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Article
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Pathogenic mutations of human phosphorylation sites affect protein–protein interactions
Here the authors characterise the impact of phosphorylation site mutations in intrinsically disordered regions (IDRs) on protein-protein interactions, highlighting the critical role of phosphorylation of IDRs in health and disease.
- Trendelina Rrustemi
- , Katrina Meyer
- & Matthias Selbach
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| Open Access
VISTA checkpoint inhibition by pH-selective antibody SNS-101 with optimized safety and pharmacokinetic profiles enhances PD-1 response
VISTA is a pH-dependent inhibitory checkpoint for T-cells that is abundant on myeloid lineage cells and antagonists of VISTA may successfully reinvigorate anti-tumour immunity. Here, the authors show that the antibody SNS-101, which is currently being investigated in humans in a clinical trial, is characterized by pH-sensitivity that endows it with favorable pharmacokinetic and safety profiles, and enhanced therapeutic effect when combined with PD-1 checkpoint inhibitors.
- Thomas Thisted
- , F. Donelson Smith
- & Edward H. van der Horst
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ReLo is a simple and rapid colocalization assay to identify and characterize direct protein–protein interactions
Characterising interactions between proteins that are large and poorly soluble remains challenging. Here, the authors describe ReLo, a rapid and versatile eukaryotic cell culture-based method for detecting and studying direct interactions between structurally complex proteins.
- Harpreet Kaur Salgania
- , Jutta Metz
- & Mandy Jeske
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| Open Access
Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis
ATGL is a key enzyme in intracellular lipolysis. Here, the authors use deep mutational scanning to define the determinants of protein interaction between ATGL and its regulatory partners, gaining insights into lipolysis mechanisms in cells.
- Johanna M. Kohlmayr
- , Gernot F. Grabner
- & Ulrich Stelzl
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Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells
Here the authors report a strategy to directly capture substrates of lysine-modifying enzymes via post-translational modification (PTM)-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner.
- Hao Hu
- , Wei Hu
- & Xiao-Hua Chen
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Proteome-Wide Identification of RNA-dependent proteins and an emerging role for RNAs in Plasmodium falciparum protein complexes
Ribonucleoprotein complexes play fundamental roles in many cellular processes. Here, the authors used a proteome-wide approach, R-DeeP, to identify protein complexes associated with RNA in the deadliest human malaria parasite, Plasmodium falciparum.
- Thomas Hollin
- , Steven Abel
- & Karine G. Le Roch
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| Open Access
Micropillar arrays, wide window acquisition and AI-based data analysis improve comprehensiveness in multiple proteomic applications
Obtaining a comprehensive proteomic profile for complex samples is still an elusive task. Here, the authors present an LC-MS/MS workflow including micropillar arrays, wide isolation windows and AI-based data analysis to boost proteomic coverage and throughput for multiple proteomic samples.
- Manuel Matzinger
- , Anna Schmücker
- & Rupert L. Mayer
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| Open Access
From interaction networks to interfaces, scanning intrinsically disordered regions using AlphaFold2
Here, the authors show that AlphaFold2 accurately predicts protein interfaces involving disordered regions. Combining different delimitations and sequence alignments increases the success rate, while scanning short overlapping fragments identifies binding sites.
- Hélène Bret
- , Jinmei Gao
- & Raphaël Guerois
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Mapping protein states and interactions across the tree of life with co-fractionation mass spectrometry
Co-fractionation mass spectrometry (CF-MS) is a powerful technique for mapping protein interactions under physiological conditions. Here, the authors uniformly re-process 411 CF-MS experiments and carry out meta-analyses of protein abundance, protein-protein interactions, and phosphorylation sites in the resulting resource.
- Michael A. Skinnider
- , Mopelola O. Akinlaja
- & Leonard J. Foster
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Proximity extracellular protein-protein interaction analysis of EGFR using AirID-conjugated fragment of antigen binding
Extracellular protein-protein interactions (exPPIs) are essential for understanding the biological function of membrane receptor proteins. Here, the authors demonstrate the FabID technology as a new proximity biotinylation approach that can analyse exPPIs dynamically modulated by drugs and ligands.
