The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. Here, to explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3 and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely, thymosin beta 4, thymosin beta 10 and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. To explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3, and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely thymosin beta 4, thymosin beta 10, and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

Biosynthesis of magnetosome-nanobody complex in Magnetospirillum gryphiswaldense MSR-1 and a magnetosome-nanobody-based enzyme-linked immunosorbent assay for the detection of tetrabromobisphenol A in water.

In this study, two mutant strains, TBC and TBC+, able to biosynthesize a novel functional magnetosome-nanobody (Nb), were derived from the magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1. The magnetosome-Nbs biosynthesized by TBC+ containing multi-copies of the Nb gene had a higher binding ability to an environmental pollutant, tetrabromobisphenol A (TBBPA), than those biosynthesized by TBC containing only one copy of the Nb gene. The magnetosome-Nbs from TBC+ can effectively bind to TBBPA in solutions with high capacity without being affected by a broad range of NaCl and methanol concentrations as well as pH. Therefore, a magnetosome-Nb-based enzyme-linked immunosorbent assay (ELISA) was developed and optimized for the detection of TBBPA, yielding a half-maximum signal inhibition concentration of 0.23 ng/mL and a limit of detection of 0.025 ng/mL. The assay was used to detect TBBPA in spiked river water samples, giving average recoveries between 90 and 120% and coefficients of variation of 2.5-6.3%. The magnetosome-Nb complex could be reused 4 times in ELISA without affecting the performance of the assay. Our results demonstrate the potential of magnetosome-Nbs produced by TBC+ as cost-effective and environment-friendly reagents for immunoassays to detect small molecules in environmental waters.

Enabling Global Analysis of Protein Citrullination via Biotin Thiol Tag-Assisted Mass Spectrometry.

Citrullination is a key post-translational modification (PTM) that affects protein structures and functions. Although it has been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize this PTM. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of citrullination via mass spectrometry. We perform global mapping of the citrullination proteome of mouse tissues. In total, we identify 691 citrullination sites from 432 proteins which represents the largest data set to date. We discover novel distribution and functions of this PTM. This study depicts a landscape of protein citrullination and lays the foundation for further deciphering their physiological and pathological roles.

Native Ion Mobility-Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level.

Protein sialylation has been closely linked to many diseases including Alzheimer's disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aß cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM-MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aß cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aß cytotoxicity. Native gel electrophoresis and IM-MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aß cytotoxicity.

PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability.

The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer cells to regulate anabolic metabolism. Although PKM2 was recently reported to regulate lipid homeostasis, the molecular mechanism remains unclear. Herein, we discovered an ER transmembrane protein 33 (TMEM33) as a downstream effector of PKM2 that regulates activation of SREBPs and lipid metabolism. Loss of PKM2 leads to up-regulation of TMEM33, which recruits RNF5, an E3 ligase, to promote SREBP-cleavage activating protein (SCAP) degradation. TMEM33 is transcriptionally regulated by nuclear factor erythroid 2-like 1 (NRF1), whose cleavage and activation are controlled by PKM2 levels. Total plasma cholesterol levels are elevated by either treatment with PKM2 tetramer-promoting agent TEPP-46 or by global PKM2 knockout in mice, highlighting the essential function of PKM2 in lipid metabolism. Although depletion of PKM2 decreases cancer cell growth, global PKM2 knockout accelerates allografted tumor growth. Together, our findings reveal the cell-autonomous and systemic effects of PKM2 in lipid homeostasis and carcinogenesis, as well as TMEM33 as a bona fide regulator of lipid metabolism.

Proteome-wide and matrisome-specific alterations during human pancreas development and maturation.

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation.

Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1.

BACKGROUND: Magnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil. RESULTS: Three recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78-101%. CONCLUSIONS: The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

High quality and fast compressed sensing MRI reconstruction via edge-enhanced dual discriminator generative adversarial network.

Generative adversarial networks (GAN) are widely used for fast compressed sensing magnetic resonance imaging (CSMRI) reconstruction. However, most existing methods are difficult to make an effective trade-off between abstract global high-level features and edge features. It easily causes problems, such as significant remaining aliasing artifacts and clearly over-smoothed reconstruction details. To tackle these issues, we propose a novel edge-enhanced dual discriminator generative adversarial network architecture called EDDGAN for CSMRI reconstruction with high quality. In this model, we extract effective edge features by fusing edge information from different depths. Then, leveraging the relationship between abstract global high-level features and edge features, a three-player game is introduced to control the hallucination of details and stabilize the training process. The resulting EDDGAN can offer more focus on edge restoration and de-aliasing. Extensive experimental results demonstrate that our method consistently outperforms state-of-the-art methods and obtains reconstructed images with rich edge details. In addition, our method also shows remarkable generalization, and its time consumption for each 256 × 256 image reconstruction is approximately 8.39 ms.

Hyphenation of strong cation exchange chromatography to native mass spectrometry for high throughput online characterization of charge heterogeneity of therapeutic monoclonal antibodies.

Characterization of charge heterogeneity in monoclonal antibodies (mAbs) is needed during developability assessment and downstream development of drug candidates. Charge heterogeneity can come from post-translational modifications like deamidation, isomerization, and sialylation. Elucidation of charge variants with mass spectrometry (MS) has historically been challenging. Due to the nonvolatility and high ionic strength of conventional buffer systems, labor-intensive offline fractionation followed by MS analysis is routinely used. Here, we describe an alternative strategy that directly couples strong cation exchange (SCX) chromatography to high-resolution Orbitrap MS for online native MS analysis (SCX-MS). A combined pH and salt gradient was used for universal separation of mAbs from a wide range of pI values (6.38 ~ 9.2), including infliximab (Remicade®, chimeric IgG1/kappa), NISTmab (humanized IgG1/kappa) and trastuzumab (Herceptin®, humanized IgG1/kappa), without tailoring of chromatographic profiles. Liquid chromatography and MS parameters were optimized to achieve high-quality spectra and enhanced detection of low abundant species under high flow rate conditions. Genedata Expressionist, a vendor agnostic software, was used for data processing. This integrated strategy allows unbiased characterization of numerous charge variant species and low molecular weight fragments (<0.05%) without post-column flow splitting. The application was further expanded with middle-up approaches for subdomain analysis, which demonstrated the versatility of the strategy for analysis of various construct types. With our analysis of mAbs during developability assessment and forced degradation studies, which aimed at assessing potential critical quality attributes in antibody drug molecules, we provide, for the first time, direct visualization of molecular alterations of mAbs at intact level. Furthermore, strong correlation was observed between this novel MS approach and analysis by capillary isoelectric focusing.

Nanosecond photochemically promoted click chemistry for enhanced neuropeptide visualization and rapid protein labeling.

Comprehensive protein identification and concomitant structural probing of proteins are of great biological significance. However, this is challenging to accomplish simultaneously in one confined space. Here, we develop a nanosecond photochemical reaction (nsPCR)-based click chemistry, capable of structural probing of proteins and enhancing their identifications through on-demand removal of surrounding matrices within nanoseconds. The nsPCR is initiated using a photoactive compound, 2-nitrobenzaldehyde (NBA), and is examined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Benefiting from the on-demand matrix-removal effect, this nsPCR strategy enables enhanced neuropeptide identification and visualization from complex tissue samples such as mouse brain tissue. The design shows great promise for structural probing of proteins up to 155 kDa due to the exclusive accessibility of nsPCR to primary amine groups, as demonstrated by its general applicability using a series of proteins with various lysine residues from multiple sample sources, with accumulated labeling efficiencies greater than 90%.

In Depth Quantification of Extracellular Matrix Proteins from Human Pancreas.

Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates ß cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal ß cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal ß cell maturation.

Visualization and Identification of Neurotransmitters in Crustacean Brain via Multifaceted Mass Spectrometric Approaches.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has emerged as a label-free analytical tool for fast biomolecule profiling on tissue sections. Among various functional molecules, mapping neurotransmitters and related metabolites is of tremendous significance, as these compounds are critical to signaling in the central nervous system. Here, we demonstrated the use of both derivatization and reaction-free approaches that greatly reduced signal complexity and thus enabled complementary signaling molecule visualization on crab brain sections via MALDI-LTQ-Orbitrap XL platform. Pyrylium salt served as a primary amine derivatization reagent and produced prominent signal enhancement of multiple neurotransmitters, including dopamine, serotonin, γ-aminobutyric acid, and histamine that were not detected in underivatized tissues. Molecules with other functional groups, such as acetylcholine and phosphocholine, were directly imaged after matrix application. The identities of discovered neurotransmitters were verified by standards using LC-MS/MS. This study broadens our understanding of metabolic signaling in the crustacean nervous system and highlights potential of multifaceted MS techniques for unambiguous neurotransmitter characterization.

HOTMAQ: A Multiplexed Absolute Quantification Method for Targeted Proteomics.

Absolute quantification in targeted proteomics is challenging due to a variety of factors, including low specificity in complex backgrounds, limited analytical throughput, and wide dynamic range. To address these problems, we developed a hybrid offset-triggered multiplex absolute quantification (HOTMAQ) strategy that combines cost-effective mass difference and isobaric tags to enable simultaneous construction of an internal standard curve in the MS1 precursor scan, real-time identification of peptides at the MS2 level, and mass offset-triggered accurate quantification of target proteins in synchronous precursor selection (SPS)-MS3 spectra. This approach increases the analytical throughput of targeted quantitative proteomics by up to 12-fold. The HOTMAQ strategy was employed to verify candidate protein biomarkers in preclinical Alzheimer's disease with high accuracy. The greatly enhanced throughput and quantitative performance, paired with sample flexibility, makes HOTMAQ broadly applicable to targeted peptidomics, proteomics, and phosphoproteomics.

Urinary Metabolomic and Proteomic Analyses in a Mouse Model of Prostatic Inflammation.

Lower urinary tract symptoms (LUTS) are common among aging men. Since prostatic inflammation is one of its etiologies, it is plausible that urinary metabolite and protein biomarkers could be identified and used to diagnose inflammation-induced LUTS. We characterized the urine metabolome and proteome in a mouse model of bacterial-induced prostatic inflammation. Mass Spectrometry (MS)-based multi-omics analysis was employed to discover urinary protein and metabolite-based biomarkers. The investigation of isobaric dimethylated leucine (DiLeu) labeling on metabolites allowed metabolomics and proteomics analysis on the same liquid chromatography (LC)-MS platform. In total, 143 amine-containing metabolites and 1058 urinary proteins were identified and quantified (data are available via ProteomeXchange with identifier PXD018023); among them, 14 metabolites and 168 proteins were significantly changed by prostatic inflammation. Five metabolic pathways and four inflammation-related biological processes were potentially disrupted. By comparing our findings with urinary biomarkers identified in a mouse model of genetic-induced prostate inflammation and with those previously found to be associated with LUTS in older men, we identified creatine, haptoglobin, immunoglobulin kappa constant and polymeric Ig receptor as conserved biomarkers for prostatic inflammation associated with LUTS. These data suggest that these putative biomarkers could be used to identify men in which prostate inflammation is present and contributing to LUTS.

Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas.

Extracellular matrix (ECM) plays an important developmental role by regulating cell behaviour through structural and biochemical stimulation. Tissue-specific ECM, attained through decellularization, has been proposed in several strategies for tissue and organ replacement. Decellularization of animal pancreata has been reported, but the same methods applied to human pancreas are less effective due to higher lipid content. Moreover, ECM-derived hydrogels can be obtained from many decellularized tissues, but methods have not been reported to obtain human pancreas-derived hydrogel. Using novel decellularization methods with human pancreas we produced an acellular, 3D biological scaffold (hP-ECM) and hydrogel (hP-HG) amenable to tissue culture, transplantation and proteomic applications. The inclusion of a homogenization step in the decellularization protocol significantly improved lipid removal and gelation capability of the resulting ECM, which was capable of gelation at 37 °C in vitro and in vivo, and is cytocompatible with a variety of cell types and islet-like tissues in vitro. Overall, this study demonstrates the characterisation of a novel protocol for the decellularization and delipidization of human pancreatic tissue for the production of acellular ECM and ECM hydrogel suitable for cell culture and transplantation applications. We also report a list of 120 proteins present within the human pancreatic matrisome.

Surfactant and Chaotropic Agent Assisted Sequential Extraction/On-Pellet Digestion (SCAD) for Enhanced Proteomics.

As a popular sample preparation approach, filter-aided sample preparation (FASP) has been widely used in proteomic analysis. However, several limitations have been noted, including sample loss during filtration, repetitive centrifugation steps, and the possibility of breakage of filtration membrane. Extraction bias among different sample preparation strategies presents another challenge. To overcome these limitations and address remaining challenges, we developed a novel surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion (SCAD) protocol. The new strategy resulted in higher protein yield and improved peptide recovery and protein coverage compared to two conventional sample preparation methods (FASP and urea). In combination of three strategies, more than 10,000 distinct protein groups were identified with 1% FDR from MDA-MB-231 cells without any prefractionation. This in-depth proteome analysis was accomplished by optimization of protein extraction, enzymatic digestion, LC gradient, and peptide sequencing method. Ingenuity Pathways Analysis (IPA) of proteins exclusively identified in SCAD revealed several crucial signaling pathways that regulate breast cancer progression. SCAD also enabled an unbiased extraction of different categories of proteins (membrane, intracellular, nuclear) associated with tumorigenesis, which integrates the advantages of FASP and urea extraction. This novel strategy expedites comprehensive protein identification, which is applicable for biomarker discovery in various types of cancers.

Large-Scale Differentiation and Site Specific Discrimination of Hydroxyproline Isomers by Electron Transfer/Higher-Energy Collision Dissociation (EThcD) Mass Spectrometry.

3- and 4-Hydroxyprolines (HyP) are regioisomers that play different roles in various species and organs. Despite their distinct functions inside cells, they are generally considered indistinguishable using mass spectrometry due to their identical masses. Here, we demonstrate, for the first time, that characteristic w ions can be produced by electron-transfer/higher energy collision dissociation (EThcD) dual fragmentation technique to confidently discriminate 3-HyP/4-HyP isomers. An integrated and high throughput strategy was developed which combined online LC separation with EThcD for large-scale differentiation of 3-HyP/4-HyP in complex samples. An automated algorithm was developed for charge state dependent characterization of 3-HyP/4-HyP isomers. Using this combined discrimination approach, we identified 108 3-HyP sites and 530 4-HyP sites from decellularized pancreas, allowing more than 5-fold increase of both 3-HyP and 4-HyP identifications compared to previous reports. This approach outperformed ETD and HCD in the analysis of HyP-containing peptides with unique capacity to generate w ions for HyP discrimination, improved fragmentation of precursor ions, as well as unambiguous localization of modifications. A high content of 3-HyP was observed in the C-terminal (GPP)n domain of human CO1A1, which was previously only identified in vertebrate fibrillar collagens from tendon. Unexpectedly, some unusual HyP sites at Xaa position in Gly-HyP-Ala, Gly-HyP-Val, Gly-HyP-Gln, Gly-HyP-Ser, and Gly-HyP-Arg were also confirmed to be 3-hydroxylated, whose functions and enzymes are yet to be discovered. Overall, this novel discrimination strategy can be readily implemented into de novo sequencing or other proteomic search engines.

Erratum: PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis.

This corrects the article DOI: 10.1038/ncb3630.

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis.

Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP3Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP3R-dependent mitochondrial functions.