Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting.

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. Rather than a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike glycopeptides. This database allows any accurate mass LC-MS laboratory to reliably identify and quantify Spike glycopeptides from a single overnight elastase digest in less than 90 minutes.

Reconstitution of the core of the malaria parasite glideosome with recombinant Plasmodium class XIV myosin A and Plasmodium actin.

Motility of the apicomplexan malaria parasite Plasmodium falciparum is enabled by a multiprotein glideosome complex, whose core is the class XIV myosin motor, PfMyoA, and a divergent Plasmodium actin (PfAct1). Parasite motility is necessary for host-cell invasion and virulence, but studying its molecular basis has been hampered by unavailability of sufficient amounts of PfMyoA. Here, we expressed milligram quantities of functional full-length PfMyoA with the baculovirus/Sf9 cell expression system, which required a UCS (UNC-45/CRO1/She4p) family myosin chaperone from Plasmodium spp. In addition to the known light chain myosin tail interacting protein (MTIP), we identified an essential light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. The speed at which PfMyoA moved actin was fastest with both light chains bound, consistent with the light chain-binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfELC binding to the heavy chain required that MTIP also be bound to the heavy chain, unlike MTIP that bound the heavy chain independently of PfELC. Neither the presence of calcium nor deletion of the MTIP N-terminal extension changed the speed of actin movement. Of note, PfMyoA moved filaments formed from Sf9 cell-expressed PfAct1 at the same speed as skeletal muscle actin. Duty ratio estimates suggested that as few as nine motors can power actin movement at maximal speed, a feature that may be necessitated by the dynamic nature of Plasmodium actin filaments in the parasite. In summary, we have reconstituted the essential core of the glideosome, enabling drug targeting of both of its core components to inhibit parasite invasion.