- Kohdai Yamada
- , Ryouhei Shioya
- & Tatsuya Sawasaki
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The SPOC proteins DIDO3 and PHF3 co-regulate gene expression and neuronal differentiation
Death-inducer obliterator 3 (DIDO3) and PHD finger protein 3 (PHF3) are paralogue proteins that regulate transcription elongation by docking onto phosphorylated serine-2 in the C-terminal domain (CTD) of Pol II through their SPOC domains. Here the authors characterize the interplay of these proteins and show that they coregulate neuronal target genes.
- Johannes Benedum
- , Vedran Franke
- & Dea Slade
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| Open Access
The protein interactome of the citrus Huanglongbing pathogen Candidatus Liberibacter asiaticus
Research on the biology and pathogenicity of ‘Candidatus Liberibacter asiaticus’ (CLas), the bacterium that causes citrus Huanglongbing disease, is hampered by our inability to cultivate it in artificial media. Here, Carter et al. use a high-throughput yeast-two-hybrid screen to identify thousands of interactions between CLas proteins, thus providing insights into their potential functions.
- Erica W. Carter
- , Orlene Guerra Peraza
- & Nian Wang
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| Open Access
Proteomic characterization of epithelial ovarian cancer delineates molecular signatures and therapeutic targets in distinct histological subtypes
The molecular phenotypic features of epithelial ovarian cancer (EOC) remain elusive. Here, the authors perform mass spectrometry-based proteomic profiling for 269 EOC patients and reveal molecularly distinct features and potential therapeutic targets among the histological subtypes of EOC.
- Ting-Ting Gong
- , Shuang Guo
- & Qi-Jun Wu
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Improved in situ characterization of protein complex dynamics at scale with thermal proximity co-aggregation
Vast majority of cellular activities are carried out by protein complexes that assembled dynamically in response to cellular needs and environmental cues. Here, the authors present Slim-TPCA, an effective and readily deployable strategy to unravel the functional roles of protein complexes en masse across various cellular processes.
- Siyuan Sun
- , Zhenxiang Zheng
- & Chris Soon Heng Tan
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BioE3 identifies specific substrates of ubiquitin E3 ligases
Here, the authors describe BioE3, a biotin-based method to discriminate direct substrates of ubiquitin E3 ligases of interest from mere interactors using proximity proteomics. BioE3 responds to chemical treatments, and works with RING- and HECT-type E3s, as well as ubiquitin-likes (e.g., SUMO).
- Orhi Barroso-Gomila
- , Laura Merino-Cacho
- & James D. Sutherland
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An adaptive stress response that confers cellular resilience to decreased ubiquitination
Hunt et al. identify the protein sets that are modulated by RNAi for each E2 ubiquitin-conjugating enzyme in human cells. By analyzing the UBA1/E2-sensitive proteome, they report an adaptive stress response that preserves peroxisomal protein import in cells with decreased ubiquitination capacity.
- Liam C. Hunt
- , Vishwajeeth Pagala
- & Fabio Demontis
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Article
| Open Access
Local membrane source gathering by p62 body drives autophagosome formation
Phase separated p62 body plays pivotal roles in autophagy. Here, the authors describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation.
- Xuezhao Feng
- , Daxiao Sun
- & Na Mi
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Article
| Open Access
Multiple E3 ligases control tankyrase stability and function
The poly(ADP-ribosyl)transferases, tankyrase 1 and 2, are regulated by RNF146-mediated K48-linked polyubiquitylation and degradation. Here the authors show that this is opposed by K11-linked diubiquitylation by RING-UIM E3 ligases RNF114 and 166 and further impacted by several PAR-binding E3 ligases.
- Jerome Perrard
- & Susan Smith
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A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation
The authors show that dynamics of protein phosphorylation in the vertebrate cell cycle is largely attributable to CDK-mediated regulation of intrinsically disordered proteins that are involved in biomolecular condensate formation.
