Suppression of PKR promotes network excitability and enhanced cognition by interferon-γ-mediated disinhibition.

The double-stranded RNA-activated protein kinase (PKR) was originally identified as a sensor of virus infection, but its function in the brain remains unknown. Here, we report that the lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability. In addition, loss of PKR increases the late phase of long-lasting synaptic potentiation (L-LTP) in hippocampal slices. These effects are caused by an interferon-γ (IFN-γ)-mediated selective reduction in GABAergic synaptic action. Together, our results reveal that PKR finely tunes the network activity that must be maintained while storing a given episode during learning. Because PKR activity is altered in several neurological disorders, this kinase presents a promising new target for the treatment of cognitive dysfunction. As a first step in this direction, we show that a selective PKR inhibitor replicates the Pkr(-/-) phenotype in WT mice, enhancing long-term memory storage and L-LTP.

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

A Density Functional Theory Analysis of Electrochemical Oxidation of Methane to Alcohol over High-Entropy Oxide (CoCrFeMnNi)3O4 Catalysts.

The direct conversion of methane into alcohol is a promising approach for achieving a low-carbon future, yet it remains a major challenge. In this study, we utilize density functional theory to explore the potential of the (CoCrFeMnNi)3O4 (CCFMNO) high entropy oxide (HEO) for electrochemical oxidation of methane to methanol and ethanol, alongside their competition with CO2 production. Our primary focus in this study is on thermodynamics, enabling a prompt analysis of the catalyst's potential, with the calculation of electrochemical barriers falling beyond our scope. Among all potential active sites within CCFMNO HEO, we identify Co as the most active site for methane activation when using carbonate ions as oxidants. This results in methanol production with a limiting potential of 1.4 VCHE, and ethanol and CO2 productions with a limiting potential of 1.2 VCHE. Additionally, our findings suggest that the occupied p-band center of O* on CCFMNO HEO is a potential descriptor for identifying the most active site within CCFMNO HEO. Overall, our results indicate that CCFMNO HEO holds promise as catalysts for methane oxidation to alcohols, employing carbonate ions as oxidants.

Transplanting Gold Active Sites into Non-Precious-Metal Nanoclusters for Efficient CO2-to-CO Electroreduction.

Electrocatalytic CO2 reduction reaction (CO2RR) is greatly facilitated by Au surfaces. However, large fractions of underlying Au atoms are generally unused during the catalytic reaction, which limits mass activity. Herein, we report a strategy for preparing efficient electrocatalysts with high mass activities by the atomic-level transplantation of Au active sites into a Ni4 nanocluster (NC). While the Ni4 NC exclusively produces H2, the Au-transplanted NC selectively produces CO over H2. The origin of the contrasting selectivity observed for this NC is investigated by combining operando and theoretical studies, which reveal that while the Ni sites are almost completely blocked by the CO intermediate in both NCs, the Au sites act as active sites for CO2-to-CO electroreduction. The Au-transplanted NC exhibits a remarkable turnover frequency and mass activity for CO production (206 molCO/molNC/s and 25,228 A/gAu, respectively, at an overpotential of 0.32 V) and high durability toward the CO2RR over 25 h.

Spatial and temporal diversity of glycome expression in mammalian brain.

Mammalian brain glycome remains a relatively poorly understood area compared to other large-scale "omics" studies, such as genomics and transcriptomics due to the inherent complexity and heterogeneity of glycan structure and properties. Here, we first performed spatial and temporal analysis of glycome expression patterns in the mammalian brain using a cutting-edge experimental tool based on liquid chromatography-mass spectrometry, with the ultimate aim to yield valuable implications on molecular events regarding brain functions and development. We observed an apparent diversity in the glycome expression patterns, which is spatially well-preserved among nine different brain regions in mouse. Next, we explored whether the glycome expression pattern changes temporally during postnatal brain development by examining the prefrontal cortex (PFC) at different time point across six postnatal stages in mouse. We found that glycan expression profiles were dynamically regulated during postnatal developments. A similar result was obtained in PFC samples from humans ranging in age from 39 d to 49 y. Novel glycans unique to the brain were also identified. Interestingly, changes primarily attributed to sialylated and fucosylated glycans were extensively observed during PFC development. Finally, based on the vast heterogeneity of glycans, we constructed a core glyco-synthesis map to delineate the glycosylation pathway responsible for the glycan diversity during the PFC development. Our findings reveal high levels of diversity in a glycosylation program underlying brain region specificity and age dependency, and may lead to new studies exploring the role of glycans in spatiotemporally diverse brain functions.

Total Knee Replacement After Arthroscopic Meniscectomy in Knee Osteoarthritis: A Nationwide Population-Based Cohort Study.

