Single-Cell and Subcellular Analysis Using Ultrahigh Resolution 21 T MALDI FTICR Mass Spectrometry.

The mammalian brain contains ∼20,000 distinct lipid species that contribute to its structural organization and function. The lipid profiles of cells change in response to a variety of cellular signals and environmental conditions that result in modulation of cell function through alteration of phenotype. The limited sample material combined with the vast chemical diversity of lipids makes comprehensive lipid profiling of individual cells challenging. Here, we leverage the resolving power of a 21 T Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer for chemical characterization of individual hippocampal cells at ultrahigh mass resolution. The accuracy of the acquired data allowed differentiation of freshly isolated and cultured hippocampal cell populations, as well as finding differences in lipids between the soma and neuronal processes of the same cell. Differences in lipids include TG 42:2 observed solely in the cell bodies and SM 34:1;O2 found only in the cellular processes. The work represents the first mammalian single cells analyzed at ultrahigh resolution and is an advance in the performance of mass spectrometry (MS) for single-cell research.

New Pathways of Mutational Change in SARS-CoV-2 Proteomes Involve Regions of Intrinsic Disorder Important for Virus Replication and Release.

The massive worldwide spread of the SARS-CoV-2 virus is fueling the COVID-19 pandemic. Since the first whole-genome sequence was published in January 2020, a growing database of tens of thousands of viral genomes has been constructed. This offers opportunities to study pathways of molecular change in the expanding viral population that can help identify molecular culprits of virulence and virus spread. Here we investigate the genomic accumulation of mutations at various time points of the early pandemic to identify changes in mutationally highly active genomic regions that are occurring worldwide. We used the Wuhan NC_045512.2 sequence as a reference and sampled 15 342 indexed sequences from GISAID, translating them into proteins and grouping them by month of deposition. The per-position amino acid frequencies and Shannon entropies of the coding sequences were calculated for each month, and a map of intrinsic disorder regions and binding sites was generated. The analysis revealed dominant variants, most of which were located in loop regions and on the surface of the proteins. Mutation entropy decreased between March and April of 2020 after steady increases at several sites, including the D614G mutation site of the spike (S) protein that was previously found associated with higher case fatality rates and at sites of the NSP12 polymerase and the NSP13 helicase proteins. Notable expanding mutations include R203K and G204R of the nucleocapsid (N) protein inter-domain linker region and G251V of the viroporin encoded by ORF3a between March and April. The regions spanning these mutations exhibited significant intrinsic disorder, which was enhanced and decreased by the N-protein and viroporin 3a protein mutations, respectively. These results predict an ongoing mutational shift from the spike and replication complex to other regions, especially to encoded molecules known to represent major ß-interferon antagonists. The study provides valuable information for therapeutics and vaccine design, as well as insight into mutation tendencies that could facilitate preventive control.