- Juan Manuel Valverde
- , Geronimo Dubra
- & Maarten Altelaar
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FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice
Hepatic ischemia-reperfusion injury (IRI) is a common complication that occurs during hepatic resection and transplantation. Here the authors show that Hepatic stellate cells secrete FGF18 which alleviates hepatocyte injury by USP16/KEAP1/Nrf2 signaling.
- Gaozan Tong
- , Yiming Chen
- & XiaoKun Li
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| Open Access
AlphaFold-Multimer predicts cross-kingdom interactions at the plant-pathogen interface
AlphaFold-Multimer was used to screen of 1,879 small secreted proteins from plant pathogens to be inhibitors of six tomato defense enzymes. Four of these inhibit subtilase P69B, showing the use of AI to predict cross-kingdom protein interactions.
- Felix Homma
- , Jie Huang
- & Renier A. L. van der Hoorn
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| Open Access
Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism
Motile cilia are hair-like structures that are found on the surface of eukaryotic cells providing cell motility. Here, authors reveal the twelve components of nexin-dynein regulatory complex and associated proteins in cilia from Tetrahymena thermophila.
- Avrin Ghanaeian
- , Sumita Majhi
- & Khanh Huy Bui
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| Open Access
A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development
The molecular mechanisms ensuring early face, brain, and skin formation are unclear. Here, the authors uncover a posttranslational pathway that controls cytoskeletal signaling circuits to coordinate ectodermal patterning and neurulation.
- Anthony J. Asmar
- , Shaun R. Abrams
- & Achim Werner
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| Open Access
Deciphering intercellular signaling complexes by interaction-guided chemical proteomics
Systematic profiling of the indirect cell–cell interactions remains challenging. Here, the authors report a chemical proteomics method to identify ligand-receptor complexes formed between cell surface receptors and secreted proteins from neighboring cells.
- Jiangnan Zheng
- , Zhendong Zheng
- & Ruijun Tian
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| Open Access
Symbiont-host interactome mapping reveals effector-targeted modulation of hormone networks and activation of growth promotion
Pathogens secrete effectors to promote disease, symbionts might use them to confer benefits. Here, the authors identify 106 candidate effectors from the symbiont Serendipita indica, characterise their interactions, and reveal their roles in regulating phytohormone signalling and promoting growth.
- Rory Osborne
- , Laura Rehneke
- & Patrick Schäfer
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| Open Access
Targeted cross-linker delivery for the in situ mapping of protein conformations and interactions in mitochondria
Current methods for analysing protein structures and interactions generally require the separation of specific organelles or changes to the intracellular environment. Here, authors developed nanocarrier-based cross-linking mass spectrometry techniques to assess mitochondrial proteins within living cells.
- Yuwan Chen
- , Wen Zhou
- & Yukui Zhang
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| Open Access
A pharmacoproteomic landscape of organotypic intervention responses in Gram-negative sepsis
Sepsis can cause organ damage through disparate immunological and metabolic processes. Here the authors demonstrate a proteomics-based scoring strategy for quantifying quantitative and organotypic changes in relationship to dosing, timing, and potential synergistic intervention combinations during sepsis.
- Tirthankar Mohanty
- , Christofer A. Q. Karlsson
- & Johan Malmström
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| Open Access
PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell Complex
The WAVE regulatory complex activates Arp2/3 at the cell cortex and in membrane protrusions to generate persistent cell migration. Here authors show that PPP2R1A, a scaffold subunit of protein phosphatase 2, associates with an alternative form of the WAVE complex where WAVE, the subunit that activates Arp2/3, is replaced by NHSL1.
- Yanan Wang
- , Giovanni Chiappetta
- & Alexis M. Gautreau
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| Open Access
Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health
Daly, Danson and colleagues employ a multi-omic approach in neuroglioma cells to characterise endolysosomal dysfunction caused by perturbation of the evolutionarily conserved Retromer complex, highlighting Retromer’s neuroprotective function.
- James L. Daly
- , Chris M. Danson
- & Peter J. Cullen
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| Open Access
Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs
Protein-protein interactions underlie all aspects of a viral infection. Here the authors employ a pan-viral approach for systematic identification of motif-mediated interactions between viral and human proteins and show that the information can be used to find targets for antiviral drug development.
- Filip Mihalič
- , Leandro Simonetti
- & Ylva Ivarsson
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| Open Access
Next-generation large-scale binary protein interaction network for Drosophila melanogaster
Maps of protein-protein interactions (PPIs) help identify new components of pathways, complexes, and processes. In this work, state-of-the-art methods are used to identify binary Drosophila PPIs, generating broadly useful physical and data resources.
- Hong-Wen Tang
- , Kerstin Spirohn
- & Stephanie E. Mohr
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| Open Access
Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub
The 16p11.2 duplication confers risk for autism and schizophrenia, but the disease mechanisms are unknown. Here, the authors use proteomics to show dysregulation of synaptic and epilepsy-associated protein networks in the cortex of model mice, and demonstrate that correcting Prrt2 gene dosage rescues circuit hypersynchrony and behavioural phenotypes.
- Marc P. Forrest
- , Marc Dos Santos
- & Peter Penzes
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| Open Access
The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators
Here the authors establish the SPOC domain as a universal reader of the RNA Pol II CTD code and a versatile reader of phosphoserine marks found in co- and post-transcriptional regulators such as m6A writer and reader proteins.
- Lisa-Marie Appel
- , Vedran Franke
- & Dea Slade
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| Open Access
Enhanced access to the human phosphoproteome with genetically encoded phosphothreonine
Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. Here the authors optimize genetically encoded phosphothreonine to study the regulation of CHK2 kinase using large-scale DNA arrays that enable phosphoproteome expression techniques to identify sitespecific overlap between CHK2 substrates and 14-3-3 interactions.
- Jack M. Moen
- , Kyle Mohler
- & Jesse Rinehart
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| Open Access
An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation
Specificity in signaling activated by receptor tyrosine kinases is typically attributed to characteristics of their intracellular domains. Here, the authors demonstrate that an extracellular receptor sequence motif controls intracellular signaling as a result of extracellular glycan interactions.
- Katri Vaparanta
- , Anne Jokilammi
- & Klaus Elenius
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| Open Access
CDK12 is hyperactivated and a synthetic-lethal target in BRAF-mutated melanoma
In patients with melanoma, increased RAS/mitogen-activated protein kinase (MAPK) pathway activity is known to drive chemotherapy resistance. Here, the authors identify CDK12 as a downstream effector of the RAS/MAPK pathway and therapeutic target which mediates chemotherapy resistance through increased expression of DNA repair associated genes.
- Thibault Houles
- , Geneviève Lavoie
- & Philippe P. Roux
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| Open Access
A slit-diaphragm-associated protein network for dynamic control of renal filtration
The slit-diaphragm is a cellular junction that is crucial for blood filtration in the kidney. Kocylowski et al. show that the junction-spanning components are embedded in a protein network for dynamic control of filtration; network disturbance leads to severe filtration defects with proteinuria.
- Maciej K. Kocylowski
- , Hande Aypek
- & Florian Grahammer
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| Open Access
Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo
Protein complexes play a decisive role for lysosomal function. Here, the authors use cross-linking mass spectrometry and integrative modeling to investigate lysosomes and early endosomes; characterizing protein interactions, structures, and the cargo of flotillin-mediated endocytosis.
- Jasjot Singh
- , Hadeer Elhabashy
- & Dominic Winter
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Article
| Open Access
Phosphatase protector alpha4 (α4) is involved in adipocyte maintenance and mitochondrial homeostasis through regulation of insulin signaling
The insulin signalling cascade can be inhibited by phosphatases, including Ser/Thr protein phosphatase 2A (PP2A). Here the authors show that Alpha4, a regulator of the PP2A catalytic subunit, modulates insulin receptor tyrosine phosphorylation via the YBX-1/PTP1B pathway and is involved in maintenance of adipose tissue homeostasis and systemic metabolism.
- Masaji Sakaguchi
- , Shota Okagawa
- & Eiichi Araki
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| Open Access
Quantitative fragmentomics allow affinity mapping of interactomes
Protein networks have been widely explored but most binding affinities remain unknown, limiting the quantitative interpretation of interactomes. Here the authors measure affinities of 65,000 interactions involving human PDZ domains and target sequence motifs relevant for viral infection and cancer.
