9-Fluorenylmethyl Chloroformate Labeling for O-Glycan Analysis.

Structural analysis of O-glycans from mucins and characterization of the interaction of these glycans with other biomolecules are essential for a full understanding of mucins. Various techniques have been developed for the structural and functional analysis of glycans. While 9-fluorenylmethyl chloroformate (Fmoc-Cl) is generally used to protect amino groups in peptide synthesis, it can also be used as a glycan-labeling reagent for structural analysis. Fmoc-labeled glycans are strongly fluorescent and can be analyzed with high sensitivity using liquid chromatography-fluorescence detection (LC-FD) analysis as well as being analyzed with high sensitivity by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Fmoc-labeled glycans can be easily delabeled and converted to glycosylamine-form or free (hemiacetal or aldehyde)-form glycans that can be used to fabricate glycan arrays or synthesize glycosyl dendrimers. This derivatization allows for the isolation from biological samples of glycans that are difficult to synthesize chemically, as well as the fabrication of immobilized-glycan devices. The Fmoc labeling method promises to be a tool for accelerating O-glycan structural analysis and an understanding of molecular interactions. In this chapter, we introduce the Fmoc labeling method for analysis of O-glycans and fabrication of O-glycan arrays.

Liquid Chromatography and Capillary Electrophoresis Analysis of 2AA-Labeled O-Glycans.

To reveal O-glycan structures in mucins, it is necessary to release covalently bound O-glycans from the polypeptide backbone and derivatize to a form suitable for structural analysis. Various derivatization methods can now be applied in the analysis of O-glycans following the development of O-glycan release methods. Among the many derivatization methods available, we prefer to use fluorescent labeling with 2-aminobenzoic acid (anthranilic acid, 2AA). 2AA-labeled O-glycans can be detected with high sensitivity using liquid chromatography fluorescence detection (LC-FD) analysis because of the strong fluorescence. In addition, as 2AA has a carboxyl group that carries a negative charge, 2AA-labeled O-glycans can be analyzed with high sensitivity in negative ion mode mass spectrometry. Furthermore, because the negative charge of 2AA provides a driving force for electrophoresis, 2AA-labeled O-glycans can be analyzed using capillary electrophoresis (CE) and capillary affinity electrophoresis. High detection sensitivity and versatility are key advantages of the 2AA-labeling method. Here we present our preferred LC-FD and CE analytical methods for 2AA-labeled O-glycans.

Application of elemental analysis-coupled isotope ratio mass spectrometry for protein turnover analysis using deuterium labeling: Purification and analysis of hydrogen isotope ratio of non-derivatized protein-bound alanine.

RATIONALE: Heavy water can be used as a tracer for the evaluation of protein turnover. By adding heavy water (D2 O) to the precursor pool, nonessential amino acids, including alanine, can be isotopically labeled in vivo. Protein turnover can then be quantified by measuring the hydrogen isotope ratio of protein-bound alanine. METHODS: In this study, we constructed a novel method to apply deuterium labeling of alanine to the evaluation of protein turnover using elemental analysis-coupled isotope ratio mass spectrometry (EA-IRMS). We established a preparative high-performance liquid chromatography method to isolate alanine from protein hydrolysates. EA-IRMS was then used to determine the hydrogen isotope ratio of alanine isolated from hydrolysates of protein from mouse myoblast C2C12 cells that had been treated with D2 O over the course of 72 h. RESULTS: In cells treated with 4% D2 O, the deuterium enrichment of alanine increased to approximately 0.9% over time, while that of cells treated with 0.017% D2 O increased to approximately 0.006%. The rate of protein synthesis calculated by fitting the increase of deuterium excess to rise-to-plateau kinetics was similar regardless of the concentration of D2 O. When C2C12 cells treated with insulin and rapamycin were analyzed 24 h after the addition of 0.017% D2 O, protein turnover was found to be accelerated by insulin, but this effect was offset by co-treatment with rapamycin. CONCLUSION: The derivative-free measurement of the hydrogen isotope ratio of protein-bound alanine using EA-IRMS can be applied to the evaluation of protein turnover. The proposed method is an accessible option for many laboratories to perform highly sensitive IRMS-based evaluations of protein metabolic turnover.

Noble gases and nitrogen in samples of asteroid Ryugu record its volatile sources and recent surface evolution.

The near-Earth carbonaceous asteroid (162173) Ryugu is expected to contain volatile chemical species that could provide information on the origin of Earth's volatiles. Samples of Ryugu were retrieved by the Hayabusa2 spacecraft. We measured noble gas and nitrogen isotopes in Ryugu samples and found that they are dominated by presolar and primordial components, incorporated during Solar System formation. Noble gas concentrations are higher than those in Ivuna-type carbonaceous (CI) chondrite meteorites. Several host phases of isotopically distinct nitrogen have different abundances among the samples. Our measurements support a close relationship between Ryugu and CI chondrites. Noble gases produced by galactic cosmic rays, indicating a ~5 million year exposure, and from implanted solar wind record the recent irradiation history of Ryugu after it migrated to its current orbit.

Predictive prognostic biomarkers in patients with COVID­19 infection.

The present study aimed to identify useful biomarkers to predict deterioration in patients with coronavirus disease 2019 (COVID­19). A total of 201 COVID­19 patients were classified according to their disease severity into non­severe (n=125) and severe (n=76) groups, and the behavior of laboratory biomarkers was examined according to the prognosis. Neutrophil count, aspartate aminotransferase (AST), alanine aminotransferase, lactate dehydrogenase (LDH), C­reactive protein (CRP), sialylated carbohydrate antigen KL­6 (KL­6), procalcitonin (PCT), presepsin (PSP) and D­dimer levels were significantly higher, and lymphocyte count and platelet count were significantly lower in the non­severe group compared with the severe group. In the non­severe group, ROC analysis demonstrated that only four biomarkers, CRP, PSP, AST and LDH were useful for differentiating the prognosis between improvement and deterioration subgroups. No strong correlation was revealed for any of the markers. Multivariate analysis identified CRP as a significant prognostic factor in non­severe cases (odds ratio, 41.45; 95% confidence interval, 4.91­349.24; P<0.001). However, there were no blood biomarkers that could predict the outcome of patients in the severe group. Overall, several blood markers changed significantly according to disease severity in the course of COVID­19 infection. Among them, CRP, PSP, LDH and AST were the most reliable markers for predicting the patient's prognosis in non­severe COVID­19 cases.

First asteroid gas sample delivered by the Hayabusa2 mission: A treasure box from Ryugu.

The Hayabusa2 spacecraft returned to Earth from the asteroid 162173 Ryugu on 6 December 2020. One day after the recovery, the gas species retained in the sample container were extracted and measured on-site and stored in gas collection bottles. The container gas consists of helium and neon with an extraterrestrial 3He/4He and 20Ne/22Ne ratios, along with some contaminant terrestrial atmospheric gases. A mixture of solar and Earth's atmospheric gas is the best explanation for the container gas composition. Fragmentation of Ryugu grains within the sample container is discussed on the basis of the estimated amount of indigenous He and the size distribution of the recovered Ryugu grains. This is the first successful return of gas species from a near-Earth asteroid.

Recent advances and trends in sample preparation and chemical modification for glycan analysis.

Growing significance of glycosylation in protein functions has accelerated the development of methodologies for detection, identification, and characterization of protein glycosylation. In the past decade, glycobiology research has been advanced by innovative techniques with further progression in the post-genome era. Although significant technical progress has been made in terms of analytical throughput, comprehensiveness, and sensitivity, most methods for glycosylation analysis still require laborious and time-consuming sample preparation tasks. Additionally, sample preparation methods that are focused on specific glycan(s) require an in-depth understanding of various issues in glycobiology. In this review, modern sample preparation and chemical modification methods for the structural and quantitative glycan analyses together with the challenges and advantages of recent sample preparation methods are summarized. The techniques presented herein can facilitate the exploration of biomarkers, understanding of unknown glycan functions, and development of biopharmaceuticals.

A rapid and facile preparation of APTS-labeled N-glycans by combination of ion pair-assisted extraction and HILIC-SPE for routine glycan analysis.

Glycoanalytical technology is required for a wide variety of scientific research, including basic glycobiological pharmaceutical, and biomarker research. Although several innovative analytical techniques have been developed for these purposes, quantitative glycan analysis based on electrophoretic separation, has often been impeded by the lack of cost-effective and facile sample preparation approaches. Here, we developed a rapid and facile sample preparation workflow for cost-effective glycan analysis and demonstrated its use with fully automated microchip electrophoresis (ME). Purification of 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans was based on the combination of ion-pair assisted extraction (IPAE) with hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE). Compared to commonly used sample preparation methods, the IPAE/HILIC-SPE method undergoes minimal nonspecific loss and undesirable degradation of N-glycans during the purification step. Furthermore, our method required only 10 min, and the entire workflow, including glycan release, labeling, and concentration processes was completed within 4 h. Although the present system should be improved to enable analysis of more complex mixtures, ME-based separation of APTS-labeled N-glycans offers a fully automated operation including conditioning, sample loading, separation, and can be analyzed with a sample-to-sample throughput of 120 s in parallel processes. The present workflow is easy to implement, does not require expensive reagents and instruments and may be useful for glycoscientists across disciplines.

