Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens.

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.

R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation.

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.

Proteome-pI 2.0: proteome isoelectric point database update.

Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric point-the pH at which a particular molecule carries no net electrical charge-is an important parameter for many analytical biochemistry and proteomics techniques. Additionally, it can be obtained directly from the pKa values of individual charged residues of the protein. The Proteome-pI 2.0 database includes data for over 61 million protein sequences from 20 115 proteomes (three to four times more than the previous release). The isoelectric point for proteins is predicted by 21 methods, whereas pKa values are inferred by one method. To facilitate bottom-up proteomics analysis, individual proteomes were digested in silico with the five most commonly used proteases (trypsin, chymotrypsin, trypsin + LysC, LysN, ArgC), and the peptides' isoelectric point and molecular weights were calculated. The database enables the retrieval of virtual 2D-PAGE plots and customized fractions of a proteome based on the isoelectric point and molecular weight. In addition, isoelectric points for proteins in NCBI non-redundant (nr), UniProt, SwissProt, and Protein Data Bank are available in both CSV and FASTA formats. The database can be accessed at http://isoelectricpointdb2.org.

The Human Proteoform Atlas: a FAIR community resource for experimentally derived proteoforms.

The Human Proteoform Atlas (HPfA) is a web-based repository of experimentally verified human proteoforms on-line at http://human-proteoform-atlas.org and is a direct descendant of the Consortium of Top-Down Proteomics' (CTDP) Proteoform Atlas. Proteoforms are the specific forms of protein molecules expressed by our cells and include the unique combination of post-translational modifications (PTMs), alternative splicing and other sources of variation deriving from a specific gene. The HPfA uses a FAIR system to assign persistent identifiers to proteoforms which allows for redundancy calling and tracking from prior and future studies in the growing community of proteoform biology and measurement. The HPfA is organized around open ontologies and enables flexible classification of proteoforms. To achieve this, a public registry of experimentally verified proteoforms was also created. Submission of new proteoforms can be processed through email vianrtdphelp@northwestern.edu, and future iterations of these proteoform atlases will help to organize and assign function to proteoforms, their PTMs and their complexes in the years ahead.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

LectomeXplore, an update of UniLectin for the discovery of carbohydrate-binding proteins based on a new lectin classification.

Lectins are non-covalent glycan-binding proteins mediating cellular interactions but their annotation in newly sequenced organisms is lacking. The limited size of functional domains and the low level of sequence similarity challenge usual bioinformatics tools. The identification of lectin domains in proteomes requires the manual curation of sequence alignments based on structural folds. A new lectin classification is proposed. It is built on three levels: (i) 35 lectin domain folds, (ii) 109 classes of lectins sharing at least 20% sequence similarity and (iii) 350 families of lectins sharing at least 70% sequence similarity. This information is compiled in the UniLectin platform that includes the previously described UniLectin3D database of curated lectin 3D structures. Since its first release, UniLectin3D has been updated with 485 additional 3D structures. The database is now complemented by two additional modules: PropLec containing predicted ß-propeller lectins and LectomeXplore including predicted lectins from sequences of the NBCI-nr and UniProt for every curated lectin class. UniLectin is accessible at https://www.unilectin.eu/.

Pfam: The protein families database in 2021.

The Pfam database is a widely used resource for classifying protein sequences into families and domains. Since Pfam was last described in this journal, over 350 new families have been added in Pfam 33.1 and numerous improvements have been made to existing entries. To facilitate research on COVID-19, we have revised the Pfam entries that cover the SARS-CoV-2 proteome, and built new entries for regions that were not covered by Pfam. We have reintroduced Pfam-B which provides an automatically generated supplement to Pfam and contains 136 730 novel clusters of sequences that are not yet matched by a Pfam family. The new Pfam-B is based on a clustering by the MMseqs2 software. We have compared all of the regions in the RepeatsDB to those in Pfam and have started to use the results to build and refine Pfam repeat families. Pfam is freely available for browsing and download at http://pfam.xfam.org/.

Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion.

Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase ß (PDEß) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEß-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEß plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.

Integrated analysis of transcriptome and proteome to explore the genes related to steroid-induced femoral head necrosis.

PURPOSE: Femoral head necrosis (FHN) is a common disease of hip. However, the pathogenesis of FHN is not well understood. This study attempted to explore the potentially important genes and proteins involved in FHN. METHODS: We integrated the transcriptomic and proteomic methods to quantitatively screen the differentially expressed genes (DEGs) and proteins (DEPs) between Control and FHN groups. Gene ontology (GO) terms and KEGG pathway enrichment analysis were used to assess the roles of DEGs and DEPs. qRT-PCR and western blot were performed to verify the key genes/proteins in FHN. CCK-8 assay was performed to measure cell viability. The protein expression of Bax and Bcl-2 were used to evaluate cell apoptosis. RESULTS: Transcriptome and proteome studies indicated 758 DEGs and 1097 DEPs between Control and FHN groups, respectively. Cell division, extracellular exosome, and serine-type endopeptidase activity were the most common terms in biological process (BP), cellular component (CC), and molecular function (MF) enrichment, respectively. DEPs were mainly enriched in cellular process, cell, and binding for BP, CC, and MF categories, respectively. DEGs were mainly involved in PI3K-Akt pathway and DEPs were mainly focused in glycolysis/gluconeogenesis pathway. Notably, 14 down-regulated and 22 up-regulated genes/proteins were detected at both the transcript and protein level. LRG1, SERPINE2, STMN1, COL14A1, SLC37A2, and MMP2 were determined as the key genes/proteins in FHN. SERPINE2/STMN1 overexpression increased viability and decreased apoptosis of dexamethasone-treated MC3T3-E1 cells. CONCLUSIONS: Our study investigated some pivotal regulatory genes/proteins in the pathogenesis of FHN, providing novel insight into the genes/proteins involved in FHN.

IDPology of the living cell: intrinsic disorder in the subcellular compartments of the human cell.

Intrinsic disorder can be found in all proteomes of all kingdoms of life and in viruses, being particularly prevalent in the eukaryotes. We conduct a comprehensive analysis of the intrinsic disorder in the human proteins while mapping them into 24 compartments of the human cell. In agreement with previous studies, we show that human proteins are significantly enriched in disorder relative to a generic protein set that represents the protein universe. In fact, the fraction of proteins with long disordered regions and the average protein-level disorder content in the human proteome are about 3 times higher than in the protein universe. Furthermore, levels of intrinsic disorder in the majority of human subcellular compartments significantly exceed the average disorder content in the protein universe. Relative to the overall amount of disorder in the human proteome, proteins localized in the nucleus and cytoskeleton have significantly increased amounts of disorder, measured by both high disorder content and presence of multiple long intrinsically disordered regions. We empirically demonstrate that, on average, human proteins are assigned to 2.3 subcellular compartments, with proteins localized to few subcellular compartments being more disordered than the proteins that are localized to many compartments. Functionally, the disordered proteins localized in the most disorder-enriched subcellular compartments are primarily responsible for interactions with nucleic acids and protein partners. This is the first-time disorder is comprehensively mapped into the human cell. Our observations add a missing piece to the puzzle of functional disorder and its organization inside the cell.

Assessment of separation methods for extracellular vesicles from human and mouse brain tissues and human cerebrospinal fluids.

Extracellular vesicle (EV) is a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous system including animal and human brain tissues as well as cerebrospinal fluids, and the low yield of proteins in the separated EVs. To understand their exact molecular composition and their functional roles, development of the reliable protocols for EV separation is necessary. Here we report the methods for EV separation from human and mouse unfixed frozen brain tissues by a sucrose step gradient ultracentrifugation method, and from human cerebrospinal fluids by an affinity capture method. The separated EVs were assessed for morphological, biophysical and proteomic properties of separated EVs by nanoparticle tracking analysis, transmission electron microscopy, and labeled and label-free mass spectrometry for protein profiling with step-by-step protocols for each assessment.

The hunt for sORFs: A multidisciplinary strategy.

Growing evidence illustrates the shortcomings on the current understanding of the full complexity of the proteome. Previously overlooked small open reading frames (sORFs) and their encoded microproteins have filled important gaps, exerting their function as biologically relevant regulators. The characterization of the full small proteome has potential applications in many fields. Continuous development of techniques and tools led to an improved sORF discovery, where these can originate from bioinformatics analyses, from sequencing routines or proteomics approaches. In this mini review, we discuss the ongoing trends in the three fields and suggest some strategies for further characterization of high potential candidates.

Non-AUG start codons: Expanding and regulating the small and alternative ORFeome.

Recent ribosome profiling and proteomic studies have revealed the presence of thousands of novel coding sequences, referred to as small open reading frames (sORFs), in prokaryotic and eukaryotic genomes. These genes have defied discovery via traditional genomic tools not only because they tend to be shorter than standard gene annotation length cutoffs, but also because they are, as a class, enriched in sequence properties previously assumed to be unusual, including non-AUG start codons. In this review, we summarize what is currently known about the incidence, efficiency, and mechanism of non-AUG start codon usage in prokaryotes and eukaryotes, and provide examples of regulatory and functional sORFs that initiate at non-AUG codons. While only a handful of non-AUG-initiated novel genes have been characterized in detail to date, their participation in important biological processes suggests that an improved understanding of this class of genes is needed.

Dependence of Graphene Oxide (GO) Toxicity on Oxidation Level, Elemental Composition, and Size.

The mass production of graphene oxide (GO) unavoidably elevates the chance of human exposure, as well as the possibility of release into the environment with high stability, raising public concern as to its potential toxicological risks and the implications for humans and ecosystems. Therefore, a thorough assessment of GO toxicity, including its potential reliance on key physicochemical factors, which is lacking in the literature, is of high significance and importance. In this study, GO toxicity, and its dependence on oxidation level, elemental composition, and size, were comprehensively assessed. A newly established quantitative toxicogenomic-based toxicity testing approach, combined with conventional phenotypic bioassays, were employed. The toxicogenomic assay utilized a GFP-fused yeast reporter library covering key cellular toxicity pathways. The results reveal that, indeed, the elemental composition and size do exert impacts on GO toxicity, while the oxidation level exhibits no significant effects. The UV-treated GO, with significantly higher carbon-carbon groups and carboxyl groups, showed a higher toxicity level, especially in the protein and chemical stress categories. With the decrease in size, the toxicity level of the sonicated GOs tended to increase. It is proposed that the covering and subsequent internalization of GO sheets might be the main mode of action in yeast cells.

Characterization of Extracellular Vesicle Cargo in Sjögren's Syndrome through a SWATH-MS Proteomics Approach.

Primary Sjögren's syndrome (pSS) is a complex heterogeneous disease characterized by a wide spectrum of glandular and extra-glandular manifestations. In this pilot study, a SWATH-MS approach was used to monitor extracellular vesicles-enriched saliva (EVs) sub-proteome in pSS patients, to compare it with whole saliva (WS) proteome, and assess differential expressed proteins between pSS and healthy control EVs samples. Comparison between EVs and WS led to the characterization of compartment-specific proteins with a moderate degree of overlap. A total of 290 proteins were identified and quantified in EVs from healthy and pSS patients. Among those, 121 proteins were found to be differentially expressed in pSS, 82% were found to be upregulated, and 18% downregulated in pSS samples. The most representative functional pathways associated to the protein networks were related to immune-innate response, including several members of S100 protein family, annexin A2, resistin, serpin peptidase inhibitors, azurocidin, and CD14 monocyte differentiation antigen. Our results highlight the usefulness of EVs for the discovery of novel salivary-omic biomarkers and open novel perspectives in pSS for the identification of proteins of clinical relevance that could be used not only for the disease diagnosis but also to improve patients' stratification and treatment-monitoring. Data are available via ProteomeXchange with identifier PXD025649.

Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L.

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 µM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Proteome Analysis and In Vitro Antiviral, Anticancer and Antioxidant Capacities of the Aqueous Extracts of Lentinula edodes and Pleurotus ostreatus Edible Mushrooms.

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

Isolation of Nucleoid Fraction from Mycoplasma gallisepticum Cells with Synchronized Cell Division.

It is assumed that unknown mechanisms can be involved in adaptation Mycoplasma gallisepticum to unfavorable factors, one of these can be local rearrangements of the structure and spatial organization of the chromosome. To study these mechanisms, we obtained a culture of M. gallisepticum with synchronized division and isolated the nucleoid fraction from this culture by the method of mild cell lysis and centrifugation in a sucrose gradient. Liquid chromatography-mass spectrometry analysis of the proteome showed that in comparison with the cell lysate, the nucleoid fraction was enriched with DNA-binding proteins. This analysis will help to find new nucleoid-associated proteins and to study their dynamics, distribution, and their role during infection and under stress conditions.

APEX, a Master Key To Resolve Membrane Topology in Live Cells.

Determining the topology of the membrane proteome is fundamental for understanding its function at the membrane. However, conventional methods involving test tube reactions often lead to unreliable results, which do not accurately reflect membrane topology under physiological conditions, as perturbations occur during lysis. In this Perspective, we introduce a new method using engineered ascorbate peroxidase (APEX) for revealing membrane topological information in live cells without performing complicated sample preparation. We also discuss several examples of clearly resolved membrane topologies of various important mitochondrial proteins (e.g., LETM1, NDUFB10, MCU, SFXN1, and EXD2) and endoplasmic reticulum proteins (e.g., HMOX1) determined by using APEX-based methods.

METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies-Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses.

We present METATRYP version 2 software that identifies shared peptides across the predicted proteomes of organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine METATRYP database with predicted proteomes from environmental sequencing confirms a low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed to facilitate ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa analyzed, sharing <1% peptides with taxa outside of the betacoronavirus group, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches.