lipids turn over to protect the hydrophilic surface. The off-center scenario conforms to a hybrid model of the charged residue model and chain ejection model. The nanodisc migrates to the water/air interface, leading to lipids escaping from the dissociated dimer and the partial ejection of one monomer of MSP. A diversity of morphologies of product gaseous ions was observed, as evidenced by their structural details, collision cross sections and moments of inertia. Our results are in reasonable agreement with results of mass spectrometry in mass-to-charge ratio, charge state and collision cross section. Our work displays the first atomistic view of a heterogeneous system, a lipid nanodisc, releasing from highly charged nano-droplets into the gas phase, which may provide structural supports for the interpretation of mass spectra of complicated systems. What we observe in our simulations could also be generalized to other protein-lipid systems.

Prostate-specific membrane antigen (PSMA) overexpressed on prostate cancer (PCa) cells is a satisfactory theranostic target in PCa. To seek novel non-glutamate-ureabased PSMA inhibitors by the strategy of bioisosterism, 10 ligands were designed, synthesized, and characterized. Among them, ligands 17, 18, and 21−24 bearing the squaramic acid moiety proved to be potent PSMA inhibitors, with K i values ranging from 0.40 to 2.49 nM, which are comparable or higher in inhibitory potency compared to previously reported glutamate-urea-based inhibitors. Docking studies of 15, 17, and 19 were carried out to explore their binding mode in the active site of PSMA. Two nearinfrared (NIR) probes, 23 (λ EM = 650 nm) and 24 (λ EM = 1088 nm), displayed favorable in vivo NIR imaging and successful NIR-II image-guided tumor resection surgery in PSMA-positive tumor-bearing mice, which demonstrated the effectiveness of these new squaramic acid-based inhibitors.