Network analysis and functional estimation of the microbiome reveal the effects of cashew nut shell liquid feeding on methanogen behaviour in the rumen.

The effects of cashew nut shell liquid (CNSL) feeding on the methane (CH4 ) emission and the ruminal microbiome of Lai Sind beef cattle were investigated. Changes in the methane production and rumen microbiome by CNSL feeding were monitored by a respiration chamber and 16S rRNA gene amplicon sequencing respectively. The results demonstrated that CNSL feeding mitigated 20.2%-23.4% of the CH4 emission in vivo without apparent adverse effects on feed intake and feed digestibility. The rumen fluid analysis revealed a significant increase in the proportion of propionate in the total short-chain fatty acids. The relative abundance of methanogen (order Methanobacteriales) decreased significantly, indicating the direct inhibitory effect of CNSL on methanogens. The predicted function of the rumen microbiome indicated that carbohydrate and lipid metabolisms including propionate production were upregulated by CNSL feeding, whereas CH4 metabolism was downregulated. A network analysis revealed that methanogen changed its partner bacteria after CNSL feeding. The δ13 C of CH4 ranged from -74.2‰ to -66.6‰ with significant fluctuation by CNSL feeding, in agreement with the shift of the rumen microbiome. Our findings demonstrate that CNSL feeding can mitigate the CH4 emission from local cattle production systems in South-East Asia by modifying the rumen microbiome and its function.

Analysis of Minor Acidic N-Glycans in Human Serum.

Prior investigations by our research group focused on the method development for the simultaneous analysis of sulfated and phosphorylated glycans. Herein, the developed method was applied to analyze minor acidic N-glycans including sulfated and phosphorylated N-glycans in human serum. First, 2-aminobenzoic acid-labeled minor acidic N-glycans were enriched from the serum using a serotonin-immobilized column and were then separated into groups using hydrophilic interaction liquid chromatography, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phosphorylated hybrid-type and sulfated bi-antennary N-glycans were detected in the serum. In addition, we observed that multiple types of glucuronidated N-glycans were present. These results indicate that the developed method is applicable to the analysis of glucuronidated as well as sulfated and phosphorylated N-glycans. It was also applied to the sera obtained from 17 healthy subjects and 15 pancreatic cancer patients, and the profiles of sulfated, phosphorylated, and glucuronidated N-glycans were compared. The expressed amount of glucuronidated N-glycans was significantly decreased in some pancreatic cancer patients. Numerous examples of the N-glycan analysis in human serum were reported, but phosphorylated and glucuronidated glycans were not investigated. The methods described herein allow the analysis of minor acidic glycans that are typically difficult to detect.

Screening for Components/Compounds with Anti-Rotavirus Activity: Detection of Interaction Between Viral Spike Proteins and Glycans.

Rotaviruses are the major etiologic agents of acute gastroenteritis. Viral attachment to the cell surface is crucial to initiate infection. The VP8∗ domain, the trypsinized cleavage fragment of the outermost spike protein VP4 of rotavirus, has a galectin-like structure required for binding to the cell surface. We used the evanescent-field fluorescence-assisted assay to understand the complex mechanism underlying the virus-glycan/glycoprotein interaction. Besides, we have described virus infection assays, neutralization assay, and pretreatment assay, using cell culture. These approaches using rotavirus particles will provide novel information that has been difficult to obtain from glycan microarray using recombinant VP8∗.

Circulating natural antibodies against 3'-sialyllactose complement the diagnostic performance of CA19-9 for the early detection of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma is a devastating malignancy with an extremely poor prognosis. Although the most widely used biomarker for pancreatic cancer is carbohydrate antigen CA19-9, it is elevated mainly in the late stage of pancreatic cancer. Some serum natural antibodies against carbohydrates have been shown to be possible diagnostic markers for cancer. OBJECTIVE: This study was conducted to determine whether the level of natural antibodies against carbohydrates fluctuates in pancreatic ductal adenocarcinoma. METHODS: Serum from pancreatic cancer subjects (n= 55) and 43 subjects free of malignant disease were studied. The contents of natural antibodies against sialyl glycans and CA19-9 in serum were determined by enzyme-linked immunosorbent assay. RESULTS: The level of serum anti-3'-sialyllactose antibodies in pancreatic cancer subjects was significantly lower than that in healthy controls. In contrast, the amounts of serum antibodies against other sialyl glycans were comparable between the two groups. Concentration of serum anti-3'-sialyllactose IgG provided excellent AUC of 0.86, with sensitivity 82%, specificity 81%, and accuracy 82%. The combination of serum anti-3'-sialyllactose IgG with CA19-9 improved the sensitivity of pancreatic cancer detection at an early stage. CONCLUSIONS: Natural antibodies against 3'-sialyllactose constitute a promising biomarker for pancreatic cancer detection. The measurement of serum anti-3'-sialyllactose antibodies could play a supportive role in diagnostics and complement the performance of CA19-9 for the early detection of pancreatic ductal adenocarcinoma.

