Recent efforts in gut microbiome studies have highlighted the importance of explicitly describing the ecological processes beyond correlative analysis. However, we are still at the early stage of understanding the organizational principles of the gut ecosystem, partially because of the limited information provided by currently used analytical tools in ecological modeling practices. Proteomics and metaproteomics can provide a number of insights for ecological studies, including biomass, matter and energy flow, and functional diversity. In this Mini Review, we discuss proteomics and metaproteomics-based experimental strategies that can contribute to studying the ecology, in particular at the mucosal-luminal interface (MLI) where the direct host-microbiome interaction happens. These strategies include isolation protocols for different MLI components, enrichment methods to obtain designated array of proteins, probing for specific pathways, and isotopic labeling for tracking nutrient flow. Integration of these technologies can generate spatiotemporal and site-specific biological information that supports mathematical modeling of the ecosystem at the MLI.

In-depth coverage of proteomic analysis could enhance our understanding to the mechanism of the protein functions. Unfortunately, many highly hydrophobic proteins and low-abundance proteins, which play critical roles in signaling networks, are easily lost during sample preparation, mainly attributed to the fact that very few extractants can simultaneously satisfy the requirements on strong solubilizing ability to membrane proteins and good enzyme compatibility. Thus, it is urgent to screen out ideal extractant from the huge compound libraries in a fast and effective way. Herein, by investigating the interior mechanism of extractants on the membrane proteins solubilization and trypsin compatibility, a molecular dynamics simulation system was established as complement to the experimental procedure to narrow down the scope of candidates for proteomics analysis. The simulation data shows that the van der Waals interaction between cation group of ionic liquid and membrane protein is the dominant factor in determining protein solubilization. In combination with the experimental data, 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) is on the shortlist for the suitable candidates from comprehensive aspects. Inspired by the advantages of C12Im-Cl, an ionic liquid-based filter-aided sample preparation (i-FASP) method was developed. Using this strategy, over 3,300 proteins were confidently identified from 10³ HeLa cells (~100 ng proteins) in a single run, an improvement of 53% over the conventional FASP method. Then the i-FASP method was further successfully applied to the label-free relative quantitation of human liver cancer and para-carcinoma tissues with obviously improved accuracy, reproducibility and coverage than the commonly used urea-based FASP method. The above results demonstrated that the i-FASP method could be performed as a versatile tool for the in-depth coverage proteomic analysis of biological samples.

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [³⁵S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

High-speed analysis of large (prote)omics sample sets at the rate of thousands or millions of samples per day on a single platform has been a challenge since the beginning of proteomics. For many years, ESI-based MS methods have dominated proteomics because of their high sensitivity and great depth in analyzing complex proteomes. However, despite improvements in speed, ESI-based MS methods are fundamentally limited by their sample introduction, which excludes off-line sample preparation/fractionation because of the time required to switch between individual samples/sample fractions, and therefore being dependent on the speed of on-line sample preparation methods such as liquid chromatography. Laser-based ionization methods have the advantage of moving from one sample to the next without these limitations, being mainly restricted by the speed of modern sample stages, i.e. 10 ms or less between samples. This speed matches the data acquisition speed of modern high-performing mass spectrometers whereas the pulse repetition rate of the lasers (>1 kHz) provides a sufficient number of desorption/ionization events for successful ion signal detection from each sample at the above speed of the sample stages. Other advantages of laser-based ionization methods include the generally higher tolerance to sample additives and contamination compared with ESI MS, and the contact-less and pulsed nature of the laser used for desorption, reducing the risk of cross-contamination. Furthermore, new developments in MALDI have expanded its analytical capabilities, now being able to fully exploit high-performing hybrid mass analyzers and their strengths in sensitivity and MS/MS analysis by generating an ESI-like stable yield of multiply charged analyte ions. Thus, these new developments and the intrinsically high speed of laser-based methods now provide a good basis for tackling extreme sample analysis speed in the omics.

MS-based proteome profiling has become increasingly comprehensive and quantitative, yet a persistent shortcoming has been the relatively large samples required to achieve an in-depth measurement. Such bulk samples, typically comprising thousands of cells or more, provide a population average and obscure important cellular heterogeneity. Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells, a level of coverage that required an input of thousands of cells just a few years ago. This review discusses important factors and parameters that should be optimized across the workflow for single-cell and other low-input measurements. It also highlights recent developments that have advanced the field and opportunities for further development.

