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Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by a lack of high-throughput, unbiased, and quantitative methods to identify protein-calcium engagement. To address this, we adapted protein thermostability assays in budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2,884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-coenzyme A (CoA) reductase DECR1 binds calcium at physiologically relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These discovery-based proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes., Competing Interests: Declaration of interests D.K.S. is a consultant and/or collaborator with Thermo Fisher Scientific, AI Proteins, Genentech, and Matchpoint Therapeutics., (Copyright © 2024. Published by Elsevier Inc.)

Epigenetic inhibitors exhibit powerful antiproliferative and anticancer activities. However, cellular responses to small-molecule epigenetic inhibition are heterogenous and dependent on factors such as the genetic background, metabolic state, and on-/off-target engagement of individual small-molecule compounds. The molecular study of the extent of this heterogeneity often measures changes in a single cell line or using a small number of compounds. To more comprehensively profile the effects of small-molecule perturbations and their influence on these heterogeneous cellular responses, we present a molecular resource based on the quantification of chromatin, proteome, and transcriptome remodeling due to histone deacetylase inhibitors (HDACi) in non-isogenic cell lines. Through quantitative molecular profiling of 10,621 proteins, these data reveal coordinated molecular remodeling of HDACi treated cancer cells. HDACi-regulated proteins differ greatly across cell lines with consistent (JUN, MAP2K3, CDKN1A) and divergent (CCND3, ASF1B, BRD7) cell-state effectors. Together these data provide valuable insight into cell-type driven and heterogeneous responses that must be taken into consideration when monitoring molecular perturbations in culture models., Competing Interests: Conflict of Interest: We acknowledge our ongoing collaborations and sponsored agreements with Thermo Fisher Scientific, Genentech, and AI Proteins.

Gastrulation is the highly coordinated process by which the early embryo breaks symmetry, establishes germ layers and a body plan, and sets the stage for organogenesis. As early mammalian development is challenging to study in vivo, stem cell-derived models have emerged as powerful surrogates, e.g. human and mouse gastruloids. However, although single cell RNA-seq (scRNA-seq) and high-resolution imaging have been extensively applied to characterize such in vitro embryo models, a paucity of measurements of protein dynamics and regulation leaves a major gap in our understanding. Here, we sought to address this by applying quantitative proteomics to human and mouse gastruloids at four key stages of their differentiation (naïve ESCs, primed ESCs, early gastruloids, late gastruloids). To the resulting data, we perform network analysis to map the dynamics of expression of macromolecular protein complexes and biochemical pathways, including identifying cooperative proteins that associate with them. With matched RNA-seq and phosphosite data from these same stages, we investigate pathway-, stage- and species-specific aspects of translational and post-translational regulation, e.g. finding peri-gastrulation stages of human and mice to be discordant with respect to the mitochondrial transcriptome vs. proteome, and nominating novel kinase-substrate relationships based on phosphosite dynamics. Finally, we leverage correlated dynamics to identify conserved protein networks centered around congenital disease genes. Altogether, our data (https://gastruloid.brotmanbaty.org/) and analyses showcase the potential of intersecting in vitro embryo models and proteomics to advance our understanding of early mammalian development in ways not possible through transcriptomics alone.

The accuracy of crucial nuclear processes such as transcription, replication, and repair, depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA-protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed "DNA O-MAP", which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins. We show that DNA O-MAP can be coupled with sample multiplexed quantitative proteomics and next-generation sequencing to quantify DNA-protein and DNA-DNA interactions at specific genomic loci., Competing Interests: Competing Interest Statement D.K.S. is a collaborator with Thermo Fisher Scientific, Genentech, Calico Labs, and AI Proteins. C.K.C., A.F.T., E.K., D.M.S., and B.J.B. have filed a patent application covering aspects of this work. B.J.B. is listed as an inventor on patent applications related to the SABER technology related to this work.

Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localized in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.

Oncogenic mutations in KRAS can be recognized by T cells on specific class I human leukocyte antigen (HLA-I) molecules, leading to tumor control. To date, the discovery of T cell targets from KRAS mutations has relied on occasional T cell responses in patient samples or the use of transgenic mice. To overcome these limitations, we have developed a systematic target discovery and validation pipeline. We evaluate the presentation of mutant KRAS peptides on individual HLA-I molecules using targeted mass spectrometry and identify 13 unpublished KRAS G12C/D/R/V mutation/HLA-I pairs and nine previously described pairs. We assess immunogenicity, generating T cell responses to nearly all targets. Using cytotoxicity assays, we demonstrate that KRAS-specific T cells and T cell receptors specifically recognize endogenous KRAS mutations. The discovery and validation of T cell targets from KRAS mutations demonstrate the potential for this pipeline to aid the development of immunotherapies for important cancer targets., Competing Interests: J.C., S.P.G., B.P.C., J.L.D., J.K., D.L., D.A.R., P.J.T., J.R.S., K.C.F., M.S.R., M.M.v.B., R.B.G., T.A.A., and V.R.J. are employees and shareholders of BioNTech SE. C.D.McG., A.B., and J.G.A. are former employees of Neon Therapeutics (acquired by BioNTech SE). C.D.McG. is a graduate student at the University of Washington. A.B. is an employee and shareholder of TScan Therapeutics. J.G.A. is an employee of the Broad Institute of MIT and Harvard. R.B.G. is a member of the board of directors at Alkermes plc and Infinity Pharmaceuticals, and a member of the scientific advisory board at Leap Therapeutics., (© 2021 The Authors.)

Increasing evidence indicates CD4 + T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies., (Copyright © 2019 Elsevier Inc. All rights reserved.)