CXCR4 expression is associated with proneural-to-mesenchymal transition in glioblastoma.

Glioblastoma (GBM) is the most common primary intracranial malignant tumor and consists of three molecular subtypes: proneural (PN), mesenchymal (MES) and classical (CL). Transition between PN to MES subtypes (PMT) is the glioma analog of the epithelial-mesenchymal transition (EMT) in carcinomas and is associated with resistance to therapy. CXCR4 signaling increases the expression of MES genes in glioma cell lines and promotes EMT in other cancers. RNA sequencing (RNAseq) data of PN GBMs in The Cancer Genome Atlas (TCGA) and secondary high-grade gliomas (HGGs) from an internal cohort were examined for correlation between CXCR4 expression and survival as well as expression of MES markers. Publicly available single-cell RNA sequencing (scRNAseq) data was analyzed for cell type specific CXCR4 expression. These results were validated in a genetic mouse model of PN GBM. Higher CXCR4 expression was associated with significantly reduced survival and increased expression of MES markers in TCGA and internal cohorts. CXCR4 was expressed in immune and tumor cells based on scRNAseq analysis. Higher CXCR4 expression within tumor cells on scRNAseq was associated with increased MES phenotype, suggesting a cell-autonomous effect. In a genetically engineered mouse model, tumors induced with CXCR4 exhibited a mesenchymal phenotype and shortened survival. These results suggest that CXCR4 signaling promotes PMT and shortens survival in GBM and highlights its inhibition as a potential therapeutic strategy.

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor response to standard therapies and therefore unfavorable clinical outcomes. Better understanding of TNBC and new therapeutic strategies are urgently needed. ROR nuclear receptors are multifunctional transcription factors with important roles in circadian pathways and other processes including immunity and tumorigenesis. Nobiletin (NOB) is a natural compound known to display anticancer effects, and our previous studies showed that NOB activates RORs to enhance circadian rhythms and promote physiological fitness in mice. Here, we identified several TNBC cell lines being sensitive to NOB, by itself or in combination. Cell and xenograft experiments showed that NOB significantly inhibited TNBC cell proliferation and motility in vitro and in vivo. ROR loss- and gain-of-function studies showed concordant effects of the NOB-ROR axis on MDA-MB-231 cell growth. Mechanistically, we found that NOB activates ROR binding to the ROR response elements (RRE) of the IκBα promoter, and NOB strongly inhibited p65 nuclear translocation. Consistent with transcriptomic analysis indicating cancer and NF-κB signaling as major pathways altered by NOB, p65-inducible expression abolished NOB effects, illustrating a requisite role of NF-κB suppression mediating the anti-TNBC effect of NOB. Finally, in vivo mouse xenograft studies showed that NOB enhanced the antitumor efficacy in mammary fat pad implanted TNBC, as a single agent or in combination with the chemotherapy agent Docetaxel. Together, our study highlights an anti-TNBC mechanism of ROR-NOB via suppression of NF-κB signaling, suggesting novel preventive and chemotherapeutic strategies against this devastating disease.

Depletion of ST6GALNACIII retards A549 non-small cell lung cancer cell proliferation by downregulating transferrin receptor protein 1 expression.

Alterations in sialylation of terminal residues of glycoproteins have been implicated in forming tumor-associated glycans. ST6GALNAC transfers sialyl moiety to N-acetylgalactosamine residue via α2,6 linkage. Although the oncogenic characteristics of ST6GALNACI or II have been demonstrated in various cancer cells, the impact of ST6GALNACIII on tumor progression remains undefined. In this study, we evaluated the effect of ST6GALNACIII knockdown on the growth of A549 non-small cell lung cancer cells. ST6GALNACIII depletion resulted in significant retardation in growth of A549 cells under various culture conditions, including collagen-supported 3D culture and anchorage-independent soft agar culture conditions. Liquid chromatography with tandem mass spectrometry revealed that two glycopeptides of transferrin receptor protein 1 (TFR1) containing N-acetylhexosamine-sialic acid were not detected in ST6GALNACIII-depleted A549 cells compared with control cells. Subsequent lectin binding assay, western blotting, and real-time RT-PCR indicated that TFR1 sialylation was not significantly changed, but TFR1 protein and mRNA expressions were decreased after ST6GALNACIII knockdown. However, cell growth retardation by ST6GALNACIII knockdown was partially rescued by TFR1 overexpression. Additionally, TFR1 mRNA degradation was accelerated following ST6GALNACIII knockdown with concomitant reduction in mRNA levels of iron regulatory protein 1 and 2, the upstream regulators of TFR1 mRNA stability. Therefore, our results indicated an important role of ST6GALNACIII in promoting A549 cell growth through quantitative regulation of TFR1 expression and provided therapeutic implications for ST6GALNACIII targeting in tumor growth suppression in vivo.

The Significance of Planned Fertility Preservation for Women With Endometrioma Before an Expected Ovarian Cystectomy.

Endometrioma is known to reduce the ovarian reserve and the extent of the decrease is more severe when ovarian surgery is performed. Therefore, to prevent this decline in fertility, patients with endometrioma are considered candidates for preoperative fertility preservation (FP). In this study, we evaluate the efficacy of FP in women with endometrioma before planned ovarian surgery. A total of 95 cycles in 62 patients with endometrioma, undergoing controlled ovarian stimulation (COS) for FP using a gonadotropin-releasing hormone (GnRH) antagonist protocol before an expected ovarian surgery, were enrolled retrospectively. COS outcomes were compared according to endometrioma laterality. Additionally, first COS cycle outcomes in patients with endometrioma were compared with those in infertile patients, or in patients with a benign ovarian cyst using propensity score matching. When multiple COS cycles were performed, the results of cumulative cycles were analyzed. Embryo quality was worse in the bilateral endometrioma group. Compared with the infertile patient group, the patients with endometrioma had significantly lower Anti-Müllerian Hormone (AMH) and fewer numbers of oocytes retrieved (median, 3.3 vs. 1.2, p<0.001; 7.0 vs. 4.0, p=0.009, respectively). Compared with mature oocytes in infertile patients or patients with a benign cyst, mature oocytes were fewer in patients with endometrioma, but this was not statistically significant (median, 4.0 vs. 3.0, p=0.085; 5.5 vs. 3.0, p=0.052, respectively). The median value of the cumulative number of cryopreserved oocytes or embryos was 14.5 up to the fourth cycle compared to 3 up to the first cycle, with cumulative effect. Women with endometrioma should be counseled for FP before planned ovarian cystectomy. The number of cryopreserved oocytes or embryos can be increased by repeated cycles.

Self-assembling multidomain peptide hydrogels accelerate peripheral nerve regeneration after crush injury.

Multidomain peptide (MDP) hydrogels are a class of self-assembling materials that have been shown to elicit beneficial responses for soft tissue regeneration. However, their capacity to promote nervous system regeneration remains unknown. The peripheral nervous system (PNS) substantially recovers after injury, partly due to the abundance of extracellular matrix (ECM) components in its basal lamina. However, severe peripheral nerve injuries that significantly damage the ECM continue to be a major clinical challenge as they occur at a high rate and can be extremely detrimental to patients' quality of life. In this study, a panel of eight MDPs were designed to contain various motifs mimicking extracellular matrix components and growth factors and successfully self-assembled into injectable, nanofibrous hydrogels. Using an in vitro screening system, various lysine based MDPs were found to enhance neurite outgrowth. To test their capacity to promote nerve regeneration in vivo, rat sciatic nerve crush injury was performed with MDP hydrogels injected directly into the injury sites. MDP hydrogels were found to enhance macrophage recruitment to the injury site and degrade efficiently over time. Rats that were injected with the MDP hydrogel K2 and laminin motif-containing MDPs K2-IIKDI and K2-IKVAV were found to have significantly accelerated functional recovery and remyelination compared to those injected with HBSS or other MDPs. These results demonstrate that MDPs enhance neurite outgrowth and promote a multicellular pro-regenerative response in peripheral nerve injury. This study provides important insights into the potential of MDPs as biomaterials for nerve regeneration and other clinical applications.

The Daam2-VHL-Nedd4 axis governs developmental and regenerative oligodendrocyte differentiation.

Dysregulation of the ubiquitin-proteasomal system (UPS) enables pathogenic accumulation of disease-driving proteins in neurons across a host of neurological disorders. However, whether and how the UPS contributes to oligodendrocyte dysfunction and repair after white matter injury (WMI) remains undefined. Here we show that the E3 ligase VHL interacts with Daam2 and their mutual antagonism regulates oligodendrocyte differentiation during development. Using proteomic analysis of the Daam2-VHL complex coupled with conditional genetic knockout mouse models, we further discovered that the E3 ubiquitin ligase Nedd4 is required for developmental myelination through stabilization of VHL via K63-linked ubiquitination. Furthermore, studies in mouse demyelination models and white matter lesions from patients with multiple sclerosis corroborate the function of this pathway during remyelination after WMI. Overall, these studies provide evidence that a signaling axis involving key UPS components contributes to oligodendrocyte development and repair and reveal a new role for Nedd4 in glial biology.

Circadian regulation of chemotherapy-induced peripheral neuropathic pain and the underlying transcriptomic landscape.

Growing evidence demonstrates circadian rhythms of pain hypersensitivity in various chronic disorders. In chemotherapy-induced peripheral neuropathy (CIPN), agents such as paclitaxel are known to elicit chronic neuropathic pain in cancer patients and seriously compromise their quality of life. Here, we report that the mechanical threshold for allodynia in paclitaxel-treated rats exhibited a robust circadian oscillation, reaching the nadir during the daytime (inactive phase). Using Per2::LucSV circadian reporter mice expressing a PER2::LUC fusion protein, we isolated dorsal root ganglia (DRG), the primary sensory cell body for peripheral nerve injury generated hypersensitivity, and monitored ex vivo reporter bioluminescence. We observed strong circadian reporter rhythms in DRG neurons which are highly entrainable by external cues. Paclitaxel treatment significantly lengthened DRG circadian periods, with little effects on the amplitude of oscillation. We further observed the core protein BMAL1 and PER2 in DRG neurons and satellite cells. Using DRG and dorsal horn (DH; another key structure for CIPN pain response) tissues from vehicle and paclitaxel treated rats, we performed RNA-sequencing and identified diurnal expression of core clock genes as well as clock-controlled genes in both sites. Interestingly, 20.1% and 30.4% of diurnal differentially expressed genes (DEGs) overlapped with paclitaxel-induced DEGs in the DRG and the DH respectively. In contrast, paclitaxel-induced DEGs displayed only a modest overlap between daytime and nighttime (Zeitgeber Time 8 and 20). Furthermore, paclitaxel treatment induced de novo diurnal DEGs, suggesting reciprocal interaction of circadian rhythms and chemotherapy. Our study therefore demonstrates a circadian oscillation of CIPN and its underlying transcriptomic landscape.

Computational classification of core and outer fucosylation of N-glycoproteins in human plasma using collision-induced dissociation in mass spectrometry.