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.

Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.

We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, whereas defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. Although peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were up-regulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

Stroke remains a leading cause of death and disability worldwide. Despite continuous advances, the identification of key molecular signatures in the hyper-acute phase of ischemic stroke is still a primary interest for translational research on stroke diagnosis, prognosis, and treatment. Data integration from high-throughput -omics techniques has become crucial to unraveling key interactions among different molecular elements in complex biological contexts, such as ischemic stroke. Thus, we used advanced data integration methods for a multi-level joint analysis of transcriptomics and proteomics data sets obtained from mouse brains at 2 h after cerebral ischemia. By modeling net-like correlation structures, we identified an integrated network of genes and proteins that are differentially expressed at a very early stage after stroke. We validated 10 of these deregulated elements in acute stroke, and changes in their expression pattern over time after cerebral ischemia were described. Of these, CLDN20, GADD45G, RGS2, BAG5, and CTNND2 were next evaluated as blood biomarkers of cerebral ischemia in mice and human blood samples, which were obtained from stroke patients and patients presenting stroke-mimicking conditions. Our findings indicate that CTNND2 levels in blood might potentially be useful for distinguishing ischemic strokes from stroke-mimicking conditions in the hyper-acute phase of the disease. Furthermore, circulating GADD45G content within the first 6 h after stroke could also play a key role in predicting poor outcomes in stroke patients. For the first time, we have used an integrative biostatistical approach to elucidate key molecules in the initial stages of stroke pathophysiology and highlight new notable molecules that might be further considered as blood biomarkers of ischemic stroke.

Ankylosing Spondylitis (AS) is a common, inflammatory rheumatic disease, which primarily affects the axial skeleton and is associated with sacroiliitis, uveitis and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine if plasma from patients with AS contained autoantibodies and if so, characterize and quantify this response in comparison to patients with Rheumatoid Arthritis (RA) and healthy controls. Two high-density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis in order to determine patterns of signalling cascades or tissue origin. 44% of patients with Ankylosing Spondylitis demonstrated a broad autoantibody response, as compared to 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The AS patients autoantibody responses were targeted towards connective, skeletal and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic Acid Programmable Protein Arrays constitute a powerful tool to study autoimmune diseases.

In quantitative proteomics work, the differences in expression of many separate proteins are routinely examined to test for significant differences between treatments. This leads to the multiple hypothesis testing problem: when many separate tests are performed many will be significant by chance and be false positive results. Statistical methods such as the false discovery rate (FDR) method that deal with this problem have been disseminated for more than one decade. However a survey of proteomics journals shows that such tests are not widely implemented in one commonly used technique, quantitative proteomics using two-dimensional electrophoresis (2-DE). We outline a selection of multiple hypothesis testing methods, including some that are well known and some lesser known, and present a simple strategy for their use by the experimental scientist in quantitative proteomics work generally. The strategy focuses on the desirability of simultaneous use of several different methods, the choice and emphasis dependent on research priorities and the results in hand. This approach is demonstrated using case scenarios with experimental and simulated model data.

Cover art by Julie Newdoll for MCP April issue.

The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. Firstly, it can verify the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Secondly, the toolkit can convert several XML-based data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at http://www.proteored.org/MIAPE/.

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This article has been withdrawn by the authors. This article did not comply with the editorial guidelines of MCP. Specifically, single peptide based protein identifications of 9-19% were included in the analysis and discussed in the results and conclusions. We wish to withdraw this article and resubmit a clarified, corrected manuscript for review.

Glycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass, for example HexNAc(2)Hex(5) rather than man5. Even at this level, however, glycopeptide analysis poses challenges:  finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra.  Here we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, for example, at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, for example, ± 0.01 Daltons. Also new is glycan "wildcard" search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analysed the glyco/proteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyse Sequential Window Acquisition of all THeoretical mass spectra (SWATH) Data Independent Acquisition (DIA) mass spectrometry data based on glycopeptides identified by Byonic. We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the Liqueur de Tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared to 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. This work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic SWATH/DIA workflow that is broadly applicable to other sample types.