Intramolecular isotopic evidence for bacterial oxidation of propane in subsurface natural gas reservoirs.

Microbial anaerobic oxidation of hydrocarbons is a key process potentially involved in a myriad of geological and biochemical environments yet has remained notoriously difficult to identify and quantify in natural environments. We performed position-specific carbon isotope analysis of propane from cracking and incubation experiments. Anaerobic bacterial oxidation of propane leads to a pronounced and previously unidentified 13C enrichment in the central position of propane, which contrasts with the isotope signature associated with the thermogenic process. This distinctive signature allows the detection and quantification of anaerobic oxidation of hydrocarbons in diverse natural gas reservoirs and suggests that this process may be more widespread than previously thought. Position-specific isotope analysis can elucidate the fate of natural gas hydrocarbons and provide insight into a major but previously cryptic process controlling the biogeochemical cycling of globally significant greenhouse gases.

IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts.

In skeletal muscle, myoblast differentiation results in the formation of multinucleated myofibers. Although recent studies have shown that unfolded protein responses (UPRs) play an important role in intracellular remodeling and contribute to skeletal muscle differentiation, the involvement of IRE1-XBP1 signaling, a major UPR signaling pathway, remains unclear. This study aimed to investigate the effect of the IRE1-XBP1 pathway on skeletal muscle differentiation. In C2C12 cells, knockdown of IRE1 and XBP1 in cells remarkably suppressed differentiation. In addition, apoptosis and autophagy were dramatically enhanced in the XBP1-knockdown cells, highlighting the participation of IRE1-XBP1 in cell survival maintenance with differentiation stimuli during skeletal muscle differentiation. In myogenic cells, we demonstrated that the expression of CDK5 (cyclin-dependent kinase 5) is regulated by XBP1s, and we propose that XBP1 regulates the expression of MyoD family genes via the induction of CDK5. In conclusion, this study revealed that IRE1-XBP1 signaling plays critical roles in cell viability and the expression of differentiation-related genes in predifferentiated myoblasts and during the early differentiation phase.

Simultaneous Analysis of Sulfated and Phosphorylated Glycans by Serotonin-Immobilized Column Enrichment and Hydrophilic Interaction Chromatography.

Changes in the structures and quantities of sulfated glycans play important roles in inflammatory and neurological diseases and cancer. Therefore, sulfated glycans are expected to become diagnostic markers for a variety of diseases such as Alzheimer's disease and cancer. On the other hand, structural abnormalities in the phosphorylated glycans on lysosomal enzymes cause a number of lysosomal diseases, while novel phosphorylated glycans have been found in other proteins. As with sulfated glycans, structural and quantitative changes in these phosphorylated glycans and their associations with disease are also of interest. In this article, we introduce a new method for the simultaneous analysis of sulfated and phosphorylated glycans. We first employ a serotonin-immobilized column to enrich these glycans. Glycans obtained in this manner were sequentially subjected to other analytical techniques without desalting. We employed hydrophilic interaction chromatography to distinguish the sulfate and phosphate groups of the glycans and were able to analyze sulfated and phosphorylated N-glycans expressed on thyroglobulin, ovalbumin, and myozyme. We showed that our method not only analyzes sulfated and phosphorylated glycans, but also glycans containing the GlcNAc-HPO3 residue. We comparatively analyzed sulfated glycans, phosphorylated glycans, and GlcNAc-HPO3-residue-containing glycans expressed on MKN7 cells (well-differentiated gastric cancer cells) and MKN45 cells (poorly differentiated gastric cancer cells). To the best of our knowledge, this is the first report of a method for the simultaneous and quantitative analysis of sulfated and phosphorylated glycans.

Position-specific 13 C/12 C analysis of amino acid carboxyl groups - automated flow-injection analysis based on reaction with ninhydrin.