RATIONALE: Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. METHODS: The relative intensities of the Y1 F, Y2 F, Y3 F, and Y4 F product ions in the CID-MS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation. The Core Fucose Index (CFI) was then calculated by multiplication of the relative intensities with a weight factor from logistic regression to differentiate between the core and none fucosylation. From the relative intensities of the B2 F and B3 SF ions of the MS/MS spectra of the AGP N-glycopeptides for outer fucosylation, the Outer Fucose Index (OFI) was calculated to differentiate between the outer and none fucosylation. RESULTS: In order to classify core and/or outer fucosylation of N-glycoproteins, we defined the fucosylation score (F-score) by a sigmoidal equation using a combination of the CFI and the OFI. For application, we classified the fucosylation types of N-glycoproteins in human plasma with 99.7% accuracy from the F-score. Human plasma samples showed 54.4%, 33.3%, 10.3%, and 1.6% for none, core, outer, and dual fucosylated N-glycopeptides, respectively. Core fucosylation was abundant at mono- and bi-antennary N-glycopeptides. Outer fucosylation was abundant at tri- and tetra-antennary N-glycopeptides. In total, 113 N-glycopeptides of 29 glycoproteins from 3365 glycopeptide spectral matches (GPSMs) were classified for different types of fucosylation. CONCLUSIONS: We established an F-score to classify three different fucosylation types: core, outer, and dual types of N-glycopeptides. The fucosylation types of 20 new N-glycopeptides from 11 glycoproteins in human plasma were classified using the F-score. Therefore, the F-score can be useful for the automatic classification of different types of fucosylation in N-glycoproteins of biological fluids including plasma, serum, and urine.

Classification of Mucin-Type O-Glycopeptides Using Higher-Energy Collisional Dissociation in Mass Spectrometry.

Changes in mucin-type O-glycosylation of human proteins affect protein function, immune response, and cancer progression. Since O-glycoproteins are characterized by the microheterogeneity of diverse O-glycans with no conserved sequence and the macroheterogeneity of multiple glycosylation sites on serine and/or threonine in human proteins, the assessment of different mucin types, such as Tn-antigen, core 1, and core 2, and their extended core types in O-glycopeptides, is extremely challenging. Here, we present an O-GlycoProteome Analyzer (O-GPA) that automatically classifies mucin-type O-glycosylation using higher-energy collisional dissociation (HCD) in mass spectrometry. First, we estimated the number of GlcNAc residues using the intensity ratio of GlcNAc-specific fragment ions (HexNAc-CH6O3 and HexNAc-2H2O) over GalNAc-specific fragment ions (HexNAc-C2H6O3 and HexNAc-C2H4O2) in the HCD spectrum. Furthermore, we classified the different mucin types of O-glycopeptides from characteristic B2 (HexNAc2) or Y2α (PEP + HexNAc2), and Y2ß (PEP + HexNAcHex) fragment ions, along with the number of GlcNAc. Furthermore, O-GPA automatically determined single or multiple O-glycosylation, regardless of the mucin types. The mucin type of O-glycopeptides from human urine and plasma was confirmed with an overall accuracy of 96%. We found 97 core 1, 56 core 2, 13 extended core 1, and 12 extended core 2 glycopeptides from urine; and 22 core 1, 13 core 2, 7 extended core 1, 1 extended core 2, and 1 Tn-antigen from plasma. Our strategy can be used to successfully characterize specific mucin types of O-glycoproteins in human biological samples.

Genetic characterization of canine parvovirus type 2c from domestic dogs in Korea.

Canine parvovirus type 2 (CPV-2) is an aetiological agent that causes acute haemorrhagic enteritis and fatal myocarditis in dogs. Since CPV-2 first emerged in the late 1970s, its rapid evolution has resulted in three antigenic variants: CPV-2a, CPV-2b and CPV-2c. Here, we report, for the first time in Korea, two cases of CPV-2c infection in two dogs with severe diarrhoea. The complete open reading frame (4,269nt) of CPV-2, encoding both non-structural (NS) and structural (VP) proteins, was sequenced. Based on the amino acid Gln present at residue 426 of the VP2 gene, these strains were typed as CPV-2c, and were named Korea CPV-2c_1 and Korea CPV-2c_2. These strains shared 99.48% reciprocal nucleotide sequence identity and had the highest nucleotide identity (99.77%-99.34%) with Asian CPV strains isolated in China, Italy (found in a dog imported from Thailand), and Vietnam from 2013 to 2017. Phylogenetic analysis based on the non-structural (NS1) and capsid (VP2) genes revealed that Korean CPV-2c strains clustered closely to Asian CPV strains, and separately from strains isolated in Europe, South America and North America. Amino acid changes never reported before were observed in NS1 (Thr70Pro, Cys287Tyr), VP1 (Lys17Arg, Phe33Leu) and VP2 (Gln365His, Ala516Val). Additional observed mutations, including Phe267Tyr, Tyr324Ile and Gln370Arg, have been previously reported in the recent CPV-2c strains with Asian origins. These results suggest that the Korean CPV-2c strains were potentially introduced via neighbouring Asian countries.

Comparison of microRNA expressions for the identification of chemical hazards in in vivo and in vitro hepatic injury models.

Biofluid-based biomarkers provide an efficient tool for hazard identification of chemicals. Here, we explored the potential of microRNAs (miRNAs) as biomarkers for hepatotoxicity of chemicals by linking in vitro to in vivo animal models. A search of the literature identified candidate circulating miRNA biomarkers of chemical-induced hepatotoxicity. The expression of candidate miRNAs (miR-122, miR-151a, miR-192, miR-193a, miR-194, miR-21, miR-29c), was determined by real-time reverse transcription-polymerase chain reaction in in vivo acute liver injury induced by acetaminophen, and then were further compared with those of in vitro cell assays. Candidate miRNAs, except miR-29c, were significantly or biologically upregulated by acetaminophen, at a dose that caused acute liver injury as confirmed by hepatocellular necrosis. Except miR-122 and miR-193a, other miRNAs elevated in in vivo models were confirmed by in vitro models using HepG2 cells, whereas they failed by in vitro models using human primary hepatocytes. These findings indicate that certain miRNAs may still have the potential of toxicological biomarkers in linking in vitro to in vivo hepatotoxicity.

Identification of Missing Proteins in Human Olfactory Epithelial Tissue by Liquid Chromatography-Tandem Mass Spectrometry.

We performed proteomic analyses of human olfactory epithelial tissue to identify missing proteins using liquid chromatography-tandem mass spectrometry. Using a next-generation proteomic pipeline with a < 1.0% false discovery rate at the peptide and protein levels, we identified 3731 proteins, among which five were missing proteins (P0C7M7, P46721, P59826, Q658L1, and Q8N434). We validated the identified missing proteins using the corresponding synthetic peptides. No olfactory receptor (OR) proteins were detected in olfactory tissue, suggesting that detection of ORs would be very difficult. We also identified 49 and 50 alternative splicing variants mapped at the neXtProt and GENCODE databases, respectively, and 2000 additional single amino acid variants. This data set is available at the ProteomeXchange consortium via PRIDE repository (PXD010025).

Steroid receptor coactivator-2 (SRC-2) coordinates cardiomyocyte paracrine signaling to promote pressure overload-induced angiogenesis.

Pressure overload-induced cardiac stress induces left ventricular hypertrophy driven by increased cardiomyocyte mass. The increased energetic demand and cardiomyocyte size during hypertrophy necessitate increased fuel and oxygen delivery and stimulate angiogenesis in the left ventricular wall. We have previously shown that the transcriptional regulator steroid receptor coactivator-2 (SRC-2) controls activation of several key cardiac transcription factors and that SRC-2 loss results in extensive cardiac transcriptional remodeling. Pressure overload in mice lacking SRC-2 induces an abrogated hypertrophic response and decreases sustained cardiac function, but the cardiomyocyte-specific effects of SRC-2 in these changes are unknown. Here, we report that cardiomyocyte-specific loss of SRC-2 (SRC-2 CKO) results in a blunted hypertrophy accompanied by a rapid, progressive decrease in cardiac function. We found that SRC-2 CKO mice exhibit markedly decreased left ventricular vasculature in response to transverse aortic constriction, corresponding to decreased expression of the angiogenic factor VEGF. Of note, SRC-2 knockdown in cardiomyocytes decreased VEGF expression and secretion to levels sufficient to blunt in vitro tube formation and proliferation of endothelial cells. During pressure overload, both hypertrophic and hypoxic signals can stimulate angiogenesis, both of which stimulated SRC-2 expression in vitro Furthermore, SRC-2 coactivated the transcription factors GATA-binding protein 4 (GATA-4) and hypoxia-inducible factor (HIF)-1α and -2α in response to angiotensin II and hypoxia, respectively, which drive VEGF expression. These results suggest that SRC-2 coordinates cardiomyocyte secretion of VEGF downstream of the two major angiogenic stimuli occurring during pressure overload bridging both hypertrophic and hypoxia-stimulated paracrine signaling.

Daam2 driven degradation of VHL promotes gliomagenesis.

Von Hippel-Landau (VHL) protein is a potent tumor suppressor regulating numerous pathways that drive cancer, but mutations in VHL are restricted to limited subsets of malignancies. Here we identified a novel mechanism for VHL suppression in tumors that do not have inactivating mutations. Using developmental processes to uncover new pathways contributing to tumorigenesis, we found that Daam2 promotes glioma formation. Protein expression screening identified an inverse correlation between Daam2 and VHL expression across a host of cancers, including glioma. These in silico insights guided corroborating functional studies, which revealed that Daam2 promotes tumorigenesis by suppressing VHL expression. Furthermore, biochemical analyses demonstrate that Daam2 associates with VHL and facilitates its ubiquitination and degradation. Together, these studies are the first to define an upstream mechanism regulating VHL suppression in cancer and describe the role of Daam2 in tumorigenesis.

Glia-specific enhancers and chromatin structure regulate NFIA expression and glioma tumorigenesis.

Long-range enhancer interactions critically regulate gene expression, yet little is known about how their coordinated activities contribute to CNS development or how this may, in turn, relate to disease states. By examining the regulation of the transcription factor NFIA in the developing spinal cord, we identified long-range enhancers that recapitulate NFIA expression across glial and neuronal lineages in vivo. Complementary genetic studies found that Sox9-Brn2 and Isl1-Lhx3 regulate enhancer activity and NFIA expression in glial and neuronal populations. Chromatin conformation analysis revealed that these enhancers and transcription factors form distinct architectures within these lineages in the spinal cord. In glioma models, the glia-specific architecture is present in tumors, and these enhancers are required for NFIA expression and contribute to glioma formation. By delineating three-dimensional mechanisms of gene expression regulation, our studies identify lineage-specific chromatin architectures and associated enhancers that regulate cell fate and tumorigenesis in the CNS.

Direct analysis of site-specific N-glycopeptides of serological proteins in dried blood spot samples.

Dried blood spot (DBS) samples have a number of advantages, especially with respect to ease of collection, transportation, and storage and to reduce biohazard risk. N-glycosylation is a major post-translational modification of proteins in human blood that is related to a variety of biological functions, including metastasis, cell-cell interactions, inflammation, and immunization. Here, we directly analyzed tryptic N-glycopeptides from glycoproteins in DBS samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without centrifugation of blood samples, depletion of major proteins, desalting of tryptic peptides, and enrichment of N-glycopeptides. Using this simple method, we identified a total of 41 site-specific N-glycopeptides from 16 glycoproteins in the DBS samples, from immunoglobulin gamma 1 (IgG-1, 10 mg/mL) down to complement component C7 (50 µg/mL). Of these, 32 N-glycopeptides from 14 glycoproteins were consistently quantified over 180 days stored at room temperature. The major abundant glycoproteins in the DBS samples were IgG-1 and IgG-2, which contain nine asialo-fucosylated complex types of 16 different N-glycopeptide isoforms. Sialo-non-fucosylated complex types were primarily detected in the other glycoproteins such as alpha-1-acid glycoprotein 1, 2, alpha-1-antitypsin, alpha-2-macroglobulin, haptoglobin, hemopexin, Ig alpha 1, 2 chain C region, kininogen-1, prothrombin, and serotransferrin. We first report the characterization of site-specific N-glycoproteins in DBS samples by LC-MS/MS with minimal sample preparation.

Identification of diverse astrocyte populations and their malignant analogs.

Astrocytes are the most abundant cell type in the brain, where they perform a wide array of functions, yet the nature of their cellular heterogeneity and how it oversees these diverse roles remains shrouded in mystery. Using an intersectional fluorescence-activated cell sorting-based strategy, we identified five distinct astrocyte subpopulations present across three brain regions that show extensive molecular diversity. Application of this molecular insight toward function revealed that these populations differentially support synaptogenesis between neurons. We identified correlative populations in mouse and human glioma and found that the emergence of specific subpopulations during tumor progression corresponded with the onset of seizures and tumor invasion. In sum, we have identified subpopulations of astrocytes in the adult brain and their correlates in glioma that are endowed with diverse cellular, molecular and functional properties. These populations selectively contribute to synaptogenesis and tumor pathophysiology, providing a blueprint for understanding diverse astrocyte contributions to neurological disease.

Analysis of fucosylation in liver-secreted N-glycoproteins from human hepatocellular carcinoma plasma using liquid chromatography with tandem mass spectrometry.

Fucosylation of N-glycoproteins has been implicated in various diseases, such as hepatocellular carcinoma (HCC). However, few studies have performed site-specific analysis of fucosylation in liver-secreted proteins. In this study, we characterized the fucosylation patterns of liver-secreted proteins in HCC plasma using a workflow to identify site-specific N-glycoproteins, where characteristic B- and/or Y-ion series with and without fucose in collision-induced dissociation were used in tandem mass spectrometry. In total, 71 fucosylated N-glycopeptides from 13 major liver-secreted proteins in human plasma were globally identified by LC-MS/MS. Additionally, 37 fucosylated N-glycopeptides were newly identified from nine liver-secreted proteins, including alpha-1-antichymotrypsin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, ceruloplasmin, alpha-1-acid glycoprotein 1/2, alpha-2-macroglobulin, serotransferrin, and beta-2-glycoprotein 1. Of the fucosylated N-glycopeptides, bi- and tri-antennary glycoforms were the most common ones identified in liver-secreted proteins from HCC plasma. Therefore, we suggest that this analytical method is effective for characterizing fucosylation in liver-secreted proteins. Graphical abstract A global map of fucosylated and non-fucosylated glycopeptides from 13 liver-secreted glycoproteins in hepatocellular carcinoma plasma.

Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.

Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project.

In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation.

The characterization of site-specific microheterogeneity in glycoprotein is very important for understanding cell biology and disease processes. Vitronectin is well known to be a multifunctional glycoprotein in the blood and the extracellular matrix, which is related to hepatocellular carcinoma (HCC). Here, we systematically analyzed the site-specific N-glycopeptides of vitronectin in human plasma by tandem mass spectrometry combined with immunoprecipitation and hydrophilic interaction liquid chromatography (HILIC) enrichment. Vitronectin was purified with immunoprecipitation by monoclonal antibody from plasma and digested to tryptic N-glycopeptides.Then, enrichment with HILIC materials was used and followed by analysis with nano-LC/MS/MS. The sequences of N-glycopeptides were identified from the mass spectra by high-energy C-trap dissociation (HCD) and collision-induced dissociation (CID). In HCD mode, oxonium ions were used for recognizing glycopeptides and y ions for sequencing the peptide backbone. In CID mode, Y ions were used for characterizing their glycoforms. As a result, a total of 17 site-specific N-glycopeptides were completely identified in all of the three N-glycosylation sites of vitronectin in human plasma, including 12 N-glycopeptides first reported. Finally, we specifically found that three hybrid and four complex glycopeptides of triantennary forms with outer fucosylation increased in HCC human plasma.