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.

Glycosylation is a highly diverse set of co- and post-translational modification of proteins. For mammalian glycoproteins, glycosylation is often site-, tissue- and species-specific, and diversified by microheterogeneity. Multitudinous biochemical, cellular, physiological and organismic effects of their glycans have been revealed, either intrinsic to the carrier proteins or mediated by endogenous reader proteins with carbohydrate recognition domains. Furthermore, glycans frequently form the first line of access by or defense from foreign invaders, and new roles for nucleocytoplasmic glycosylation are blossoming. We now know enough to conclude that the same general principles apply in invertebrate animals and unicellular eukaryotes – different branches of which spawned the plants or fungi and animals. The two major driving forces for exploring the glycomes of invertebrates and protists are (i) to understand the biochemical basis of glycan-driven biology in these organisms, especially of pathogens, and (ii) to uncover the evolutionary relationships between glycans, their biosynthetic enzyme genes, and biological functions for new glycobiological insights. With an emphasis on emerging areas of protist glycobiology, here we offer an overview of glycan diversity and evolution, to promote future access to this treasure trove of glycobiological processes.

Regulation of gene expression is essential for the functioning of all eukaryotic organisms. Understanding gene expression regulation requires determining which proteins interact with regulatory elements in chromatin. Mass spectrometry-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation, as it allows studying protein function and protein complex formation in their in vivo chromatin-bound context. Total chromatin isolated from cells can be directly analysed using mass spectrometry or further fractionated into transcriptionally active and inactive chromatin prior to MS-based analysis. Newly formed chromatin that is assembled during DNA replication can also be specifically isolated and analysed. Furthermore, capturing specific chromatin domains facilitates the identification of previously unknown transcription factors interacting with these domains. Finally, in recent years, advances have been made towards identifying proteins that interact with a single genomic locus of interest. In this review, we highlight the power of chromatin proteomics approaches and how these provide complementary alternatives compared to conventional affinity purification methods. Furthermore, we discuss the biochemical challenges that should be addressed to consolidate and expand the role of chromatin proteomics as a key technology in the context of gene expression regulation and epigenetics research in health and disease.

The complexity and dynamics of the immensely heterogeneous glycoproteome of the prostate cancer (PCa) tumour micro-environment remain incompletely mapped, a knowledge gap that impedes our molecular-level understanding of the disease. To this end, we have used sensitive glycomics and glycoproteomics to map the protein-, cell- and tumour grade-specific N- and O-glycosylation in surgically-removed PCa tissues spanning five histological grades (n = 10/grade) and tissues from patients with benign prostatic hyperplasia (n = 5). Quantitative glycomics revealed PCa grade-specific alterations of the oligomannosidic-, paucimannosidic- and branched sialylated complex-type N-glycans, and dynamic remodelling of the sialylated core 1- and core 2-type O-glycome. Deep quantitative glycoproteomics identified ~7,400 unique N-glycopeptides from 500 N-glycoproteins and ~500 unique O-glycopeptides from nearly 200 O-glycoproteins. With reference to a recent Tissue and Blood Atlas, our data indicate that paucimannosidic glycans of the PCa tissues arise mainly from immune cell-derived glycoproteins. Further, the grade-specific PCa glycosylation arises primarily from dynamics in the cellular makeup of the PCa tumour microenvironment across grades involving increased oligomannosylation of prostate-derived glycoproteins and decreased bisecting GlcNAcylation of N-glycans carried by the extracellular matrix proteins. Further, elevated expression of several oligosaccharyltransferase subunits and enhanced N-glycoprotein site occupancy were observed associated with PCa progression. Finally, correlations between the protein-specific glycosylation and PCa progression were observed including increased site-specific core 2-type O-glycosylation of collagen VI. In conclusion, integrated glycomics and glycoproteomics have enabled new insight into the complexity and dynamics of the tissue glycoproteome associated with PCa progression generating an important resource to explore the underpinning disease mechanisms.

Malaria elimination is still pending on the development of novel tools that rely on a deep understanding of parasite biology. Proteins of all living cells undergo a myriad number of posttranslational modifications (PTMs) that are critical to multifarious life processes. An extensive proteome-wide dissection revealed a fine PTM map of most proteins in both Plasmodium falciparum, the causative agent of severe malaria, and the infected red blood cells. More than two-thirds of proteins of the parasite and its host cell underwent extensive and dynamic modification throughout the erythrocytic developmental stage. PTMs critically modulate the virulence factors involved in the host-parasite interaction and pathogenesis. Furthermore, P. falciparum stabilized the supporting proteins of erythrocyte origin by selective de-modification. Collectively, our multiple omic analyses, apart from having furthered a deep understanding of the systems biology of P. falciparum and malaria pathogenesis, provide a valuable resource for mining new antimalarial targets.

Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging since changes can occur simultaneously at protease, their inhibitor and substrate levels. Here, we present a pipeline that combines peptidomics, proteomics and peptidase predictions for studying proteolytic events in the saliva of seventy-nine patients and their association with oral squamous cell carcinoma (OSCC) prognosis. Our findings revealed differences in the saliva peptidome of patients with (pN+) or without (pN0) lymph node metastasis and delivered a panel of ten endogenous peptides correlated with poor prognostic factors plus five molecules able to classify pN0 and pN+ patients (ROC-AUC>0.85). In addition, endo- and exopeptidases putatively implicated in the processing of differential peptides were investigated using cancer tissue gene expression data from publicly repositories reinforcing their association with poorer survival rates and prognosis in oral cancer. The dynamics of the OSCC-related proteolysis was further explored via the proteomic profiling of saliva. This revealed that peptidase/endopeptidase inhibitors exhibited reduced levels in the saliva of pN+ patients, as confirmed by SRM-MS, whilst minor changes were detected in the level of saliva proteases. Taken together, our results indicated that proteolytic activity is accentuated in the saliva of OSCC patients with lymph node metastasis and, at least in part, this is modulated by reduced levels of salivary peptidase inhibitors. Therefore, this integrated pipeline provided better comprehension and discovery of molecular features with implications in the oral cancer metastasis prognosis.

Hirschsprung disease (HSCR) is a heterogeneous group of neurocristopathy characterized by the absence of the enteric ganglia along a variable length of the intestine. Genetic defects play a major role in the pathogenesis of HSCR while family studies of pathogenic variants in all the known genes (loci) only demonstrate incomplete penetrance and variable expressivity for unknown reasons. Here, we applied large-scale, quantitative proteomics of human colon tissues from 21 patients using iTRAQ method followed by bioinformatics analysis. Selected findings were confirmed by parallel reaction monitoring (PRM) verification. At last the interesting differentially expressed proteins were confirmed by western blot. A total of 5341 proteins in human colon tissues were identified. Among them, 664 proteins with >1.2-fold difference were identified in 6 groups: groups A1 and A2 pooled protein from the ganglionic and aganglionic colon of male, long-segment HSCR patients (L-HSCR, n=7); groups B1 and B2 pooled protein from the ganglionic and aganglionic colon of male, short-segment HSCR patients (S-HSCR, n=7); and groups C1 and C2 pooled protein from the ganglionic and aganglionic colon of female, S-HSCR patients (n=7). Based on these analyses, 49 proteins from 5 pathways were selected for PRM verification, including ribosome, endocytosis, spliceosome, oxidative phosphorylation and cell adhesion. The downregulation of three neuron projection development genes ARF4, KIF5B and RAB8A in the aganglionic part of the colon were verified in 15 paired colon samples using WB. The findings of this study will shed new light on the pathogenesis of HSCR and facilitate the development of therapeutic targets.

The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution mass spectrometry. By combining meiotic labels annotated from the MGI mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder, was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of ten genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore,  mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.

Extracellular vesicle (EV) proteins from acute myeloid leukemia (AML) cell lines were analyzed using mass spectrometry. The analyses identified 2450 proteins, including 461 differentially expressed proteins (290 upregulated and 171 downregulated). CD53 and CD47 were upregulated and were selected as candidate biomarkers. The association between survival of patients with AML and the expression levels of CD53 and CD47 at diagnosis was analyzed using mRNA expression data from The Cancer Genome Atlas database. Patients with higher expression levels showed significantly inferior survival than those with lower expression levels. Enzyme-linked immunosorbent assay results of the expression levels of CD53 and CD47 from EVs in the bone marrow of patients with AML at diagnosis and at the time of complete remission with induction chemotherapy revealed that patients with downregulated CD53 and CD47 expression appeared to relapse less frequently. Network model analysis of EV proteins revealed several upregulated kinases, including LYN, CSNK2A1, SYK, CSK, and PTK2B. The potential cytotoxicity of several clinically applicable drugs that inhibit these kinases was tested in AML cell lines. The drugs lowered the viability of AML cells. The collective data suggest that AML-derived EVs could reflect essential leukemia biology.

We have previously shown that multimers of plasma pentraxin-3 (PTX3) were predictive of survival in patients with sepsis. To characterize the release kinetics and cellular source of plasma protein changes in sepsis, serial samples were obtained from healthy volunteers (n=10, 3 time-points) injected with low-dose endotoxin (LPS) and analyzed using data-independent acquisition (DIA) MS. The human plasma proteome response was compared to an LPS-induced endotoxemia model in mice. Proteomic analysis of human plasma revealed a rapid neutrophil degranulation signature, followed by a rise in acute phase proteins. Changes in circulating PTX3 correlated with increases in neutrophil-derived proteins following LPS injection. Time course analysis of the plasma proteome in mice showed a time-dependent increase in multimeric PTX3, alongside increases in neutrophil-derived myeloperoxidase (MPO) upon LPS treatment. The mechanisms of oxidation-induced multimerisation of PTX3 were explored in two genetic mouse models: MPO global knock-out mice and LysM CreNox2KO mice, in which NADPH oxidase 2 (Nox2) is only deficient in myeloid cells. Nox2 is the enzyme responsible for the oxidative burst in neutrophils. Increases in plasma multimeric PTX3 were not significantly different between wildtype and MPO or LysM CreNox2KO knock-out mice. Thus, PTX3 may already be stored and released in a multimeric form. Through in vivo neutrophil depletion and multiplexed vascular proteomics, PTX3 multimer deposition within the aorta was confirmed to be neutrophil-dependent. Proteomic analysis of aortas from LPS-injected mice returned PTX3 as the most upregulated protein, where multimeric PTX3 was deposited as early as 2 h post-LPS along with other neutrophil-derived proteins. In conclusion, the rise in multimeric PTX3 upon LPS injection correlates with neutrophil-related protein changes in plasma and in aortas. MPO and myeloid Nox2 are not required for the multimerisation of PTX3; instead, neutrophil extravasation is responsible for the LPS-induced deposition of multimeric PTX3 in the aorta.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in mature medaka testis. As for the interaction with TGFβ-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid (Y2H) screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired Y2H assay. Co-immunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastrutural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (TGFβ and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA which may be fundamentally conserved across phyla during sexual differentiation.

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of co-eluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias towards high abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or Carboxylate modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid-phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange (SCX) and reversed phase (RP)-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH RP. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low abundance bait proteins.

Data independent acquisition (DIA) is now an emerging method in bottom-up proteomics and capable of achieving deep proteome coverage and accurate label-free quantification. However, for post-translational modifications (PTM), such as glycosylation, DIA methodology is still in the early stage of development. The full characterization of glycoproteins requires site specific glycan identification as well as subsequent quantification of glycan structures at each site. The tremendous complexity of glycosylation represents a significant analytical challenge in glycoproteomics. This review focuses on the development and perspectives of DIA methodology for N- and O- glycoproteomics and posits that DIA-based glycoproteomics could be a method of choice to address some of the challenging aspects of glycoproteomics. First, the current challenges in glycoproteomics and the basic principles of DIA is briefly introduced. DIA based glycoproteomics is then summarized and described into four aspects based on the actual samples. Lastly, we discussed the important challenges and future perspectives in the field. We believe that DIA can significantly facilitate glycoproteomic studies and contribute to the development of future advanced tools and approaches in the field of glycoproteomics.

Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species (ROS) that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse lifespans including the longest-lived mammal, the bowhead whale. We show that organismal lifespan is negatively correlated with turnover rates of highly abundant proteins. In comparison to mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production and reduced ROS levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

Recent advances in MS-based proteomics have vastly increased the quality and scope of biological information that can be derived from human samples. These advances have rendered current workflows increasingly applicable in biomedical and clinical contexts. As proteomics is poised to take an important role in the clinic, associated ethical responsibilities increase in tandem with the impact on the health, privacy, and well-being of individuals. Here we conducted and report a systematic literature review of ethical issues in clinical proteomics. We add our perspectives from a background of bioethics, the results of our accompanying paper extracting individual-sensitive results from patient samples, and the literature addressing similar issues in genomics. The spectrum of potential issues ranges from patient re-identification to incidental findings of clinical significance. The latter can be divided into actionable and unactionable findings. Some of these have the potential to be employed in discriminatory or privacy-infringing ways. However, incidental findings may also have great positive potential. A plasma proteome profile, for instance, could inform on the general health or disease status of an individual regardless of the narrow diagnostic question that prompted it. We suggest that early discussion of ethical issues in clinical proteomics is important to ensure that eventual regulations reflect the considered judgment of the community as well as to anticipate opportunities and problems that may arise as the technology matures further.

Tectonic Politics: Global Political Risk in an Age of Transformation Book sysadmin 7 May 2019

Political risk now affects more markets and countries than ever before and that risk will continue to rise. But traditional methods of managing political risk are no longer legitimate or effective.

In Tectonic Politics, Nigel Gould-Davies explores the complex, shifting landscape of political risk and how to navigate it. He analyses trends in each form of political risk: the power to destroy, seize, regulate, and tax.

He shows how each of these forms reflects a deeper transformation of the global political economy that is reordering the relationship between power, wealth, and values. In a world where everything is political, the craft of engagement is as important as the science of production and the art of the deal.

The successful company must integrate that craft—the engager’s way of seeing and doing—into strategy and culture.

Drawing on a career in academia, business, and diplomacy, Gould-Davies provides corporate leaders, scholars, and engaged citizens with a groundbreaking study of the fastest-rising political risk today. ‘As tectonic plates shape the earth,’ he writes, ‘so tectonic politics forges its governance.’

The book is published as part of the Insights series.

Praise for Tectonic Politics

About the author

Nigel Gould-Davies was an associate fellow of the Russia and Eurasia Programme at Chatham House.

Purchase

• UK (via Amazon)
• Rest of world (via Brookings Institution Press)
• Students (via Browns Books)

Diagnosing social behavioural dynamics of corruption Other resource dora.popova 8 December 2021

This interactive toolkit identifies the types of social expectations which sustain selected corrupt practices and provides behaviourally-informed recommendations for changing them.

When tackling a problem as complex as corruption, it is important to understand why and how people behave in different situations where corruption occurs. In contexts where it is easier to engage in corruption than avoid it, identifying the social expectations and informal rules which sustain corrupt practices can advance corruption prevention and deepen collective action.  

Behavioural approaches to corruption offer a better understanding of diverse social settings, group dynamics, power distribution, social motivations, and expectations that contribute to a more tolerant environment for certain forms of the phenomenon. They are also highly complementary to traditional corruption measures, which tend to focus on the enforcement of legal sanctions and deterrents. Behavioural approaches, especially those inspired by social norms theory, highlight complex social characteristics and informal rules of specific corrupt practices, and effectively support implementation of more dynamic context-specific anti-corruption interventions.

Since 2016, the Chatham House Africa programme’s Social Norms and Accountable Governance (SNAG) project has adopted a behavioural approach based on social norms methodology to investigate the social beliefs which motivate different forms of corruption. Drawing on the project’s extensive evidence-gathering and analysis, this toolkit offers users navigable behavioural mapping of contextual factors, beliefs, and expectations surrounding common corrupt practices. 

It aims to support anti-corruption actors in diagnosing informal rules and social expectations which sustain corruption in some societies. It also proposes behavioural-informed guidance for developing or adapting anti-corruption interventions and activities, so they account for informal rules of behaviour such as social norms. 
 
The toolkit supports users to:

• Identify whether and how widespread corrupt practices are motivated by social beliefs and expectations.

