[Recent advances in protein precipitation-based methods for drug-target screening].

Drug targets are biological macromolecules that bind drug molecules in vivo. Therefore, the system-wide identification of drug targets plays a vital role in fully understanding the mechanism of drug action, efficacy, and side effects. The unbiased screening of drug targets may accelerate the process of drug discovery and candidate screening. Mass spectrometry is a key tool for large-scale protein identification and accurate quantification owing to its high acquisition speed, resolution, and sensitivity. Mass spectrometry-based proteomics has been widely used for drug-target screening. It can systematically identify the protein-target landscape of a drug and elucidate drug-protein interactions. Commonly used drug-target characterization methods, such as labeling-based affinity enrichment, require the chemical derivatization of drug molecules, which is not only time-consuming but may also affect the affinity of the drug towards its targets. Furthermore, the spatial effects of the derivatization groups may block interactions between the drug and its targets. Considering the disadvantages of affinity-enrichment methods, strategies that do not require chemical derivatization have received widespread attention. Proteins may undergo denaturation, unfolding, and precipitation under different conditions such as high temperatures, extreme pH, denaturants, and mechanical stress. Binding to small-molecule drugs may alter the folding balance of target proteins. The conformational stability of target proteins can be stabilized by binding with drugs, and protein-drug complexes are more resistant than free proteins to the precipitation induced by different conditions. Based on this mechanism, various large-scale drug-target identification methods using protein precipitation have been developed by combining proteomics and mass spectrometry analysis, including thermal proteome profiling and solvent-, mechanical stress-, and pH-induced protein precipitation. These methods have been successfully applied to the characterization of small-molecule drug targets. In this review, we describe the protein precipitation-based methods used for the high-throughput discovery of drug targets and elucidation of the interactions between drugs and proteins in the past decade. We also summarize the characteristics of each method and discuss their application potential in drug-efficacy evaluation and drug discovery.

O-GlcNAcase regulates pluripotency states of human embryonic stem cells.

Understanding the regulation of human embryonic stem cells (hESCs) pluripotency is critical to advance the field of developmental biology and regenerative medicine. Despite the recent progress, molecular events regulating hESC pluripotency, especially the transition between naive and primed states, still remain unclear. Here we show that naive hESCs display lower levels of O-linked N-acetylglucosamine (O-GlcNAcylation) than primed hESCs. O-GlcNAcase (OGA), the key enzyme catalyzing the removal of O-GlcNAc from proteins, is highly expressed in naive hESCs and is important for naive pluripotency. Depletion of OGA accelerates naive-to-primed pluripotency transition. OGA is transcriptionally regulated by EP300 and acts as a transcription regulator of genes important for maintaining naive pluripotency. Moreover, we profile protein O-GlcNAcylation of the two pluripotency states by quantitative proteomics. Together, this study identifies OGA as an important factor of naive pluripotency in hESCs and suggests that O-GlcNAcylation has a broad effect on hESCs homeostasis.

The OGT-c-Myc-PDK2 axis rewires the TCA cycle and promotes colorectal tumor growth.

Deregulated glucose metabolism termed the "Warburg effect" is a fundamental feature of cancers, including the colorectal cancer. This is typically characterized with an increased rate of glycolysis, and a concomitant reduced rate of the tricarboxylic acid (TCA) cycle metabolism as compared to the normal cells. How the TCA cycle is manipulated in cancer cells remains unknown. Here, we show that O-linked N-acetylglucosamine (O-GlcNAc) regulates the TCA cycle in colorectal cancer cells. Depletion of OGT, the sole transferase of O-GlcNAc, significantly increases the TCA cycle metabolism in colorectal cancer cells. Mechanistically, OGT-catalyzed O-GlcNAc modification of c-Myc at serine 415 (S415) increases c-Myc stability, which transcriptionally upregulates the expression of pyruvate dehydrogenase kinase 2 (PDK2). PDK2 phosphorylates pyruvate dehydrogenase (PDH) to inhibit the activity of mitochondrial pyruvate dehydrogenase complex, which reduces mitochondrial pyruvate metabolism, suppresses reactive oxygen species production, and promotes xenograft tumor growth. Furthermore, c-Myc S415 glycosylation levels positively correlate with PDK2 expression levels in clinical colorectal tumor tissues. This study highlights the OGT-c-Myc-PDK2 axis as a key mechanism linking oncoprotein activation with deregulated glucose metabolism in colorectal cancer.