BACKGROUND: Recent studies raise concerns that arthroscopic meniscectomy (AM) for degenerative tear may be detrimental to the maintenance of the joint structure. This study was performed to examine the rate of total knee replacement (TKR) among patients with knee osteoarthritis (OA) who underwent AM for meniscal tears and compare this rate with those who did not. METHODS: A retrospective cohort study was conducted using the National Health Insurance Database of South Korea. Among knee OA patients aged 50-79, those who were treated with AM due to meniscal damage from 2007 to 2009 were selected as the AM group while those not treated with AM despite the presence of meniscal damage were selected as control group. Both were matched based on a propensity score and followed-up until the earliest occurrence of: TKR, death, or 10 years. Cox proportional hazards models were used to compare the outcome. RESULTS: A total of 36,974 patients were included in AM groups and non-AM group after 1:1 matching. TKR occurred in 9.62% and 7.64% in AM and non-AM groups with the average duration after meniscectomy of 5.88 ± 2.77 and 5.50 ± 2.94 years, respectively. After adjustment for baseline confounders, the TKR rate in the AM group was calculated to be 25% higher than that in the non-AM group (subdistribution hazard ratio, 1.25; 95% confidence interval, 1.16-1.34). The mortality rate was 5.20%, which did not significantly differ between groups. CONCLUSION: OA patients who underwent AM for the meniscal injury had higher incidence of TKR up to 10 years of follow-up than the non-operated group. The greater TKR utilization observed in patients undergoing AM merits caution when treating OA patients with meniscal injury.

Evaluation of full-fatted and hydrolysate mealworm (Tenebrio molitor) larvae as a substitute for spray-dried plasma protein diet in weaning pigs.

The goal of this experiment was to examine the effect of dietary inclusion of full-fatted mealworm larvae (FFML) or hydrolysate mealworm larvae (HML) (Tenebrio molitor) as a substitute for spray-dried plasma protein (SDPP) as a protein source on the performance and immune status of nursery pigs. A total of 150 crossbred piglets (6.48 ± 0.01 kg) were randomly allocated to 1 of 3 dietary groups in two feeding phases: phase 1 (Days 0-14) and phase 2 (Days 15-35). Each treatment had 10 replicates with 5 pigs per replicate. The nutritious diets were: Phase 1: SDPP-CON (control-basal diet) + 6% SDPP diet; FFML-CON + 3% SDPP and 3% FFML diet; HML-CON + 3% SDPP and 3% HML diet. Phase 2: SDPP-CON (basal diet) + 3% SDPP diet; FFML-CON + 3% FFML diet; HML-CON + 3% HML diet. The inclusion of FFML or HML diet did not show significant difference but had a comparable effect as that of standard control diet containing SDPP on the growth performance, nutrient digestibility and faecal score throughout the trial. In comparison to pigs fed SDPP diet, pigs fed FFML and HML diets had similar and/or higher (p < 0.05) serum immunoglobulin (IgA and IgG) concentration at the end of phase 1 and 2. The result of the present study indictes that SDPP would be partially or fully replaced with FFML or HML to suit weaning pigs diet.

Impact of pitavastatin on new-onset diabetes mellitus compared to atorvastatin and rosuvastatin: a distributed network analysis of 10 real-world databases.

BACKGROUND: Statin treatment increases the risk of new-onset diabetes mellitus (NODM); however, data directly comparing the risk of NODM among individual statins is limited. We compared the risk of NODM between patients using pitavastatin and atorvastatin or rosuvastatin using reliable, large-scale data. METHODS: Data of electronic health records from ten hospitals converted to the Observational Medical Outcomes Partnership Common Data Model (n = 14,605,368 patients) were used to identify new users of pitavastatin, atorvastatin, or rosuvastatin (atorvastatin + rosuvastatin) for ≥ 180 days without a previous history of diabetes or HbA1c level ≥ 5.7%. We conducted a cohort study using Cox regression analysis to examine the hazard ratio (HR) of NODM after propensity score matching (PSM) and then performed an aggregate meta-analysis of the HR. RESULTS: After 1:2 PSM, 10,238 new pitavastatin users (15,998 person-years of follow-up) and 18,605 atorvastatin + rosuvastatin users (33,477 person-years of follow-up) were pooled from 10 databases. The meta-analysis of the HRs demonstrated that pitavastatin resulted in a significantly reduced risk of NODM than atorvastatin + rosuvastatin (HR 0.72; 95% CI 0.59-0.87). In sub-analysis, pitavastatin was associated with a lower risk of NODM than atorvastatin or rosuvastatin after 1:1 PSM (HR 0.69; CI 0.54-0.88 and HR 0.74; CI 0.55-0.99, respectively). A consistently low risk of NODM in pitavastatin users was observed when compared with low-to-moderate-intensity atorvastatin + rosuvastatin users (HR 0.78; CI 0.62-0.98). CONCLUSIONS: In this retrospective, multicenter active-comparator, new-user, cohort study, pitavastatin reduced the risk of NODM compared with atorvastatin or rosuvastatin.