RATIONALE: The fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off-line preparation to reveal this information. An automated position-specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids (δ13 CCARBOXYL values). This automation makes PSIA measurements easier and routine. METHODS: An existing high-performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post-column derivatisation unit between the HPLC system and the interface. The post-column reaction was optimised to yield CO2 from the carboxyl groups of amino acids by reaction with ninhydrin. RESULTS: The methodology described produced δ13 CCARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ13 CCARBOXYL values) between amino acids over a 1-year period. First estimates are presented for the δ13 CCARBOXYL values of a number of internationally available amino acid reference materials. CONCLUSIONS: The PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole-molecule level. When combined with on-line chromatographic separation or off-line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position-specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.

Expanded uncertainty associated with determination of isotope enrichment factors: Comparison of two point calculation and Rayleigh-plot.

The enrichment factor (ε) is a common way to express Isotope Effects (IEs) associated with a phenomenon. Many studies determine ε using a Rayleigh-plot, which needs multiple data points. More recent articles describe an alternative method using the Rayleigh equation that allows the determination of ε using only one experimental point, but this method is often subject to controversy. However, a calculation method using two points (one experimental point and one at t0) should lead to the same results because the calculation is derived from the Rayleigh equation. But, it is frequently asked "what is the valid domain of use of this two point calculation?" The primary aim of the present work is a systematic comparison of results obtained with these two methodologies and the determination of the conditions required for the valid calculation of ε. In order to evaluate the efficiency of the two approaches, the expanded uncertainty (U) associated with determining ε has been calculated using experimental data from three published articles. The second objective of the present work is to describe how to determine the expanded uncertainty (U) associated with determining ε. Comparative methodologies using both Rayleigh-plot and two point calculation are detailed and it is clearly demonstrated that calculation of ε using a single data point can give the same result as a Rayleigh-plot provided one strict condition is respected: that the experimental value is measured at a small fraction of unreacted substrate (f < 30%). This study will help stable isotope users to present their results in a more rigorous expression: ε ± U and therefore to define better the significance of an experimental results prior interpretation. Capsule: Enrichment factor can be determined through two different methods and the calculation of associated expanded uncertainty allows checking its significance.

Carbon isotopic signature reveals the geographical trend in methane consumption and production pathways in alpine ecosystems over the Qinghai-Tibetan Plateau.

On the Qinghai-Tibetan Plateau, isotopic signatures in soil-atmosphere CH4 fluxes were investigated in nine grasslands and three wetlands. In the grasslands, the fractionation factor for soil CH4 uptake, αsoil, was much smaller than the usually reported value of 0.9975-1.0095. Stepwise multiple variation analysis indicates that αsoil is higher for higher soil water contents but is lower for higher C/N ratios of soil surface biomass. In the three wetlands, the soil-emitted δ13C-CH4 was similar (-55.3 ± 5.5 ‰ and -53.0 ± 5.5 ‰) in two bogs separated by >1000 km but was lower (-63.4 ± 6.3 ‰) in a marsh. Environmental factors related to intrasite variations in soil-emitted δ13C-CH4 include the soil C/N ratio, oxidation-reduction potential, soil C concentration and soil water contents. Geographical isotopic surveys revealed environmental constraints on the CH4 consumption pathways in grasslands and the biome type-specific consistency in CH4 production pathways in wetlands.

Combination of SDS-PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics.

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.

Application of Monoclonal Antibodies Against Mouse Dermokine.

Dermokine is one of the most highly expressed proteins in differentiating keratinocytes. Mouse dermokine has been reported to be encoded by 22 exons, and its expression leads to three transcripts, ß, γ, and α, which are transcribed from two different transcriptional start sites. The α isoform represents the carboxyl-terminal domain of the ß isoform, whereas the γ isoform lacks this domain. To reveal the distributions and expression levels of each isoform in mice, we generated rat monoclonal antibodies against dermokine-ß/γ and dermokine-ß/α. In immunofluorescence studies, the expression levels of dermokine in the cytosol of the cultured mouse keratinocytes were significantly elevated by high levels of extracellular calcium. In Western blot analyses, the expression levels of dermokine-ß and dermokine-α were increased in the presence of high calcium. Finally, we developed a monoclonal antibody-based sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and showed that the secreted dermokine-ß into the culture medium from mouse keratinocytes was significantly increased in a manner dependent on the extracellular calcium concentration. These dermokine-specific antibodies have allowed us to gain new insights into the role of each dermokine isoform in cutaneous homeostasis.