A Computational and Chemical Design Strategy for Manipulating Glycan-Protein Recognition.

Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host-pathogen binding, and immune recognition, through interactions with glycan-binding proteins. A key driving force for glycan-protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized C─H groups of the pyranose (termed the CH-π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH-π interaction between glycans and proteins by genetically incorporating electron-rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N-linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N-acetylglucosamine-containing glycans. The approach thus provides a general and effective way to manipulate glycan-protein recognition for glycoscience applications.

Nuclear and cytoplasmic specific RNA binding proteome enrichment and its changes upon ferroptosis induction.

The key role of RNA-binding proteins (RBPs) in posttranscriptional regulation of gene expression is intimately tied to their subcellular localization. Here, we show a subcellular-specific RNA labeling method for efficient enrichment and deep profiling of nuclear and cytoplasmic RBPs. A total of 1221 nuclear RBPs and 1333 cytoplasmic RBPs were enriched and identified using nuclear/cytoplasm targeting enrichment probes, representing an increase of 54.4% and 85.7% compared with previous reports. The probes were further applied in the omics-level investigation of subcellular-specific RBP-RNA interactions upon ferroptosis induction. Interestingly, large-scale RBPs display enhanced interaction with RNAs in nucleus but reduced association with RNAs in cytoplasm during ferroptosis process. Furthermore, we discovered dozens of nucleoplasmic translocation candidate RBPs upon ferroptosis induction and validated representative ones by immunofluorescence imaging. The enrichment of Tricarboxylic acid cycle in the translocation candidate RBPs may provide insights for investigating their possible roles in ferroptosis induced metabolism dysregulation.

SPPUSM: An MS/MS spectra merging strategy for improved low-input and single-cell proteome identification.

Single and rare cell analysis provides unique insights into the investigation of biological processes and disease progress by resolving the cellular heterogeneity that is masked by bulk measurements. Although many efforts have been made, the techniques used to measure the proteome in trace amounts of samples or in single cells still lag behind those for DNA and RNA due to the inherent non-amplifiable nature of proteins and the sensitivity limitation of current mass spectrometry. Here, we report an MS/MS spectra merging strategy termed SPPUSM (same precursor-produced unidentified spectra merging) for improved low-input and single-cell proteome data analysis. In this method, all the unidentified MS/MS spectra from multiple test files are first extracted. Then, the corresponding MS/MS spectra produced by the same precursor ion from different files are matched according to their precursor mass and retention time (RT) and are merged into one new spectrum. The newly merged spectra with more fragment ions are next searched against the database to increase the MS/MS spectra identification and proteome coverage. Further improvement can be achieved by increasing the number of test files and spectra to be merged. Up to 18.2% improvement in protein identification was achieved for 1 ng HeLa peptides by SPPUSM. Reliability evaluation by the "entrapment database" strategy using merged spectra from human and E. coli revealed a marginal error rate for the proposed method. For application in single cell proteome (SCP) study, identification enhancement of 28%-61% was achieved for proteins for different SCP data. Furthermore, a lower abundance was found for the SPPUSM-identified peptides, indicating its potential for more sensitive low sample input and SCP studies.

Discovery of highly potent and selective KRASG12C degraders by VHL-recruiting PROTACs for the treatment of tumors with KRASG12C-Mutation.

Although several covalent KRASG12C inhibitors have made great progress in the treatment of KRASG12C-mutant cancer, their clinical applications are limited by adaptive resistance, motivating novel therapeutic strategies. Through drug design and structure optimization, a series of highly potent and selective KRASG12C Proteolysis Targeting Chimeras (PROTACs) were developed by incorporating AMG510 and VHL ligand VH032. Among them, degrader YN14 significantly inhibited KRASG12C-dependent cancer cells growth with nanomolar IC50 and DC50 values, and ＞ 95 % maximum degradation (Dmax). Molecular dynamics (MD) simulation showed that YN14 induced a stable KRASG12C: YN14: VHL ternary complex with low binding free energy (ΔG). Notably, YN14 led to tumor regression with tumor growth inhibition (TGI%) rates more than 100 % in the MIA PaCa-2 xenograft model with well-tolerated dose-schedules. We also found that KRASG12C degradation exhibited advantages in overcoming adaptive KRASG12C feedback resistance over KRASG12C inhibition. Furthermore, combination of RTKs, SHP2, or CDK9 inhibitors with YN14 exhibited synergetic efficacy in KRASG12C-mutant cancer cells. Overall, these results demonstrated that YN14 holds exciting prospects for the treatment of tumors with KRASG12C-mutation and boosted efficacy could be achieved for greater clinical applications via drug combination.

Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP-MS.

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma-mass spectrometry (ICP-MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP-MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.

Sensitive N-Glycopeptide Profiling of Single and Rare Cells Using an Isobaric Labeling Strategy without Enrichment.

Single-cell omics is critical in revealing population heterogeneity, discovering unique features of individual cells, and identifying minority subpopulations of interest. As one of the major post-translational modifications, protein N-glycosylation plays crucial roles in various important biological processes. Elucidation of the variation in N-glycosylation patterns at single-cell resolution may largely facilitate the understanding of their key roles in the tumor microenvironment and immune therapy. However, comprehensive N-glycoproteome profiling for single cells has not been achieved due to the extremely limited sample amount and incompatibility with the available enrichment strategies. Here, we have developed an isobaric labeling-based carrier strategy for highly sensitive intact N-glycopeptide profiling for single cells or a small number of rare cells without enrichment. Isobaric labeling has unique multiplexing properties, by which the "total" signal from all channels triggers MS/MS fragmentation for N-glycopeptide identification, while the reporter ions provide quantitative information. In our strategy, a carrier channel using N-glycopeptides obtained from bulk-cell samples significantly improved the "total" signal of N-glycopeptides and, therefore, promoted the first quantitative analysis of averagely 260 N-glycopeptides from single HeLa cells. We further applied this strategy to study the regional heterogeneity of N-glycosylation of microglia in mouse brain and discovered region-specific N-glycoproteome patterns and cell subtypes. In conclusion, the glycocarrier strategy provides an attractive solution for sensitive and quantitative N-glycopeptide profiling of single/rare cells that cannot be enriched by traditional workflows.

A three-stage search strategy combining database reduction and retention time filtering to improve the sensitivity of low-input and single-cell proteomic analysis.

When performing proteome profiling of low-input and single-cell samples, achieving deep protein coverage is very challenging due to the sensitivity limitation of current proteomic methods. Herein, we introduce a three-stage search strategy that combines the advantages of database reduction and Δ retention time (ΔRT) filtering. The strategy improves peptide/protein identification and reproducibility by retaining more correct identifications and filtering out incorrect identifications. The raw data were first merged and searched against a Uniprot database with a relaxed false discovery rate (FDR) of 40% to identify the possible detectable proteins. The identified proteins were then used as a new database to search the raw data against with a tighter FDR of 10%. After this, the results were filtered using ΔRT (the difference between the measured and predicted RT) to reduce the incorrect identifications and maintain the FDR below 1%. This strategy resulted in over 30% improvement in proteome coverage for single-cells and samples of similar size. The reproducibility of identification and quantification was also enhanced for the low-input samples. Moreover, the 50% higher number of differential proteins found in the two types of single neurons indicates the application potential of this strategy.

A Novel Strategy for Liposomal Drug Separation in Plasma by TiO2 Microspheres and Application in Pharmacokinetics.

Purpose: Liposomes are nano-scale materials with a biofilm-like structure. They have excellent biocompatibility and are increasingly useful in drug delivery systems. However, the in vivo fate of liposomal drugs is still unclear because existing bioanalytical methods for quantitation of total and liposomal-encapsulated drugs have limits. A novel strategy for liposomal-encapsulated drug separation from plasma was developed via the specific coordinate binding interaction of TiO2 microspheres with the phosphate groups of liposomes. Methods: Liposomal-encapsulated docetaxel was separated from plasma by TiO2 microspheres and analyzed by the UPLC-MS/MS method. The amount of TiO2, pH of the dilutions, plasma dilution factors and incubation time were optimized to improve extraction recovery. The characterization of the adsorption of liposome-encapsulated drugs by TiO2 microspheres was observed by electron microscopy. For understanding the mechanism, pseudo-first and the pseudo-second order equations were proposed for the adsorption process. The study fully validated the method for quantitation of liposomal-encapsulated in plasma and the method was applied to the pharmacokinetic study of docetaxel liposomes. Results: The encapsulated docetaxel had a concentration range of 15-4000 ng/mL from the plasma sample using a TiO2 extraction method. Successful method validation proved the method was sensitive, selective and stable, and was suitable for quantitation of docetaxel liposomes in plasma samples. Extraction recovery of this method was higher than that of SPE method. As shown in electron microscopy, the liposomes adsorbed on TiO2 microspheres were intact and there was no drug leakage. The study proposed pseudo-first and the pseudo-second order equations to facilitate the adsorption of liposomal drugs with TiO2 microspheres. The proposed strategy supports the pharmacokinetic study of docetaxel liposomes in rats. Conclusion: TiO2 extraction method was stable, reproducible, and reliable for quantitation of encapsulated docetaxel. Because of versatility of lipids, it is expected to a universal bioanalysis method for the pharmacokinetic study of liposomes.

A rapid and sensitive single-cell proteomic method based on fast liquid-chromatography separation, retention time prediction and MS1-only acquisition.

Single-cell analysis has received much attention in recent years for elucidating the widely existing cellular heterogeneity in biological systems. However, the ability to measure the proteome in single cells is still far behind that of transcriptomics due to the lack of sensitive and high-throughput mass spectrometry methods. Herein, we report an integrated strategy termed "SCP-MS1" that combines fast liquid chromatography (LC) separation, deep learning-based retention time (RT) prediction and MS1-only acquisition for rapid and sensitive single-cell proteome analysis. In SCP-MS1, the peptides were identified via four-dimensional MS1 feature (m/z, RT, charge and FAIMS CV) matching, therefore relieving MS acquisition from the time consuming and information losing MS2 step and making this method particularly compatible with fast LC separation. By completely omitting the MS2 step, all the MS analysis time was utilized for MS1 acquisition in SCP-MS1 and therefore led to 65%-138% increased MS1 feature collection. Unlike "match between run" methods that still needed MS2 information for RT alignment, SCP-MS1 used deep learning-based RT prediction to transfer the measured RTs in long gradient bulk analyses to short gradient single cell analyses, which was the key step to enhance both identification scale and matching accuracy. Using this strategy, more than 2000 proteins were obtained from 0.2 ng of peptides with a 14-min active gradient at a false discovery rate (FDR) of 0.8%. Comparing with the DDA method, improved quantitative performance was also observed for SCP-MS1 with approximately 50% decreased median coefficient of variation of quantified proteins. For single-cell analysis, 1715 ± 204 and 1604 ± 224 proteins were quantified in single 293T and HeLa cells, respectively. Finally, SCP-MS1 was applied to single-cell proteome analysis of sorafenib resistant and non-resistant HepG2 cells and revealed clear cellular heterogeneity in the resistant population that may be masked in bulk studies.

Tandem enrichment of serum exosomes and exosomal RNA with titanium dioxide.

Exosomes have great potential as biomarker carriers for disease diagnosis and prognosis. In recent years, exosomal RNA (exoRNA) has become a promising candidate for the early diagnosis and prognosis of cancers, and its pathophysiological roles in various diseases have been revealed. For example, exosome-derived mRNAs, miRNAs, circRNAs, and lncRNAs function as signalling molecules to regulate tumour growth, angiogenesis, invasion, metastasis, and the response to chemotherapy. However, the isolation of exosomes and exoRNA with high quality and purity remains challenging due to the relatively small size of exosomes and the limited amount of RNA in exosomes. In this work, we developed a novel tandem enrichment method to isolate exoRNA from serum based on the specific interaction between titanium dioxide (TiO2) and the phosphate groups on the lipid bilayer of exosomes and of the exoRNA. TiO2-based RNA isolation was first demonstrated and optimized in HeLa cells. A total of 130.9 ± 8.34 µg of RNA was rapidly enriched from approximately 5 × 106 HeLa cells within 10 min. This was a 41.5% higher yield than that using a commercial Ultrapure RNA Kit. TiO2-based tandem enrichment of exoRNA was then performed using human serum, obtaining 64.53±3.41 ng of exoRNA from 500 µL of human serum within 30 min. A total of 2,137,902 reads, including seven types of exoRNAs, were identified from the exosomes. This method is compatible with various downstream RNA processing techniques and does not use toxic or irritating reagents, such as phenol or chloroform, providing a simple, economical, rapid, and safe approach for exoRNA extraction from biological samples.

An angled-shape tip-based strategy for highly sensitive proteomic profiling of a low number of cells.

Profiling proteins plays an essential role in understanding the functions and dynamic networks in biological systems. Mass spectrometry-based proteomic analysis commonly requires multistep sample processing, which results in severe sample loss. Although the recently developed microproteomic strategies have substantially reduced sample loss via droplet microfluidic technology, specialized equipment and well-trained personnel are needed, which may limit their wide adoption. Here, we report an angled-shape tip-based strategy for rapid sample preparation and sensitive proteomic profiling of small cell populations (<1000 cells). The angled-shape tip provided a 'reactor' for the entire proteomic sample processing workflow, from cell capture and lysis to protein digestion, eliminating the sample transfer-induced protein loss. The angled-shape tip was surface-treated for anti-protein adsorption which further reduced the sample loss. Using this strategy, 1241 ± 38-4110 ± 37 protein groups and 4010 ± 700-34 879 ± 575 peptides were identified from 10-1000 HeLa cells with high quantification reproducibility in only 4.5 h sample processing time, which was superior to the reported methods and commercial kits, especially for <100 cells. This approach was easily accessible, straightforward to operate, and compatible with flow cytometry-based cell sorting. It showed great potential for in-depth proteomic profiling of rare cells (<1000 cells) in both basic biological research and clinical application.

A new integrated method for tissue extracellular vesicle enrichment and proteome profiling.

Extracellular vesicles (EVs) are membranous vesicles released by cells that carry a number of biologically important components such as lipids, proteins, and mRNAs. EVs can mediate cancer cell migration, invasion, angiogenesis, and cell survival, greatly contributing to cell-to-cell communication in the tumor microenvironment. Additionally, EVs have been found to have diagnostic and prognostic significance in various cancers. However, the direct isolation of pure EVs remains challenging, especially from tissue samples. Currently available EV isolation approaches, e.g., ultracentrifugation, are time-consuming, instrumental dependent, and have a low recovery rate with limited purity. It is urgent to develop rapid and efficient methods for enriching tissue EVs for biological and clinical studies. Here, we developed a novel isolation approach for tissue EVs using an extraction kit combined with TiO2 microspheres (kit-TiO2). The EVs were first precipitated from the tissue fluid using a precipitation agent and then further enriched using microspheres based on the specific interaction between TiO2 and the phosphate groups on the lipid bilayer of the EVs. Kit-TiO2 approach led to improved purity and enrichment efficiency of the isolated EVs, as demonstrated by western blot and proteomic analysis, compared with previously reported methods. A total of 1966 protein groups were identified from the tissue EVs. We compared the proteomic profiles of the liver tissue EVs from healthy and hepatocellular carcinoma (HCC) bearing-mice. Twenty-five significantly upregulated and 75 downregulated protein groups were found in the HCC EVs. Among the differentially expressed proteins, Atic, Copa, Cont3, Me1, Anxa3, Fth1, Anxa5, Phb1, Acaa2, ATPD, and Glud1 were reported to be highly relevant to HCC. This novel isolation strategy has provided a powerful tool for enriching EVs directly from tissues, and may be applied in biomarker discovery and drug screening of HCC.

Synthesis of Surface-Functionalized Molybdenum Disulfide Nanomaterials for Efficient Adsorption and Deep Profiling of the Human Plasma Proteome by Data-Independent Acquisition.

Blood is one of the most important clinical samples for protein biomarker discovery, as it provides rich physiological and pathological information and is easy to obtain with low invasiveness. However, the discovery of protein biomarkers in the blood by mass spectrometry (MS)-based proteomic strategies has been shown to be highly challenging due to the particularly large concentration range of proteins and the strong interference by the high-abundant proteins in the blood. Therefore, developing sensitive methods for low-abundant biomarker protein identification is a key issue that has received great attention. Here, we report the synthesis and characterization of surface-functionalized magnetic molybdenum disulfide (MoS2) for the large-scale adsorption of low-abundant plasma proteins and deep profiling by MS. MoS2 nanomaterials resulted in the coverage of more than 3400 proteins (including a single-peptide hit) in a single LC-MS analysis without peptide prefractionation using pooled plasma samples, which were five times more than those obtained by the direct analysis of the plasma proteome. A detection limit in the low ng L-1 range was obtained, which is rare compared with previous reports.

O-GlcNAcylation promotes pancreatic tumor growth by regulating malate dehydrogenase 1.

Oncogenic Kras-activated pancreatic ductal adenocarcinoma (PDAC) cells highly rely on an unconventional glutamine catabolic pathway to sustain cell growth. However, little is known about how this pathway is regulated. Here we demonstrate that Kras mutation induces cellular O-linked ß-N-acetylglucosamine (O-GlcNAc), a prevalent form of protein glycosylation. Malate dehydrogenase 1 (MDH1), a key enzyme in the glutamine catabolic pathway, is positively regulated by O-GlcNAcylation on serine 189 (S189). Molecular dynamics simulations suggest that S189 glycosylation on monomeric MDH1 enhances the stability of the substrate-binding pocket and strengthens the substrate interactions by serving as a molecular glue. Depletion of O-GlcNAcylation reduces MDH1 activity, impairs glutamine metabolism, sensitizes PDAC cells to oxidative stress, decreases cell proliferation and inhibits tumor growth in nude mice. Furthermore, O-GlcNAcylation levels of MDH1 are elevated in clinical PDAC samples. Our study reveals that O-GlcNAcylation contributes to pancreatic cancer growth by regulating the metabolic activity of MDH1.

Chemically labeled ThUBD permits rapid and super-sensitive imaging of polyubiquitination signals.

Polyubiquitination signal deliver diverse cellular signal, which have been recognized as a sophisticated ubiquitin code. The perception and transduction of ubiquitination signal depend on the specificity and sensitivity of the ubiquitin-binding domain. Accurate and sensitive detection of polyubiquitination signal is crucial for revealing the dynamic cellular ubiquitin-regulated events. Western blotting (WB) and immunohistochemistry (IHC) are the most widely used biochemical strategies to detect ubiquitination signal on substrates under diverse physiological and pathological conditions. However, anti-ubiquitin antibodies fail to reflect polyubiquitination signal unbiasedly because of their strong preference for K63-linked ubiquitin chains. Herein, we demonstrated that our previously developed tandem hybrid ubiquitin-binding domain (ThUBD) chemically labeled with a reporter group such as horseradish peroxidase (ThUBD-HRP) could significantly improve the robustness and sensitivity of polyubiquitination signal detection. This advanced method was named TUF-WB Plus (TUF-WB+). The TUF-WB+ method significantly increases the sensitivity and accuracy of ubiquitin detection and requires a shorter experimental operation time. Furthermore, it enables the ThUBD-HRP probe to function as a powerful tool for spatial in situ polyubiquitination detection in cells by immunohistochemistry. Our newly developed ThUBD-HRP probe and TUF-WB+ method provide a robust and powerful tool for ubiquitination signal detection with hypersensitivity in an unbiased manner.

A New Strategy for High-Efficient Tandem Enrichment and Simultaneous Profiling of N-Glycopeptides and Phosphopeptides in Lung Cancer Tissue.

N-glycosylation and phosphorylation, two common posttranslational modifications, play important roles in various biological processes and are extensively studied for biomarker and drug target screening. Because of their low abundance, enrichment of N-glycopeptides and phosphopeptides prior to LC-MS/MS analysis is essential. However, simultaneous characterization of these two types of posttranslational modifications in complex biological samples is still challenging, especially for tiny amount of samples obtained in tissue biopsy. Here, we introduced a new strategy for the highly efficient tandem enrichment of N-glycopeptides and phosphopeptides using HILIC and TiO2 microparticles. The N-glycopeptides and phosphosites obtained by tandem enrichment were 21%-377% and 22%-263% higher than those obtained by enriching the two PTM peptides separately, respectively, using 160-20 µg tryptic digested peptides as the starting material. Under the optimized conditions, 2798 N-glycopeptides from 434 N-glycoproteins and 5130 phosphosites from 1986 phosphoproteins were confidently identified from three technical replicates of HeLa cells by mass spectrometry analysis. Application of this tandem enrichment strategy in a lung cancer study led to simultaneous characterization of the two PTM peptides and discovery of hundreds of differentially expressed N-glycosylated and phosphorylated proteins between cancer and normal tissues, demonstrating the high sensitivity of this strategy for investigation of dysregulated PTMs using very limited clinical samples.

O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.

SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.