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Modified vaccinia Ankara (MVA) virus is the vaccine deployed to curb mpox. Here the authors conduct a multiplexed proteomic analysis to quantify cellular and viral proteins throughout MVA virus infection of human fibroblasts and macrophages and see substantial remodelling of the host proteome.

Targeted proteomics enables robust hypothesis-driven research. Here, Yu et al. present a multiplexed approach for targeted pathway proteomics and apply it to quantify protein families across 480 fully genotyped Diversity Outbred mice, revealing impacts of genetic variation on protein expression and lipid metabolism.

A proteome-wide atlas of the effect of 875 compounds reveals mechanisms of action and off-target effects.

Deeper proteome coverage empowers new biological insights. Here, the authors establish a standard analysis workflow for TMT-based data acquired on the Orbitrap Astral mass spectrometer and leverage it for aging research, distinguishing protein dynamics of early development and adulthood in mice.

The functional role of protein phosphorylation is determined not just by whether a particular site is phosphorylated or not but also by the site's stoichiometry. A method to determine the absolute stoichiometries of protein phosphorylation on a proteomic scale is described.

A global map of human subcellular architecture yields protein complex structures, reveals protein functions, identifies assemblies with multiple localizations or cell-type specificity and decodes paediatric cancer genomes.

Chou et al. generate transdifferentiated human neurons from fibroblasts from young or aged individuals and patients with Alzheimer’s disease. Studies of endolysosomes and proteostasis suggest a role for lysosomal quality control in Alzheimer’s disease pathology.

The effect of natural genetic diversity on the proteome is characterized using an outbred mouse model with extensive variation; both transcripts and proteins from mouse livers are quantified to identify a large set of protein quantitative trait loci (pQTL), and mediation analysis identifies causal protein intermediates of distant pQTL.

Solid organ transplant recipients are at increased risk of infectious disease and have unique molecular pathophysiology. Here the authors use host-microbe profiling to assess SARS-CoV-2 infection and immunity in solid organ transplant recipients, showing enhanced viral abundance, impaired clearance, and increased expression of innate immunity genes.

A disulfide tethering screen identified a molecule that covalently interacts with pro-apoptotic BAX at C126, inhibiting its activation.

A set of isobaric labeling reagents called TMTpro enables deep quantitative comparisons of proteome measurements across 16 samples.

Design of cysteine-targeting analogs of a reversible SETDB1 triple Tudor ___domain (3TD) ligand, UNC6535, led to UNC10013, a potent covalent ligand with high selectivity. UNC10013 demonstrated allosteric inhibition of SETDB1-mediated Akt methylation in cells, a promising approach to SETDB1 therapeutics.

An improved workflow enables a 42-fold higher throughput of activity-based protein profiling.

Latorre-Muro et al. show that the cytosolic chaperone PPID drives insertion of the mitochondrial import receptor TOM70 into the mitochondrial outer membrane, thereby regulating body temperature, glucose homeostasis and body weight in obese mice.

This work reports a dual transposase–peroxidase fusion to survey the accessible chromatin regions and the proximal proteome in one assay, providing a tool to capture both the genomic and proteomic contents of open chromatin.

In one of two studies, a technique known as SILAC is used to measure, on a large scale, changes in protein level as a function of expression of endogenous and exogenous miRNAs. It is found that although miRNAs directly repress the translation of hundreds of genes, additional indirect effects result in changes in expression of thousands of genes.

Kubota et al. describe a sensitive mass spectrometric method for generating signatures of kinase activity characteristic of specific cell types. They also identify kinases responsible for phosphorylating substrates of interest.

REEP ___domain-containing proteins generate membrane curvature, and some are known to shape the endoplasmic reticulum (ER). Here, the authors show that four understudied members, REEPs 1-4, localize to vesicles that appear to bud out of, and fuse back with, ER.

Castro, Shindyapina et al. explore how aging promotes B cell lymphoma in mice, identifying a population of age-associated clonal B cells that expands through mutation, c-Myc activation and epigenetic alterations to drive age-associated malignancy.

Post-acute sequelae of SARS-CoV-2 (PASC) is still not well understood. Here the authors provide patient reported outcomes from 590 hospitalized COVID-19 patients and show association of PASC with higher respiratory SARS-CoV-2 load and circulating antibody titers, and in some an elevation in circulating fibroblast growth factor 21.

Protein immunofluorescence imaging and affinity purification–mass spectrometry are combined to create a unified map of human cell architecture across scales, which the authors call the multi-scale integrated cell (MuSIC).

Targeted protein degradation uses small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation, but only a few human E3 ligases are amenable to this strategy. Here, the authors identify and characterize SP3N, a specific degrader of the prolyl isomerase FKBP12, containing an FKBP12 ligand appended with a flexible alkylamine tail that is metabolized to an active aldehyde species which recruits the SCF^FBXO22 ligase for FKBP12 degradation.

Myddosomes, in which MyD88 forms barrel-like scaffold structures for effector protein recruitment and activation, contain proteins that act at all stages and regulate all effector responses of the TLR signalling pathways.

Iterative Synthetically Phosphorylated Isomers (iSPI) is a proteome-scale library of human-derived phosphoserine-containing phosphopeptides with precisely known positions of phosphorylation. This multi-purpose resource is available for optimization, standardization, and benchmarking of key steps in phosphoproteomics workflows.

The role of IgG glycosylation in the immune response has been studied, but less is known about IgM glycosylation. Here the authors characterize glycosylation of SARS-CoV-2 spike specific IgM and show that it correlates with COVID-19 severity and affects complement deposition.

An in vivo approach to identify proteins whose enrichment near cardiac Ca_V1.2 channels changes upon β-adrenergic stimulation finds the G protein Rad, which is phosphorylated by protein kinase A, thereby relieving channel inhibition by Rad and causing an increased Ca²⁺ current.

In shotgun proteomics experiments, modified peptides account for a large part of the unassigned spectra and can be identified using ultra-tolerant database searches.

Paterson and Yu et al. demonstrate that loss of the RNA alternative splicing factor RBFOX2 in the liver during a lipogenic diet leads to dysregulation of liver lipid and cholesterol homeostasis through a specific alternative splicing programme, which includes a splice switch of the high-density lipoprotein receptor gene Scarb1.

The proteasome is a large proteolytic machine that degrades ubiquitin-tagged proteins. Substrates are recognized and unfolded by the regulatory particle (RP) and translocated into a central proteolytic chamber, called the core particle (CP), where degradation takes place. Here, and in a complimentary study, the pathway of RP assembly is elucidated, demonstrating that RP assembly is a highly orchestrated process.

The proteasome is a large proteolytic machine that degrades ubiquitin-tagged proteins. Substrates are recognized and unfolded by the regulatory particle (RP) and translocated into a central proteolytic chamber, called the core particle (CP), where degradation takes place. Here, and in a complimentary study, the pathway of RP assembly is elucidated, demonstrating that RP assembly is a highly orchestrated process.

Although cyclin D1 is frequently overexpressed in human cancers, the full range of its functions in normal development and oncogenesis is unclear. Here, tagged cyclin D1 knock-in mouse strains are developed to allow a search for cyclin D1-binding proteins in different mouse organs using high-throughput mass spectrometry. The results show that, in addition to its established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development.

A triple-stage mass spectrometry (MS3)-based method is used to remove ratio interference, resulting in accurate, large-scale, multiplexed quantitative proteomics measurements using isobaric labeling. Also in this issue, Wenger et al. provide a different solution to the same problem.

Affinity purification–mass spectrometry elucidates protein interaction networks and co-complexes to build, to our knowledge, the largest experimentally derived human protein interaction network so far, termed BioPlex 2.0.