Proteome Map Research Articles

Decoding the Virtual 2D Map of the Chloroplast Proteomes

BackgroundThe chloroplast is a semi-autonomous organelle having its own genome and corresponding proteome. Although chloroplast genomes have been reported, no reports exist on their corresponding proteomes. Therefore, a proteome-wide analysis of the chloroplast proteomes of 2893 species was conducted, and a virtual 2D map was constructed.ResultsThe resulting virtual 2D map of the chloroplast proteome exhibited a bimodal distribution. The molecular mass of the chloroplast proteome ranged from 0.448 to 616.334 kDa, and the isoelectric point (pI) ranged from 2.854 to 12.954. Chloroplast proteomes were dominated by basic pI proteins with an average pI of 7.852. The molecular weight and isoelectric point of chloroplast proteome were found to show bimodal distribution. Leu was the most abundant and Cys the least abundant amino acid in the chloroplast proteome. Notably, Trp amino acid was absent in the chloroplast protein sequences of Pilostyles aethiopica. In addition, Selenocysteine (Sec) and Pyrrolysine (Pyl) amino acids were also found to be lacking in the chloroplast proteomes.ConclusionThe virtual 2D map and amino acid composition of chloroplast proteome will enable the researchers to understand the biochemistry of chloroplast protein in detail. Further, the amino acid composition of the chloroplast proteome will also allow us to understand the codon usage bias. The codon usage bias and amino acid usage bias of chloroplast will be crucial to understanding their relationship.

Multielemental, Nutritional, and Proteomic Characterization of Different Lupinus spp. Genotypes: A Source of Nutrients for Dietary Use.

Among grain pulses, lupins have recently gained considerable interest for a number of attractive nutritional attributes relating to their high protein and dietary fiber and negligible starch contents. The seeds of Lupinus albus (cv. Multitalia and Luxor, and the Modica ecotype); L. luteus (cv. Dukat, Mister, and Taper); and L. angustifolius (cv. Sonet) analyzed in this study were deposited within the germplasm collection of the Research Centre for Cereal and Industrial Crops of Acireale and were sowed in East Sicily in 2013/14. The collected seeds were analyzed for their multielemental micro- and macronutrient profiles, resulting in a wide variability between genotypes. Lupin seed flour samples were subjected to a defatting process using supercritical CO2, with oil yields dependent on the species and genotype. We determined the fatty acid profile and tocopherol content of the lupin oil samples, finding that the total saturated fatty acid quantities of different samples were very close, and the total tocopherol content was about 1500.00 µg/g FW. The proteomic analysis of the defatted lupin seed flours showed substantial equivalence between the cultivars of the same species of Lupinus albus and L. luteus. Moreover, the L. angustifolius proteome map showed the presence of additional spots in comparison to L. albus, corresponding to α-conglutins. Lupin, in addition to being a good source of mineral elements, also contributes vitamin E and, thanks to the very high content of gamma-tocopherols, demonstrates powerful antioxidant activity.

Unsupervised Machine Learning Organization of the Functional Dark Proteome of Gram-Negative "Superbugs": Six Protein Clusters Amenable for Distinct Scientific Applications.

Uncharacterized proteins have been underutilized as targets for the development of novel therapeutics for difficult-to-treat bacterial infections. To facilitate the exploration of these proteins, 2819 predicted, uncharacterized proteins (19.1% of the total) from reference strains of multidrug Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa species were organized using an unsupervised k-means machine learning algorithm. Classification using normalized values for protein length, pI, hydrophobicity, degree of conservation, structural disorder, and %AT of the coding gene rendered six natural clusters. Cluster proteins showed different trends regarding operon membership, expression, presence of unknown function domains, and interactomic relevance. Clusters 2, 4, and 5 were enriched with highly disordered proteins, nonworkable membrane proteins, and likely spurious proteins, respectively. Clusters 1, 3, and 6 showed closer distances to known antigens, antibiotic targets, and virulence factors. Up to 21.8% of proteins in these clusters were structurally covered by modeling, which allowed assessment of druggability and discontinuous B-cell epitopes. Five proteins (4 in Cluster 1) were potential druggable targets for antibiotherapy. Eighteen proteins (11 in Cluster 6) were strong B-cell and T-cell immunogen candidates for vaccine development. Conclusively, we provide a feature-based schema to fractionate the functional dark proteome of critical pathogens for fundamental and biomedical purposes.

The First Pituitary Proteome Landscape From Matched Anterior and Posterior Lobes for a Better Understanding of the Pituitary Gland

To date, very few mass spectrometry (MS)–based proteomics studies are available on the anterior and posterior lobes of the pituitary. In the past, MS-based investigations have focused exclusively on the whole pituitary gland or anterior pituitary lobe. In this study, for the first time, we performed a deep MS-based analysis of five anterior and five posterior matched lobes to build the first lobe-specific pituitary proteome map, which documented 4090 proteins with isoforms, mostly mapped into chromosomes 1, 2, and 11. About 1446 differentially expressed significant proteins were identified, which were studied for lobe specificity, biological pathway enrichment, protein–protein interaction, regions specific to comparison of human brain and other neuroendocrine glands from Human Protein Atlas to identify pituitary-enriched proteins. Hormones specific to each lobe were also identified and validated with parallel reaction monitoring–based target verification. The study identified and validated hormones, growth hormone and thyroid-stimulating hormone subunit beta, exclusively to the anterior lobe whereas oxytocin–neurophysin 1 and arginine vasopressin to the posterior lobe. The study also identified proteins POU1F1 (pituitary-specific positive transcription factor 1), POMC (pro-opiomelanocortin), PCOLCE2 (procollagen C-endopeptidase enhancer 2), and NPTX2 (neuronal pentraxin-2) as pituitary-enriched proteins and was validated for their lobe specificity using parallel reaction monitoring. In addition, three uPE1 proteins, namely THEM6 (mesenchymal stem cell protein DSCD75), FSD1L (coiled-coil domain–containing protein 10), and METTL26 (methyltransferase-like 26), were identified using the NeXtProt database, and depicted tumor markers S100 proteins having high expression in the posterior lobe. In summary, the study documents the first matched anterior and posterior pituitary proteome map acting as a reference control for a better understanding of functional and nonfunctional pituitary adenomas and extrapolating the aim of the Human Proteome Project towards the investigation of the proteome of life.

Quantitative proteomics reveals potential anti-inflammatory protein targets of radial extracorporeal shock wave therapy in TNF-α-induced model of acute inflammation in primary human tenocytes

Tendinopathy refers to a type of tendon disease with a multifactorial spectrum. Recent research has begun to reveal the effects of inflammation on the tendinopathic process, especially in the first stage of tendinopathy. Radial extracorporeal shock wave therapy (rESWT) has been successfully used to treat orthopedic diseases. However, the molecular mechanisms underlying the anti-inflammatory effects of rESWT on tumor necrosis factor-α treated tenocytes have not been fully elucidated. In this study, we applied total protein tandem mass tag-labeled quantitative proteomics with liquid chromatography-mass spectrometer/mass spectrometer technology to identify differentially expressed proteins (DEPs) among inflammatory tenocytes, rESWT inflammatory tenocytes, and controls using three biological replicates. Human tenocytes were used and they were cultured in vitro. In total, 1028 and 40 DEPs were detected for control versus inflammatory tenocytes and for inflammatory tenocytes versus rESWT inflammatory tenocytes, respectively. Further, we identified integrin α2, selenoprotein S, and NLR family CARD domain-containing protein 4 as pivotal molecular targets of the anti-inflammatory effects of rESWT. This is the first study to provide a reference proteomic map for inflammatory tenocytes and rESWT inflammatory tenocytes. Our findings provide crucial insight into the molecular mechanisms underscoring the anti-inflammatory effects of rESWT in tendinopathy.

Protein Digestion for 2D-DIGE Analysis.

In-gel digestion of protein spots derived from two-dimensional gels and their subsequent identification by mass spectrometry is involved in a multitude of mass spectrometry-driven proteomic experiments, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). This type of proteomic methodology has been involved in the establishment of comparative proteome maps and in the identification of differentially expressed proteins and their isoforms in health and disease. Most in-gel digestion protocols follow a number of common steps including excision of the protein spots of interest, destaining, reduction and alkylation (for silver-stained gels), and dehydration and overnight digestion with the proteolytic enzyme of choice. While trypsin has been a mainstay of peptide digestion for many years, it does have its shortcomings, particularly related to incomplete peptide digestion, and this has led to a rise in popularity for other proteolytic enzymes either used alone or in combination. This chapter discusses the alternative enzymes available and describes the process of in-gel digestion using the enzyme trypsin.

Deep Phosphoproteome Landscape of Interhemispheric Functionality of Neuroanatomical Regions of the Human Brain.

Post-translational modifications (PTMs) are one of the compulsive and predominant biological processes that regulate the diverse molecular mechanism, modulate the onset of disease, and are the reason behind the functional diversity of proteins. Despite the widespread research findings in neuroproteomics, one of the key drawbacks has been the lack of proteome-level knowledge of hemispheric lateralization. We have investigated the proteome level expression in different neuroanatomical regions under the Human Brain Proteome Project (HBPP) and developed the global interhemispheric brain proteome map (Brainprot) earlier. Furthermore, this study has extended to decipher the phosphoproteome map of human brain interhemispheric regions through high-resolution mass spectrometry. The phosphoproteomics examination of 12 unique interhemispheric neurological brain regions using Orbitrap fusion liquid chromatography with tandem mass spectrometry provided comprehensive coverage of 996 phosphoproteins, 2010 phosphopeptides, and 3567 phosphosites. Moreover, interhemispheric phosphoproteome profiling has been categorized according to synaptic ontologies and interhemispheric expression to understand the functionality. Finally, we have integrated the phosphosites data under the PhosphoMap section in the Inter-Hemispheric Brain Proteome Map Portal (https://www.brainprot.org/) for the advancement and support of the ongoing neuroproteomics research worldwide. Data is available via ProteomeXchange with the identifier PXD031188.

Synaptic proteomics reveal distinct molecular signatures of cognitive change and C9ORF72 repeat expansion in the human ALS cortex

Increasing evidence suggests synaptic dysfunction is a central and possibly triggering factor in Amyotrophic Lateral Sclerosis (ALS). Despite this, we still know very little about the molecular profile of an ALS synapse. To address this gap, we designed a synaptic proteomics experiment to perform an unbiased assessment of the synaptic proteome in the ALS brain. We isolated synaptoneurosomes from fresh-frozen post-mortem human cortex (11 controls and 18 ALS) and stratified the ALS group based on cognitive profile (Edinburgh Cognitive and Behavioural ALS Screen (ECAS score)) and presence of a C9ORF72 hexanucleotide repeat expansion (C9ORF72-RE). This allowed us to assess regional differences and the impact of phenotype and genotype on the synaptic proteome, using Tandem Mass Tagging-based proteomics. We identified over 6000 proteins in our synaptoneurosomes and using robust bioinformatics analysis we validated the strong enrichment of synapses. We found more than 30 ALS-associated proteins in synaptoneurosomes, including TDP-43, FUS, SOD1 and C9ORF72. We identified almost 500 proteins with altered expression levels in ALS, with region-specific changes highlighting proteins and pathways with intriguing links to neurophysiology and pathology. Stratifying the ALS cohort by cognitive status revealed almost 150 specific alterations in cognitively impaired ALS synaptic preparations. Stratifying by C9ORF72-RE status revealed 330 protein alterations in the C9ORF72-RE +ve group, with KEGG pathway analysis highlighting strong enrichment for postsynaptic dysfunction, related to glutamatergic receptor signalling. We have validated some of these changes by western blot and at a single synapse level using array tomography imaging. In summary, we have generated the first unbiased map of the human ALS synaptic proteome, revealing novel insight into this key compartment in ALS pathophysiology and highlighting the influence of cognitive decline and C9ORF72-RE on synaptic composition.

Integration of multi-omics technologies for crop improvement: Status and prospects.

With the rapid development of next-generation sequencing (NGS), multi-omics techniques have been emerging as effective approaches for crop improvement. Here, we focus mainly on addressing the current status and future perspectives toward omics-related technologies and bioinformatic resources with potential applications in crop breeding. Using a large amount of omics-level data from the functional genome, transcriptome, proteome, epigenome, metabolome, and microbiome, clarifying the interaction between gene and phenotype formation will become possible. The integration of multi-omics datasets with pan-omics platforms and systems biology could predict the complex traits of crops and elucidate the regulatory networks for genetic improvement. Different scales of trait predictions and decision-making models will facilitate crop breeding more intelligent. Potential challenges that integrate the multi-omics data with studies of gene function and their network to efficiently select desirable agronomic traits are discussed by proposing some cutting-edge breeding strategies for crop improvement. Multi-omics-integrated approaches together with other artificial intelligence techniques will contribute to broadening and deepening our knowledge of crop precision breeding, resulting in speeding up the breeding process.

Tissue- and cell-expression of druggable host proteins provide insights into repurposing drugs for COVID-19.

Several human host proteins play important roles in the lifecycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many drugs targeting these host proteins have been investigated as potential therapeutics for coronavirus disease 2019 (COVID-19). The tissue-specific expressions of selected host proteins were summarized using proteomics data retrieved from the Human Protein Atlas, ProteomicsDB, Human Proteome Map databases, and a clinical COVID-19 study. Protein expression features in different cell lines were summarized based on recent proteomics studies. The half-maximal effective concentration or half-maximal inhibitory concentration values were collected from in vitro studies. The pharmacokinetic data were mainly from studies in healthy subjects or non-COVID-19 patients. Considerable tissue-specific expression patterns were observed for several host proteins. ACE2 expression in the lungs was significantly lower than in many other tissues (e.g., the kidneys and intestines); TMPRSS2 expression in the lungs was significantly lower than in other tissues (e.g., the prostate and intestines). The expression levels of endocytosis-associated proteins CTSL, CLTC, NPC1, and PIKfyve in the lungs were comparable to or higher than most other tissues. TMPRSS2 expression was markedly different between cell lines, which could be associated with the cell-dependent antiviral activities of several drugs. Drug delivery receptor ICAM1 and CTSB were expressed at a higher level in the lungs than in other tissues. In conclusion, the cell- and tissue-specific proteomics data could help interpret the in vitro antiviral activities of host-directed drugs in various cells and aid the transition of the in vitro findings to clinical research to develop safe and effective therapeutics for COVID-19.

MTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.

The mammalian target of rapamycin (mTOR) pathway is crucial in energy metabolism and cell proliferation. Previously, we reported transcriptome-wide 3′-untranslated region (UTR) shortening by alternative polyadenylation upon mTOR activation and its impact on the proteome. Here, we further interrogated the mTOR-activated transcriptome and found that hyperactivation of mTOR promotes transcriptome-wide exon skipping/exclusion, producing short isoform transcripts from genes. This widespread exon skipping confers multifarious regulations in the mTOR-controlled functional proteomics: AS in coding regions widely affects the protein length and functional domains. They also alter the half-life of proteins and affect the regulatory post-translational modifications. Among the RNA processing factors differentially regulated by mTOR signaling, we found that SRSF3 mechanistically facilitates exon skipping in the mTOR-activated transcriptome. This study reveals a role of mTOR in AS regulation and demonstrates that widespread AS is a multifaceted modulator of the mTOR-regulated functional proteome.

Virtual 2D map of cyanobacterial proteomes.

Cyanobacteria are prokaryotic Gram-negative organisms prevalent in nearly all habitats. A detailed proteomics study of Cyanobacteria has not been conducted despite extensive study of their genome sequences. Therefore, we conducted a proteome-wide analysis of the Cyanobacteria proteome and found Calothrix desertica as the largest (680331.825 kDa) and Candidatus synechococcus spongiarum as the smallest (42726.77 kDa) proteome of the cyanobacterial kingdom. A Cyanobacterial proteome encodes 312.018 amino acids per protein, with a molecular weight of 182173.1324 kDa per proteome. The isoelectric point (pI) of the Cyanobacterial proteome ranges from 2.13 to 13.32. It was found that the Cyanobacterial proteome encodes a greater number of acidic-pI proteins, and their average pI is 6.437. The proteins with higher pI are likely to contain repetitive amino acids. A virtual 2D map of Cyanobacterial proteome showed a bimodal distribution of molecular weight and pI. Several proteins within the Cyanobacterial proteome were found to encode Selenocysteine (Sec) amino acid, while Pyrrolysine amino acids were not detected. The study can enable us to generate a high-resolution cell map to monitor proteomic dynamics. Through this computational analysis, we can gain a better understanding of the bias in codon usage by analyzing the amino acid composition of the Cyanobacterial proteome.

Organ-specific metabolic pathways distinguish prediabetes, type 2 diabetes, and normal tissues

SummaryEnvironmental and genetic factors cause defects in pancreatic islets driving type 2 diabetes (T2D) together with the progression of multi-tissue insulin resistance. Mass spectrometry proteomics on samples from five key metabolic tissues of a cross-sectional cohort of 43 multi-organ donors provides deep coverage of their proteomes. Enrichment analysis of Gene Ontology terms provides a tissue-specific map of altered biological processes across healthy, prediabetes (PD), and T2D subjects. We find widespread alterations in several relevant biological pathways, including increase in hemostasis in pancreatic islets of PD, increase in the complement cascade in liver and pancreatic islets of PD, and elevation in cholesterol biosynthesis in liver of T2D. Our findings point to inflammatory, immune, and vascular alterations in pancreatic islets in PD that are hypotheses to be tested for potential contributions to hormonal perturbations such as impaired insulin and increased glucagon production. This multi-tissue proteomic map suggests tissue-specific metabolic dysregulations in T2D.

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis.

As the common and significant chemical modifications, post-translational modifications (PTMs) play a key role in the functional proteome. Affected by the signal interference, low concentration, and insufficient ionization efficiency of impurities, the direct detection of PTMs by mass spectrometry (MS) still faces many challenges. Therefore, sample preparation and enrichment are an indispensable link before MS analysis of PTMs in proteomics. The rapid development of functionalized materials with diverse morphologies and compositions provides an avenue for sample preparation and enrichment for PTMs analysis. In this review, we summarize recent advances in the application of novel functionalized materials in sample preparation for phosphoproteomes and glycoproteomes analysis. In addition, this review specifically discusses the design and preparation of functionalized materials based on different enrichment mechanisms, and proposes research directions and potential challenges for proteomic PTMs research.

Reaction-based fluorogenic probes for detecting protein cysteine oxidation in living cells

‘Turn-on’ fluorescence probes for detecting H2O2 in cells are established, but equivalent tools to monitor the products of its reaction with protein cysteines have not been reported. Here we describe fluorogenic probes for detecting sulfenic acid, a redox modification inextricably linked to H2O2 signaling and oxidative stress. The reagents exhibit excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. We develop a high-throughput assay for measuring S-sulfenation in cells and use it to screen a curated kinase inhibitor library. We reveal a positive association between S-sulfenation and inhibition of TK, AGC, and CMGC kinase group members including GSK3, a promising target for neurological disorders. Proteomic mapping of GSK3 inhibitor-treated cells shows that S-sulfenation sites localize to the regulatory cysteines of antioxidant enzymes. Our studies highlight the ability of kinase inhibitors to modulate the cysteine sulfenome and should find broad application in the rapidly growing field of redox medicine.

Reduced endosomal microautophagy activity in aging associates with enhanced exocyst-mediated protein secretion.

Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age‐related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub‐proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age‐related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein‐loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst‐RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst‐RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst‐RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.

Proteomic mapping and optogenetic manipulation of membrane contact sites.

Membrane contact sites (MCSs) mediate crucial physiological processes in eukaryotic cells, including ion signaling, lipid metabolism, and autophagy. Dysregulation of MCSs is closely related to various diseases, such as type 2 diabetes mellitus (T2DM), neurodegenerative diseases, and cancers. Visualization, proteomic mapping and manipulation of MCSs may help the dissection of the physiology and pathology MCSs. Recent technical advances have enabled better understanding of the dynamics and functions of MCSs. Here we present a summary of currently known functions of MCSs, with a focus on optical approaches to visualize and manipulate MCSs, as well as proteomic mapping within MCSs.

Brown Adipose Tissue Sheds Extracellular Vesicles That Carry Potential Biomarkers of Metabolic and Thermogenesis Activity Which Are Affected by High Fat Diet Intervention.

Brown adipose tissue (BAT) is a key target for the development of new therapies against obesity due to its role in promoting energy expenditure; BAT secretory capacity is emerging as an important contributor to systemic effects, in which BAT extracellular vesicles (EVs) (i.e., batosomes) might be protagonists. EVs have emerged as a relevant cellular communication system and carriers of disease biomarkers. Therefore, characterization of the protein cargo of batosomes might reveal their potential as biomarkers of the metabolic activity of BAT. In this study, we are the first to isolate batosomes from lean and obese Sprague–Dawley rats, and to establish reference proteome maps. An LC-SWATH/MS analysis was also performed for comparisons with EVs secreted by white adipose tissue (subcutaneous and visceral WAT), and it showed that 60% of proteins were exclusive to BAT EVs. Precisely, batosomes of lean animals contain proteins associated with mitochondria, lipid metabolism, the electron transport chain, and the beta-oxidation pathway, and their protein cargo profile is dramatically affected by high fat diet (HFD) intervention. Thus, in obesity, batosomes are enriched with proteins involved in signal transduction, cell communication, the immune response, inflammation, thermogenesis, and potential obesity biomarkers including UCP1, Glut1, MIF, and ceruloplasmin. In conclusion, the protein cargo of BAT EVs is affected by the metabolic status and contains potential biomarkers of thermogenesis activity.

Quantitative proteomic analysis of SARS-CoV-2 infection of primary human airway ciliated cells and lung epithelial cells demonstrates the effectiveness of SARS-CoV-2 innate immune evasion.

Background: Quantitative proteomics is able to provide a comprehensive, unbiased description of changes to cells caused by viral infection, but interpretation may be complicated by differential changes in infected and uninfected 'bystander' cells, or the use of non-physiological cellular models. Methods: In this paper, we use fluorescence-activated cell sorting (FACS) and quantitative proteomics to analyse cell-autonomous changes caused by authentic SARS-CoV-2 infection of respiratory epithelial cells, the main target of viral infection in vivo. First, we determine the relative abundance of proteins in primary human airway epithelial cells differentiated at the air-liquid interface (basal, secretory and ciliated cells). Next, we specifically characterise changes caused by SARS-CoV-2 infection of ciliated cells. Finally, we compare temporal proteomic changes in infected and uninfected 'bystander' Calu-3 lung epithelial cells and compare infection with B.29 and B.1.1.7 (Alpha) variants. Results: Amongst 5,709 quantified proteins in primary human airway ciliated cells, the abundance of 226 changed significantly in the presence of SARS-CoV-2 infection (q <0.05 and >1.5-fold). Notably, viral replication proceeded without inducing a type-I interferon response. Amongst 6,996 quantified proteins in Calu-3 cells, the abundance of 645 proteins changed significantly in the presence of SARS-CoV-2 infection (q < 0.05 and > 1.5-fold). In contrast to the primary cell model, a clear type I interferon (IFN) response was observed. Nonetheless, induction of IFN-inducible proteins was markedly attenuated in infected cells, compared with uninfected 'bystander' cells. Infection with B.29 and B.1.1.7 (Alpha) variants gave similar results. Conclusions: Taken together, our data provide a detailed proteomic map of changes in SARS-CoV-2-infected respiratory epithelial cells in two widely used, physiologically relevant models of infection. As well as identifying dysregulated cellular proteins and processes, the effectiveness of strategies employed by SARS-CoV-2 to avoid the type I IFN response is illustrated in both models.

Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling

Enzymatic-based proximity labeling approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein interactors in living cells. However, activated esters are poorly reactive which leads to a wide labeling radius and phenoxy radicals generated by peroxide treatment may disturb redox-sensitive pathways. Herein, we report a photoactivation-dependent proximity labeling (PDPL) method designed by genetically attaching photosensitizer protein miniSOG to a protein of interest. Triggered by blue light and tunned by irradiation time, singlet oxygen is generated, thereafter enabling spatiotemporally-resolved aniline probe labeling of histidine residues. We demonstrate its high-fidelity through mapping of organelle-specific proteomes. Side-by-side comparison of PDPL with TurboID reveals more specific and deeper proteomic coverage by PDPL. We further apply PDPL to the disease-related transcriptional coactivator BRD4 and E3 ligase Parkin, and discover previously unknown interactors. Through over-expression screening, two unreported substrates Ssu72 and SNW1 are identified for Parkin, whose degradation processes are mediated by the ubiquitination-proteosome pathway.