The performance of ultrasensitive liquid chromatography and tandem mass spectrometry (LC-MS/MS) methods, such as single-cell proteomics by mass spectrometry (SCoPE-MS), depends on multiple interdependent parameters. This interdependence makes it challenging to specifically pinpoint the sources of problems in the LC-MS/MS methods and approaches for resolving them. For example, a low signal at the MS2 level can be due to poor LC separation, ionization, apex targeting, ion transfer, or ion detection. We sought to specifically diagnose such problems by interactively visualizing data from all levels of bottom-up LC-MS/MS analysis. Many software packages, such as MaxQuant, already provide such data, and we developed an open source platform for their interactive visualization and analysis: Data-driven Optimization of MS (DO-MS). We found that in many cases DO-MS not only specifically diagnosed LC-MS/MS problems but also enabled us to rationally optimize them. For example, by using DO-MS to optimize the sampling of the elution peak apexes, we increased ion accumulation times and apex sampling, which resulted in a 370% more efficient delivery of ions for MS2 analysis. DO-MS is easy to install and use, and its GUI allows for interactive data subsetting and high-quality figure generation. The modular design of DO-MS facilitates customization and expansion. DO-MS v1.0.

COVID-19 CG (covidcg.org) is an open resource for tracking SARS-CoV-2 single-nucleotide variations (SNVs), lineages, and clades using the virus genomes on the GISAID database while filtering by location, date, gene, and mutation of interest. COVID-19 CG provides significant time, labor, and cost-saving utility to projects on SARS-CoV-2 transmission, evolution, diagnostics, therapeutics, vaccines, and intervention tracking. Here, we describe case studies in which users can interrogate (1) SNVs in the SARS-CoV-2 spike receptor binding domain (RBD) across different geographical regions to inform the design and testing of therapeutics, (2) SNVs that may impact the sensitivity of commonly used diagnostic primers, and (3) the emergence of a dominant lineage harboring an S477N RBD mutation in Australia in 2020. To accelerate COVID-19 efforts, COVID-19 CG will be upgraded with new features for users to rapidly pinpoint mutations as the virus evolves throughout the pandemic and in response to therapeutic and public health interventions.

Analysis by liquid chromatography and tandem mass spectrometry (LC‐MS/MS) can identify and quantify thousands of proteins in microgram‐level samples, such as those comprised of thousands of cells. Identifying proteins by LC‐MS/MS proteomics, however, remains challenging for lowly abundant samples, such as the proteomes of single mammalian cells. To increase the identification rate of peptides in such small samples, we developed DART‐ID. This method implements a data‐driven, global retention time (RT) alignment process to infer peptide RTs across experiments. DART‐ID then incorporates the global RT‐estimates within a principled Bayesian framework to increase the confidence in correct peptide‐spectrum‐matches. Applying DART‐ID to hundreds of samples prepared by the Single Cell Proteomics by Mass Spectrometry (SCoPE‐MS) design increased the peptide and proteome coverage by 30 – 50% at 1% FDR. The newly identified peptides and proteins were further validated by demonstrating that their quantification is consistent with the quantification of peptides identified from high‐quality spectra. DART‐ID can be applied to various sets of experimental designs with similar sample complexities and chromatography conditions, and is freely available online. Support or Funding Information This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial cells have a crucial role in nervous system function, and mounting evidence points to endothelial impairment as a major contributor to a wide range of neurological diseases. However, tools to genetically interrogate these cells in vivo remain limited. Here, we describe AAV-BI30, a capsid that specifically and efficiently transduces endothelial cells throughout the central nervous system. At relatively low systemic doses, this vector transduces the majority of arterial, capillary, and venous endothelial cells in the brain, retina, and spinal cord vasculature of adult C57BL/6 mice. Furthermore, we show that AAV-BI30 robustly transduces endothelial cells in multiple mouse strains and rats in vivo and human brain microvascular endothelial cells in vitro . Finally, we demonstrate AAV-BI30’s capacity to achieve efficient and endothelial-specific Cre-mediated gene manipulation in the central nervous system. This combination of attributes makes AAV-BI30 uniquely well-suited to address outstanding research questions in neurovascular biology and aid the development of therapeutics to remediate endothelial dysfunction in disease.

COVID-19 CG is an open resource for tracking SARS-CoV-2 single-nucleotide variations (SNVs) and lineages while filtering by location, date, gene, and mutation of interest. COVID-19 CG provides significant time, labor, and cost-saving utility to diverse projects on SARS-CoV-2 transmission, evolution, emergence, immune interactions, diagnostics, therapeutics, vaccines, and intervention tracking. Here, we describe case studies in which users can interrogate (1) SNVs in the SARS-CoV-2 Spike receptor binding domain (RBD) across different geographic regions to inform the design and testing of therapeutics, (2) SNVs that may impact the sensitivity of commonly used diagnostic primers, and (3) the recent emergence of a dominant lineage harboring an S477N RBD mutation in Australia. To accelerate COVID-19 research and public health efforts, COVID-19 CG will be continually upgraded with new features for users to quickly and reliably pinpoint mutations as the virus evolves throughout the pandemic and in response to therapeutic and public health interventions.

The performance of ultrasensitive LC-MS/MS methods, such as Single-Cell Proteomics by Mass Spectrometry (SCoPE-MS), depends on multiple interdependent parameters. This interdependence makes it challenging to specifically pinpoint bottlenecks in the LC-MS/MS methods and approaches for resolving them. For example, low signal at MS2 level can be due to poor LC separation, ionization, apex targeting, ion transfer, or ion detection. We sought to specifically diagnose such bottlenecks by interactively visualizing data from all levels of bottom-up LC-MS/MS analysis. Many search engines, such as MaxQuant, already provide such data, and we developed an open source platform for their interactive visualization and analysis: Data-driven Optimization of MS (DO-MS). We found that in many cases DO-MS not only specifically diagnosed bottlenecks but also enabled us to rationally optimize them. For example, we used DO-MS to diagnose poor sampling of the elution peak apex and to optimize it, which increased the efficiency of delivering ions for MS2 analysis by 370%. DO-MS is easy to install and use, and its GUI allows for interactive data subsetting and high-quality figure generation. The modular design of DO-MS facilitates customization and expansion. DO-MS is available for download from GitHub: github.com/SlavovLab/DO-MS

Animals change developmental fates in response to external cues. In the nematode Caenorhabditis elegans, unfavorable environmental conditions induce a state of diapause known as dauer by inhibiting the conserved DAF-2 insulin-like signaling (ILS) pathway through incompletely understood mechanisms. We have previously established a role for the C. elegans dosage compensation protein DPY-21 in the control of dauer arrest and DAF-2 ILS. Here, we show that the histone H4 lysine 20 methyltransferase SET-4, which also influences dosage compensation, promotes dauer arrest in part by repressing the X-linked ins-9 gene, which encodes a new agonist insulin-like peptide (ILP) expressed specifically in the paired ASI sensory neurons that are required for dauer bypass. ins-9 repression in dauer-constitutive mutants requires DPY-21, SET-4 and the FoxO transcription factor DAF-16, which is the main target of DAF-2 ILS. By contrast, autosomal genes encoding major agonist ILPs that promote reproductive development are not repressed by DPY-21, SET-4 or DAF-16/FoxO. Our results implicate SET-4 as a sensory rheostat that reinforces developmental fates in response to environmental cues by modulating autocrine and paracrine DAF-2 ILS.

9Analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS) can iden- 24. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.