Rapid-Response Water-Shrink Films with High Output Work Density Based on Polyethylene Oxide and α-Cyclodextrin for Autonomous Wound Closure.

Conventional wound closure methods, including sutures and tissue adhesives, present significant challenges for self-care treatment, particularly in the context of bleeding wounds. Existing stimuli-responsive contractile materials designed for autonomous wound closure frequently lack sufficient output work density to generate the force needed to bring the wound edges into proximity or necessitate stimuli that are not compatible with the human body. Here, we report semi-transparent, flexible, and water-responsive shrinkable films, composed of poly(ethylene oxide) and α-cyclodextrin. These films exhibit remarkable stability under ambient conditions and demonstrate significant contraction (∼50%) within 6 seconds upon exposure to water, generating substantial contractile stress (up to 6 MPa) and output work density (∼1028 kJ m-3), which is 100 times larger than that of conventional hydrogel and 25 times larger than that of skeletal muscles. Remarkably, upon hydration, these films are capable of lifting objects 10,000 times their own weight. Leveraging this technology, we further developed water-shrink tapes, which, upon contact with water, effectively constrict human skin and autonomously close bleeding wounds in animal models within 10 seconds. Our work offers a novel approach to skin wound management, showing significant potential for emergency and self-care scenarios. This article is protected by copyright. All rights reserved.

Water-responsive supercontractile polymer films for bioelectronic interfaces.

Connecting different electronic devices is usually straightforward because they have paired, standardized interfaces, in which the shapes and sizes match each other perfectly. Tissue-electronics interfaces, however, cannot be standardized, because tissues are soft1-3 and have arbitrary shapes and sizes4-6. Shape-adaptive wrapping and covering around irregularly sized and shaped objects have been achieved using heat-shrink films because they can contract largely and rapidly when heated7. However, these materials are unsuitable for biological applications because they are usually much harder than tissues and contract at temperatures higher than 90 °C (refs. 8,9). Therefore, it is challenging to prepare stimuli-responsive films with large and rapid contractions for which the stimuli and mechanical properties are compatible with vulnerable tissues and electronic integration processes. Here, inspired by spider silk10-12, we designed water-responsive supercontractile polymer films composed of poly(ethylene oxide) and poly(ethylene glycol)-α-cyclodextrin inclusion complex, which are initially dry, flexible and stable under ambient conditions, contract by more than 50% of their original length within seconds (about 30% per second) after wetting and become soft (about 100 kPa) and stretchable (around 600%) hydrogel thin films thereafter. This supercontraction is attributed to the aligned microporous hierarchical structures of the films, which also facilitate electronic integration. We used this film to fabricate shape-adaptive electrode arrays that simplify the implantation procedure through supercontraction and conformally wrap around nerves, muscles and hearts of different sizes when wetted for in vivo nerve stimulation and electrophysiological signal recording. This study demonstrates that this water-responsive material can play an important part in shaping the next-generation tissue-electronics interfaces as well as broadening the biomedical application of shape-adaptive materials.

A Leaf-Patchable Reflectance Meter for In Situ Continuous Monitoring of Chlorophyll Content.

Plant wearable sensors facilitate the real-time monitoring of plant physiological status. In situ monitoring of the plant chlorophyll content over days can provide valuable information on the photosynthetic capacity, nitrogen content, and general plant health. However, it cannot be achieved by current chlorophyll measuring methods. Here, a miniaturized and plant-wearable chlorophyll meter for rapid, non-destructive, in situ, and long-term chlorophyll monitoring is developed. The reflectance-based chlorophyll sensor with 1.5 mm thickness and 0.2 g weight (1000 times lighter than the commercial chlorophyll meter), includes a light emitting diode (LED) and two symmetric photodetectors (PDs) on a flexible substrate, and is patched onto the leaf upper epidermis with a conformal light guiding layer. A chlorophyll content index (CCI) calculated based on the sensor shows a better linear relationship with the leaf chlorophyll content (r2 > 0.9) than the traditional chlorophyll meter. This meter can wirelessly communicate with a smartphone to monitor the leaf chlorophyll change under various stresses and indicate the unhealthy status of plants for long-term application of plants under various stresses earlier than chlorophyll meter and naked-eye observation. This wearable chlorophyll sensing patch is promising in smart and precision agriculture.

Method validation and new peak detection for the liquid chromatography-mass spectrometry multi-attribute method.

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry (LC-MS) peptide mapping technique that has been proposed as a replacement for several conventional quality control (QC) methods for therapeutic proteins. In addition to quantification of multiple product quality attributes (PQAs), MAM can also monitor impurities using a new peak detection (NPD) feature. Here, results are provided from method validation and NPD studies of an MAM approach applied to rituximab as a model monoclonal antibody (mAb). Twenty-one rituximab PQAs were monitored, including oxidation, pyroglutamination, deamidation, lysine clipping, and glycosylation. The PQA monitoring aspect of the method was validated according to ICH Guidance. Accuracy, precision, specificity, detection and quantitation limits, linearity, range, and robustness were demonstrated for this MAM approach with minimal issues. All PQAs were successfully validated except for several oxidation sites, which did not pass intermediate precision criteria. The variability found in oxidation measurements was attributed to artificial oxidation during sample preparation and could likely be alleviated through several approaches. The NPD aspect of the method was also evaluated. A spike-in approach was used to assess the limits of detection and quantitation (LOD/LOQ) of the NPD feature of MAM. For NPD, the peak intensity threshold was found to be the most critical parameter for accurate detection of impurities since a low threshold can result in false positives while a high threshold can obscure the detection of true peaks. Overall, the MAM approach presented and validated here has been demonstrated to be suitable for both targeted monitoring of rituximab PQAs and non-targeted detection of new peaks that represent impurities.

Wet-Adhesive On-Skin Sensors Based on Metal-Organic Frameworks for Wireless Monitoring of Metabolites in Sweat.

Metal-organic frameworks (MOFs) with well-defined porous structures and tailored functionalities have been widely used in chemical sensing. However, the integration of MOFs with flexible electronic devices for wearable sensing is challenging because of their low electrical conductivity and fragile mechanical properties. Herein, a wearable sweat sensor for metabolite detection is presented by integrating an electrically conductive Ni-MOF with a flexible nanocellulose substrate. The MOF-based layered film sensor with inherent conductivity, highly porous structure, and active catalytic properties enables the selective and accurate detection of vitamin C and uric acid. More importantly, the lightweight sensor can conformably self-adhere to sweaty skin and exhibits high water-vapor permeability. Furthermore, a wireless epidermal nutrition tracking system for the in situ monitoring of the dynamics of sweat vitamin C is demonstrated, the results of which are comparable to those tested by high-performance liquid chromatography. This study opens a new avenue for integrating MOFs as the active layer in wearable electronic devices and holds promise for the future development of high-performance electronics with enhanced sensing, energy production, and catalytic capabilities through the implementation of multifunctional MOFs.

In vitro degradation, photo-dynamic and thermal antibacterial activities of Cu-bearing chlorophyllin-induced Ca-P coating on magnesium alloy AZ31.

Surgical failures, caused by postoperative infections of bone implants, are commonly met, which cannot be treated precisely with intravenous antibiotics. Photothermal therapy (PTT) and photodynamic therapy (PDT) have attracted widespread attention due to their non-invasive antibacterial effects on tissues and no bacterial resistance, which may be an excellent approach to solve infections related to bone implants for biodegradable magnesium alloys. Herein, a sodium copper chlorophyllin (SCC) with a porphyrin ring induced Ca-P coating was prepared on AZ31 magnesium alloy via layer-by-layer (LbL) assembly. The morphology and composition of the samples were characterized through field emission scanning electron microscope (FE-SEM) with affiliated energy dispersive spectrometer (EDS), X-ray diffractometer (XRD), and Fourier infrared spectrometer (FTIR) and X-ray photoelectron spectrometer (XPS) as well. Potentiodynamic polarization, electrochemical impedance spectroscopy (EIS) and hydrogen evolution experiments were employed to evaluate the corrosion behavior of the samples. Atomic absorption spectrophotometer was used to measure Cu elemental content of different immersion periods. Cytocompatibility and antibacterial performance of the coatings were probed using in vitro cytotoxicity tests (MTT assay), live/dead cell staining and plate counting method. The results showed that the obtained (Ca-P/SCC)10 coating exhibited good corrosion resistance, antimicrobial activity (especially under 808 nm irradiation) and biocompatibility. The antibacterial rates for E. coli and S. aureus were 99.9% and 99.8%, respectively; and the photothermal conversion efficiency was as high as 42.1%. Triple antibacterial mechanisms including photodynamic, photothermal reactions and copper-ions release were proposed. This coating exhibited a promising application for biodegradable magnesium alloys.

Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression.

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Multi-Attribute Method for Quality Control of Therapeutic Proteins.

Recent advances in high resolution mass spectrometry (MS) instrumentation and semi-automated software have led to a push toward the use of MS-based methods for quality control (QC) testing of therapeutic proteins in a cGMP environment. The approach that is most commonly being proposed for this purpose is known as the multi-attribute method (MAM). MAM is a promising approach that provides some distinct benefits compared to conventional methods currently used for QC testing of protein therapeutics, such as CEX, HILIC, and CE-SDS. Because MS-based methods have not been regularly used in this context in the past, new scientific and regulatory questions should be addressed prior to the final stages of implementation. We have categorized these questions into four major aspects for MAM implementation in a cGMP environment for both new and existing products: risk assessment, method validation, capabilities and specificities of the New Peak Detection (NPD) feature, and comparisons to conventional methods. This perspective outlines considerations for each of these main points and suggests approaches to help address potential issues.

Target identification reveals lanosterol synthase as a vulnerability in glioma.

Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain tumor for which novel therapeutic approaches are desperately needed. Previous studies have shown that the menin inhibitor MI-2 exhibits promising activity in preclinical DIPG and adult glioma models, although the mechanism underlying this activity is unknown. Here, using an integrated approach, we show that MI-2 exerts its antitumor activity in glioma largely independent of its ability to target menin. Instead, we demonstrate that MI-2 activity in glioma is mediated by disruption of cholesterol homeostasis, with suppression of cholesterol synthesis and generation of the endogenous liver X receptor ligand, 24,25-epoxycholesterol, resulting in cholesterol depletion and cell death. Notably, this mechanism is responsible for MI-2 activity in both DIPG and adult glioma cells. Metabolomic and biochemical analyses identify lanosterol synthase as the direct molecular target of MI-2, revealing this metabolic enzyme as a vulnerability in glioma and further implicating cholesterol homeostasis as an attractive pathway to target in this malignancy.

Surface plasmon resonance-assisted circularly polarized luminescent hybrid assemblies of Eu-containing polyoxometalates.

The circular dichroism and circularly polarized luminescence of achiral inorganic Eu-containing clusters in an electrostatically coassembled supramolecular assembly were induced by a cationic chiral copolymer with the help of an enhanced electromagnetic field generated by Ag nanoparticles.

Histone modification signatures in human sperm distinguish clinical abnormalities.

PURPOSE: Alternations to the paternal epigenome, specifically the components of sperm chromatin, can lead to infertility in humans and potentially transmit aberrant information to the embryo. One key component of sperm chromatin is the post-translational modification of histones (PTMs). We previously identified a comprehensive profile of histone PTMs in normozoospermic sperm; however, only specific histone PTMs have been identified in abnormal sperm by antibody-based approaches and comprehensive changes to histone PTM profiles remain unknown. Here, we investigate if sperm with abnormalities of total motility, progressive motility, and morphology have altered histone PTM profiles compared to normozoospermic sperm samples. METHODS: Discarded semen samples from 31 men with normal or abnormal semen parameters were analyzed for relative abundance of PTMs on histone H3 and H4 by "bottom-up" nano-liquid chromatography-tandem mass spectrometry. RESULTS: Asthenoteratozoospermic samples (abnormal motility, forward progression, and morphology, n = 6) displayed overall decreased H4 acetylation (p = 0.001) as well as alterations in H4K20 (p = 0.003) and H3K9 methylation (p < 0.04) when compared to normozoospermic samples (n = 8). Asthenozoospermic samples (abnormal motility and progression, n = 5) also demonstrated decreased H4 acetylation (p = 0.04) and altered H4K20 (p = 0.005) and H3K9 methylation (p < 0.04). Samples with isolated abnormal progression (n = 6) primarily demonstrated decreased acetylation on H4 (p < 0.02), and teratozoospermic samples (n = 6) appeared similar to normozoospermic samples (n = 8). CONCLUSION: Sperm samples with combined and isolated abnormalities of total motility, progressive motility, and morphology display distinct and altered histone PTM signatures compared to normozoospermic sperm. This provides evidence that alterations in histone PTMs may be important for normal sperm function and fertility.

Chiral Metal-Organic Framework Hollow Nanospheres for High-Efficiency Enantiomer Separation.

Chiral ZIF-8 hollow nanospheres with d-histidine as part of chiral ligands (denoted as H-d-his-ZIF-8) were prepared for separation of (±)-amine acids. Compared to bulk d-his-ZIF-8 without a hollow cavity, the prepared H-d-his-ZIF-8 showed 15 times higher separation capacity and higher ee values of 90.5 % for alanine, 95.2 % for glutamic acid and 92.6 % for lysine, respectively.

Metabolic labeling in middle-down proteomics allows for investigation of the dynamics of the histone code.

BACKGROUND: Middle-down mass spectrometry (MS), i.e., analysis of long (~50-60 aa) polypeptides, has become the method with the highest throughput and accuracy for the characterization of combinatorial histone posttranslational modifications (PTMs). The discovery of histone readers with multiple domains, and overall the cross talk of PTMs that decorate histone proteins, has revealed that histone marks have synergistic roles in modulating enzyme recruitment and subsequent chromatin activities. Here, we demonstrate that the middle-down MS strategy can be combined with metabolic labeling for enhanced quantification of histone proteins and their combinatorial PTMs in a dynamic manner. METHODS: We used a nanoHPLC-MS/MS system consisting of hybrid weak cation exchange-hydrophilic interaction chromatography combined with high resolution MS and MS/MS with ETD fragmentation. After spectra identification, we filtered confident hits and quantified polypeptides using our in-house software isoScale. RESULTS: We first verified that middle-down MS can discriminate and differentially quantify unlabeled from heavy labeled histone N-terminal tails (heavy lysine and arginine residues). Results revealed no bias toward identifying and quantifying unlabeled versus heavy labeled tails, even if the heavy labeled peptides presented the typical skewed isotopic pattern typical of long protein sequences that hardly get 100% labeling. Next, we plated epithelial cells into a media with heavy methionine-(methyl-13CD3), the precursor of the methyl donor S-adenosylmethionine and stimulated epithelial to mesenchymal transition (EMT). We assessed that results were reproducible across biological replicates and with data obtained using the more widely adopted bottom-up MS strategy, i.e., analysis of short tryptic peptides. We found remarkable differences in the incorporation rate of methylations in non-confluent cells versus confluent cells. Moreover, we showed that H3K27me3 was a critical player during the EMT process, as a consistent portion of histones modified as H3K27me2K36me2 in epithelial cells were converted into H3K27me3K36me2 in mesenchymal cells. CONCLUSIONS: We demonstrate that middle-down MS, despite being a more scarcely exploited MS technique than bottom-up, is a robust quantitative method for histone PTM characterization. In particular, middle-down MS combined with metabolic labeling is currently the only methodology available for investigating turnover of combinatorial histone PTMs in dynamic systems.

Characterization, Pathogenicity, and Phylogenetic Analyses of Colletotrichum Species Associated with Brown Blight Disease on Camellia sinensis in China.

Brown blight disease caused by Colletotrichum species is a common and serious foliar disease of tea (Camellia sinensis). Fungal isolates from several tea plantations causing typical brown blight symptoms were identified as belonging to the Colletotrichum acutatum species complex and the Colletotrichum gloeosporioides species complex based on morphological characteristics as well as DNA analysis of the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Colletotrichum acutatum, a new causal agent associated with C. sinensis, showed high phenotypic and genotypic diversity compared with the more commonly reported C. gloeosporioides. Phylogenetic analysis derived from individual and combined ITS and GAPDH sequences clearly clustered C. acutatum and C. gloeosporioides into separate species. Pathogenicity tests validated that both species were causal agents of tea brown blight disease and were highly pathogenic to tea leaves. However, the two groups of C. gloeosporioides with low levels of variability within their ITS and GAPDH regions differed in their virulence. This study reports for the first time the characterization of C. acutatum and C. gloeosporioides causing brown blight disease on tea (Camellia sinensis (L.) O. Kuntze) in China.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or ß GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/ß than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/ß cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity.

Co-repressor CBFA2T2 regulates pluripotency and germline development.

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2, 3, 4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2(-/-) mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

Comprehensive analysis of histone post-translational modifications in mouse and human male germ cells.

BACKGROUND: During the process of spermatogenesis, male germ cells undergo dramatic chromatin reorganization, whereby most histones are replaced by protamines, as part of the pathway to compact the genome into the small nuclear volume of the sperm head. Remarkably, approximately 90 % (human) to 95 % (mouse) of histones are evicted during the process. An intriguing hypothesis is that post-translational modifications (PTMs) decorating histones play a critical role in epigenetic regulation of spermatogenesis and embryonic development following fertilization. Although a number of specific histone PTMs have been individually studied during spermatogenesis and in mature mouse and human sperm, to date, there is a paucity of comprehensive identification of histone PTMs and their dynamics during this process. RESULTS: Here we report systematic investigation of sperm histone PTMs and their dynamics during spermatogenesis. We utilized "bottom-up" nanoliquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) to identify histone PTMs and to determine their relative abundance in distinct stages of mouse spermatogenesis (meiotic, round spermatids, elongating/condensing spermatids, and mature sperm) and in human sperm. We detected peptides and histone PTMs from all four canonical histones (H2A, H2B, H3, and H4), the linker histone H1, and multiple histone isoforms of H1, H2A, H2B, and H3 in cells from all stages of mouse spermatogenesis and in mouse sperm. We found strong conservation of histone PTMs for histone H3 and H4 between mouse and human sperm; however, little conservation was observed between H1, H2A, and H2B. Importantly, across eight individual normozoospermic human semen samples, little variation was observed in the relative abundance of nearly all histone PTMs. CONCLUSION: In summary, we report the first comprehensive and unbiased analysis of histone PTMs at multiple time points during mouse spermatogenesis and in mature mouse and human sperm. Furthermore, our results suggest a largely uniform histone PTM signature in sperm from individual humans.

Preparation of Magnetic Tubular Nanoreactors for Highly Efficient Catalysis.

We report an efficient and controllable route to prepare magnetic tubular nanoreactors composed of a mesoporous SiO2 shell with iron-noble metal nanoparticles inside. The key of this method is to design and fabricate Fe nanoparticles encapsulated in a mesoporous SiO2 shell. Making use of a galvanic replacement reaction between Fe and noble metal ions, all of the noble metal nanoparticles are loaded inside the mesoporous SiO2 shell, and thus nanoreactors are formed. Taking Pd as an example, the prepared Pd-Fe@meso-SiO2 tubular nanoreactor exhibits a high catalytic activity and excellent reusability for styrene hydrogenation under mild conditions. This study provides a facile route to fabricate magnetic nanoreactors with enhanced catalytic properties.

A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics.

Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing (13)C-labeled UDP-GlcNAc from (13)C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time.