Supercritical fluid chromatography is traditionally employed for nonpolar and moderately polar analytes, while the analysis of ionic compounds remains a recognized limitation of the technique. Moreove Show more
Supercritical fluid chromatography is traditionally employed for nonpolar and moderately polar analytes, while the analysis of ionic compounds remains a recognized limitation of the technique. Moreover, some polar lipids may contain a chromatographically challenging ionic group, which can interact with the metal surfaces of the instrument and column, resulting in poor peak shape and loss of sensitivity. Here, we introduce a novel ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method using a bioinert column, enabling the separation of lipids with a broad polarity range from nonpolar to ionic species. The UHPSFC/MS method was optimized using 79 lipid species across 41 lipid subclasses, achieving a total run time of 7.5 min, including the column equilibration. The comparison of the separation with conventional and bioinert columns revealed a substantial improvement in peak shapes for ionic lipid classes, such as PS, LPS, PA, LPA, CerP, and SPBP. Additionally, we introduce a combination of the modified chloroform-free extraction followed by a hexane elimination step. The optimized methodology was applied for the untargeted analysis of human plasma and erythrocyte-rich fraction to achieve highly confident identification of 657 lipid species across 37 lipid subclasses in human blood. The method follows the recommendations for validation of (bio)analytical methods, and its accuracy was confirmed by quantitative analysis of the reference material NIST SRM 1950, with the determined concentrations in agreement with the consensus values from ring trials. The current methodology represents a novel high-throughput and comprehensive quantitative lipidomic method for biological samples. The modified MTBE extraction enhances workflow efficiency by reducing concentrations of nonpolar lipids, which enables injection of more concentrated lipid extracts while minimizes ion source contamination. Moreover, the findings highlight the potential for the development of bioinert components specifically designed for SFC platforms, enabling broader applicability of the technique. Show less
Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance, and lipid trafficking. The exact rol Show more
Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance, and lipid trafficking. The exact role of Miro in these functions is unclear but involves binding to a variety of client proteins. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro interactors-namely, CENPF, Trak, and MYO19-all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the first calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including MTFR1/2/1L, the lipid transporters Mdm34 and VPS13D, and the ubiquitin E3-ligase Parkin. Given the shared binding mechanism of these functionally diverse clients and its conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health. Show less