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The SARS-CoV-2 Omicron variant has more than 15 mutations in the receptor binding domain of the Spike protein enabling increased transmissibility and viral escape from antibodies in vaccinated individuals. It is unclear how vaccine immunity protects against Omicron infection. Here we show that vaccinated participants at a super-spreader event have robust recall response of humoral and pre-existing cellular immunity induced by the vaccines, and an emergent de novo T cell response to non-Spike antigens. Individuals with Omicron SARS-CoV-2 breakthrough infections have significantly increased activated SARS-CoV-2 wild type Spike-specific cytotoxic T cells, activated follicular helper (T FH ) cells, functional T cell responses, boosted humoral responses, and rapid release of Spike and RBD-specific IgG + B cell plasmablasts and memory B cells into circulation. Omicron breakthrough infection affords significantly increased de novo memory T cell responses to non-Spike viral antigens. Concerted T and B cell responses may provide durable and broad immunity., (© 2022. The Author(s).)

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io ., (© 2021. The Author(s).)

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

We report a tool for the analysis of subcellular proteomics data, called MetaMass, based on the use of standardized lists of subcellular markers. We analyzed data from 11 studies using MetaMass, mapping the subcellular location of 5,970 proteins. Our analysis revealed large variations in the performance of subcellular fractionation protocols as well as systematic biases in protein annotation databases. The Excel and R versions of MetaMass should enhance transparency and reproducibility in subcellular proteomics.