• Understand how society influences the types of corrupt activity individuals engage in, or avoid, and the factors informing these choices

• Integrate empirical evidence and behavioural insights into anti-corruption strategies from diagnostics to design, and eventual implementation and evaluation

The toolkit presents evidence from SNAG’s research into three key corrupt practices – bribery, embezzlement, and electoral fraud. Each was examined in the context of typical situations in which they occur, such as law enforcement, healthcare, the power sector, voting, and education while critical factors such as religion, gender, and ethnicity were considered. 

The toolkit presents an overview of specific contexts and behavioural features of the practices and provides behavioural-informed recommendations. It also contains pop-up features with definitions and explanations of key concepts. The toolkit is designed to be expandable, allowing further content and behavioural dynamics to be added.   

In nucleotide excision repair, bulky DNA lesions such as UV-induced cyclobutane pyrimidine dimers are removed from the genome by concerted dual incisions bracketing the lesion, followed by gap filling and ligation. So far, two dual-incision patterns have been discovered: the prokaryotic type, which removes the damage in 11–13-nucleotide-long oligomers, and the eukaryotic type, which removes the damage in 24–32-nucleotide-long oligomers. However, a recent study reported that the UvrC protein of Mycobacterium tuberculosis removes damage in a manner analogous to yeast and humans in a 25-mer oligonucleotide arising from incisions at 15 nt from the 3´ end and 9 nt from the 5´ end flanking the damage. To test this model, we used the in vivo excision assay and the excision repair sequencing genome-wide repair mapping method developed in our laboratory to determine the repair pattern and genome-wide repair map of Mycobacterium smegmatis. We find that M. smegmatis, which possesses homologs of the Escherichia coli uvrA, uvrB, and uvrC genes, removes cyclobutane pyrimidine dimers from the genome in a manner identical to the prokaryotic pattern by incising 7 nt 5´ and 3 or 4 nt 3´ to the photoproduct, and performs transcription-coupled repair in a manner similar to E. coli.

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

Iran: Protests, politics and power 16 November 2022 — 6:00PM TO 7:00PM Anonymous (not verified) 18 October 2022 Online

Join Robert Macaire, UK ambassador to Iran (2018-21), and others to discuss what the protests mean for Iran’s domestic, regional and global power.

Protests in Iran, spurred after Masha Amini died in police custody, have drawn focus on how Iranians feel about state repression, a struggling economy and global isolation. Iran is facing the most adamant challenge to its power structure since the ‘green movement’ in 2009 with protests taking place in more than 50 cities and towns across the country. There is no sign that the government will back down but what will that decision mean for the power it can wield at home and abroad?

This conversation examines how the protests impact Iran’s domestic power, its regional relationships and its relations with the US.

• What do the protests demonstrate about Iran’s power domestically and regionally?

• How do the protests influence the JCPOA?

• What will the government gain if they hold a hard line on protesters?

• How do the protests impact Iran’s regional activities?

As with all members events, questions from the audience drive the conversation.

Read the transcript. 

Memory politics: the challenge of commemoration in post-Soviet Eastern Europe and the Caucasus 5 October 2021 — 1:00PM TO 2:30PM Anonymous (not verified) 21 September 2021 Online

This event explores how to address memory and commemoration in the former Soviet states, considering their role in political processes and violent conflict. 

How the past is remembered and commemorated plays a large role – perhaps too large – in contemporary political debates and in how conflicts are negotiated.

Perceptions of history influence people’s actions and are used to judge or dismiss the actions of others. Nowhere is this more so than in the political, territorial and social debates and disputes across the former Soviet Union.
 
This event examines how to address the problems caused by entrenched memory debates – and proposes a framework for ‘ethical political commemoration’ for use across historical enquiry, political processes, and conflict transformation initiatives.

The speakers explore the topic through the context of Turkey and the Armenian genocide, as well as more broadly through their own experiences in conflict transformation and peace processes.

President Maia Sandu on democracy and politics in Moldova Video jon.wallace 4 July 2022

The president covers Moldova’s challenges as it seeks closer integration with the European Union.

President Maia Sandu discusses challenges to Moldovan democracy and society during an interview at Chatham House’s 2022 London Conference.

She covers issues including corruption, the presence of Russian troops in the Transnistria region, neutrality in Moldova’s constitution, popular support for EU membership and refugees from Russian aggression in Ukraine.

How Russia’s war affects politics in southeast Europe 4 April 2023 — 11:00AM TO 12:00PM Anonymous (not verified) 24 March 2023 Online

This event will discuss how the war on Ukraine has affected southeastern Europe.

This event will discuss how the war on Ukraine has affected southeastern Europe. How have the governments and publics responded to the war?

The panel will discuss Russia’s goals and leverage in the region, including the impact of its disinformation campaigns. Are the governments reassessing their foreign policy options?

The granting of EU candidate status to Ukraine and Moldova has brought attention to the stalled accession process in the Western Balkans. What lessons from the Western Balkans can be applied in Ukraine and Moldova? Have new linkages emerged between the two regions?

Eurovision, war and the geopolitics of pop The World Today mhiggins.drupal 28 March 2023

Europe’s great song competition may strive for continental harmony, but bitter conflicts and voting blocs often drag it off key, writes Saskia Postema.

In May, the city that spawned the Beatles will add another notch to its cultural belt as Liverpool hosts the Eurovision Song Contest.

The pop competition has been celebrating extravagance, dramatic acts and lots of glitter since 1956. It is expressly designed to be non-political, with performers barred from promoting political interests, yet it exists for a specific political purpose – to promote European unity.

The organizer, the European Broadcasting Union (EBU), has certainly succeeded in part with its mission. While Europe Day on May 9 comes and goes without much fanfare, Eurovision is an eagerly anticipated fixture on the cultural calendar.

Cultural diplomacy

While some may dismiss it as simply an extravagant party, there is more to it than that: it is a form of cultural diplomacy. Beyond artistry, acts are expected to bring their cultural identity to the stage as they bid for douze points, Eurovision’s highest score, from national juries.

Like any popular forum, it reflects popular opinion. So, when war threatens the European harmony Eurovision is meant to exude, politics inevitably manifests itself.

This was evident last year. Following Russia’s invasion of Ukraine in February, Eurovision’s organizers reiterated their apolitical stance stating both countries would be allowed to participate. But when various countries threatened to withdraw if Russia was allowed to compete, the EBU changed its tune and broke off ties with Russian broadcasting agencies indefinitely. As reluctant as it was to act in this capacity, the organization still inadvertently highlighted its role as a political gatekeeper and confirmed Eurovision does function as a political arena.

Unsurprisingly, all eyes were on the Ukrainian entry that year. Kalush Orchestra’s Stefania, presenting a blend of traditional Ukrainian folk music with hip hop beats, went on to win the contest resoundingly.

And while the winning nation normally hosts next year’s competition, due to the war this honour passed to the runner-up, the United Kingdom. Britain, coincidentally, has been one of the most visible supporters of Kyiv’s war efforts. Boris Johnson, the former prime minister, was among the first leaders to visit Kyiv after the Russian invasion.

Last year’s second place ended a long spate of poor results for the UK in the competition. The first year it scored nul points was 2003 when the UK entry was Cry Baby by the duo Jemini. The result was seen widely as a backlash against Britain’s entry into the Iraq war earlier that year.

In the intervening years it has been placed last twice and received nul points again in 2021 when the entry was Embers sung by James Newman. With last year’s second place for Sam Ryder’s Space Man, the UK redeemed itself and will get to host Eurovision in Liverpool, a city twinned with Odesa since 1957.

It is not the first time that Russia’s geopolitical relations have led to Eurovision controversy. In 2008, following the Russo-Georgian war, Russia recognized the self-proclaimed independence of South Ossetia and Abkhazia which had broken away from Georgia.

As a result, Georgia initially refused to take part in the 2009 Eurovision as it was to be held in Moscow. It changed its mind, however, submitting a song by Stefane and 3G called We Don’t Wanna Put In. The submission was rejected by the EBU, which said it contained obvious references to the Russian leader, something Georgia denied.

In 2015, Ukraine withdrew from Eurovision following the Russian annexation of Crimea. The following year the EBU was faced with a similar problem when Ukraine submitted a song, 1944,sung by Jamala, which told the story of her great-grandmother who lived through the mass deportation of Crimean Tatars in the Soviet Union under Josef Stalin. This time the EBU allowed the entry, despite the Russian Duma calling for its rejection. The song went on to win.