- Gergo Gogl
- , Boglarka Zambo
- & Gilles Travé
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Article
| Open Access
Electrostatic and steric effects underlie acetylation-induced changes in ubiquitin structure and function
Ubiquitin is not only a posttranslational modifier but itself is subject to modifications, such as acetylation. Characterization of distinct acetylated ubiquitin variants reveals that each acetylation site has a particular impact on ubiquitin structure and its protein-protein interaction properties.
- Simon Maria Kienle
- , Tobias Schneider
- & Martin Scheffner
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Article
| Open Access
A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission
Trypanosomes can sense signal molecules and coordinate their movement in response to such signals, a phenomenon termed social motility (SoMo). Here, Bachmaier et al show that cyclic AMP response protein 3 (CARP3) localization to the flagellar tip and its interaction with a number of different adenylate cyclases is essential for migration to tsetse fly salivary glands and for SoMo, therewith linking SoMo and cAMP signaling to trypanosome transmission.
- Sabine Bachmaier
- , Giacomo Giacomelli
- & Michael Boshart
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| Open Access
PIM1 promotes hepatic conversion by suppressing reprogramming-induced ferroptosis and cell cycle arrest
Protein kinase-mediated phosphorylation plays a critical role in many biological processes. Here the authors develop a trans-omics-based algorithm called Central Kinase Inference to integrate quantitative transcriptomic and phosphoproteomic data, finding that PIM1 promotes hepatic conversion by suppressing reprogramming-induced ferroptosis and cell cycle arrest.
- Yangyang Yuan
- , Chenwei Wang
- & Pengyu Huang
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Article
| Open Access
Mapping autophagosome contents identifies interleukin-7 receptor-α as a key cargo modulating CD4+ T cell proliferation
Autophagy is known to impact CD4+ T cell proliferation but the identity of autophagosomal components remain unclear. Here the authors leverage a transgenic mouse model to assess the autophagosomal compartment and identify interleukin-7 receptor-α as a key cargo in proliferating CD4+ T cells.
- Dingxi Zhou
- , Mariana Borsa
- & Anna Katharina Simon
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| Open Access
Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling
Methods to identify protein interaction networks often suffer from poor spatiotemporal resolution. Here the authors present a light-activated proximity labeling method where the protein of interest is fused to the photosensitizer protein miniSOG, allowing temporally resolved labeling of interactors.
- Yansheng Zhai
- , Xiaoyan Huang
- & Gang Li
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Article
| Open Access
A method for Boolean analysis of protein interactions at a molecular level
Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification.
- Doroteya Raykova
- , Despoina Kermpatsou
- & Ola Söderberg
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| Open Access
Proteotoxicity caused by perturbed protein complexes underlies hybrid incompatibility in yeast
Hybrid incompatibility can be an important element of reproductive isolation and speciation. Using chromosome replacement lines of yeast, the authors show that perturbed proteostasis caused by destabilized hybrid protein complexes may represent a general mechanism of hybrid incompatibility.
- Krishna B. S. Swamy
- , Hsin-Yi Lee
- & Jun-Yi Leu
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Article
| Open Access
Scalable multiplex co-fractionation/mass spectrometry platform for accelerated protein interactome discovery
Co-fractionation/mass spectrometry (CF/MS) allows mapping protein interactomes but efficiency and quantitative accuracy are limited. Here, the authors develop a reproducible multiplexed CF/MS method and apply it to characterize interactome rewiring in breast cancer cells.
- Pierre C. Havugimana
- , Raghuveera Kumar Goel
- & Andrew Emili
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Article
| Open Access
Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows
Cross-linking mass spectrometry is widely used to elucidate protein structures and interactions. Here, the authors generate an extensive peptide library to benchmark the most common cross-link search engines with frequently used cross-linking reagents in low and high complex sample systems.
- Manuel Matzinger
- , Adrian Vasiu
- & Karl Mechtler
Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies