No abstract

No abstract

No abstract

The expansion of native mass spectrometry (MS) methods for both academic and industrial applications has created a substantial need for analysis of large native MS datasets. Existing software tools are poorly suited for high-throughput deconvolution of native electrospray mass spectra from intact proteins and protein complexes. The UniDec Bayesian deconvolution algorithm is uniquely well suited for high-throughput analysis due to its speed and robustness but was previously tailored towards individual spectra. Here, we optimized UniDec for deconvolution, analysis, and visualization of large data sets. This new module, MetaUniDec, centers around a hierarchical data format 5 (HDF5) format for storing datasets that significantly improves speed, portability, and file size. It also includes code optimizations to improve speed and a new graphical user interface for visualization, interaction, and analysis of data. To demonstrate the utility of MetaUniDec, we applied the software to analyze automated collision voltage ramps with a small bacterial heme protein and large lipoprotein nanodiscs. Upon increasing collisional activation, bacterial heme-nitric oxide/oxygen binding (H-NOX) protein shows a discrete loss of bound heme, and nanodiscs show a continuous loss of lipids and charge. By using MetaUniDec to track changes in peak area or mass as a function of collision voltage, we explore the energetic profile of collisional activation in an ultra-high mass range Orbitrap mass spectrometer. Graphical abstract ᅟ.

Myosin-VI has been implicated in endocytic trafficking at both the clathrin-coated and uncoated vesicle stages. The identification of alternative splice forms led to the suggestion that splicing defines the vesicle type to which myosin-VI is recruited. In contrast to this hypothesis, we find that in all cell types examined, myosin-VI is associated with uncoated endocytic vesicles, regardless of splice form. GIPC, a PDZ-domain containing adapter protein, co-assembles with myosin-VI on these vesicles. Myosin-VI is only recruited to clathrin-coated vesicles in cells that express high levels of Dab2, a clathrin-binding adapter protein. Overexpression of Dab2 is sufficient to reroute myosin-VI to clathrin-coated pits in cells where myosin-VI is normally associated with uncoated vesicles. In normal rat kidney (NRK) cells, which express high endogenous levels of Dab2, splicing of the globular tail domain further modulates targeting of ectopically expressed myosin-VI. Although myosin-VI can be recruited to clathrin-coated pits, we find no requirement for myosin-VI motor activity in endocytosis in NRK cells. Instead, our data suggest that myosin-VI recruitment to clathrin-coated pits may be an early step in the recruitment of GIPC to the vesicle surface.