Long-term proton pump inhibitor use and risk of osteoporosis and hip fractures: A nationwide population-based and multicenter cohort study using a common data model.

BACKGROUND AND AIM: Association between protonpump inhibitors (PPIs) and osteoporosis, hip fractures has not been fully elucidated. We aimed to evaluate the relationship between PPIs use and the risk of osteoporosis and hip fractures in the databases converted to a common data model (CDM) and to compare the results across the databases. METHODS: This was a population-based, propensity-matched, retrospective cohort study that included patients aged ≥ 50 years who were prescribed with PPIs for over 180 days. We compared the incidence of osteoporosis and hip fractures between new PPI user and new user of other drugs using the Cox proportional hazards model and performed meta-analysis in the electronic health record (EHR) databases. RESULTS: In the Korean National Health Insurance Service (NHIS)-CDM database, long-term PPI users had greater risk of osteoporosis [PPIs vs non-PPIs groups, 28.42/1000 person-years vs 19.29/1000 person-years; hazard ratio (HR), 1.62; 95% confidence interval (CI), 1.22-2.15; P = 0.001]. The meta-analytic results of six EHR databases also showed similar result (pooled HR, 1.57; 95% CI, 1.28-1.92). In the analysis of hip fracture, PPI use was not significantly associated with a hip fracture in the NHIS-CDM database (PPI vs non-PPI groups, 3.09/1000 person-years vs 2.26/1000 person-years; HR, 1.45; 95% CI, 0.74-2.80; P = 0.27). However, in the meta-analysis of four EHR databases, the risk of hip fractures was higher in PPI users (pooled HR, 1.82; 95% CI, 1.04-3.19). CONCLUSIONS: Long-term PPI was significantly associated with osteoporosis; however, the results of hip fractures were inconsistent. Further study based on better data quality may be needed.

Association between proton pump inhibitor use and gastric cancer: a population-based cohort study using two different types of nationwide databases in Korea.

OBJECTIVE: The association between proton pump inhibitor (PPI) use and gastric cancer related to Helicobacter pylori eradication has not been fully investigated in geographical regions with high risk of gastric cancer. We aimed to evaluate the association between PPIs and gastric cancer in Korea. DESIGN: This study analysed the original and common data model versions of the Korean National Health Insurance Service database from 2002 to 2013. We compared the incidence rates of gastric cancer after 1-year drug exposure, between new users of PPIs and other drugs excluding PPIs, by Cox proportional hazards model. We also analysed the incidence of gastric cancer among PPI users after H. pylori eradication. RESULTS: The analysis included 11 741 patients in matched PPI and non-PPI cohorts after large-scale propensity score matching. During a median follow-up of 4.3 years, PPI use was associated with a 2.37-fold increased incidence of gastric cancer (PPI≥30 days vs non-PPI; 118/51 813 person-years vs 40/49 729 person-years; HR 2.37, 95% CI 1.56 to 3.68, p=0.001). The incidence rates of gastric cancer showed an increasing trend parallel to the duration of PPI use. In H. pylori-eradicated subjects, the incidence of gastric cancer was significantly associated with PPI use over 180 days compared with the non-PPI group (PPI≥180 days vs non-PPI; 30/12 470 person-years vs 9/7814 person-years; HR 2.22, 95% CI 1.05 to 4.67, p=0.036). CONCLUSION: PPI use was associated with gastric cancer, regardless of H. pylori eradication status. Long-term PPIs should be used with caution in high-risk regions for gastric cancer.

Flashlight into the Function of Unannotated C11orf52 using Affinity Purification Mass Spectrometry.

For an enhanced understanding of the biological mechanisms of human disease, it is essential to investigate protein functions. In a previous study, we developed a prediction method of gene ontology (GO) terms by the I-TASSER/COFACTOR result, and we applied this to uPE1 in chromosome 11. Here, to validate the bioinformatics prediction of C11orf52, we utilized affinity purification and mass spectrometry to identify interacting partners of C11orf52. Using immunoprecipitation methods with three different peptide tags (Myc, Flag, and 2B8) in HEK 293T cell lines, we identified 79 candidate proteins that are expected to interact with C11orf52. The results of a pathway analysis of the GO and STRING database with candidate proteins showed that C11orf52 could be related to signaling receptor binding, cell-cell adhesion, and ribosome biogenesis. Then, we selected three partner candidates of DSG1, JUP, and PTPN11 for verification of the interaction with C11orf52 and confirmed them by colocalization at the cell-cell junctions by coimmunofluorescence experiments. On the basis of this study, we expect that C11orf52 is related to the Wnt signaling pathway via DSG1 from the protein-protein interactions, given the results of a comprehensive analysis of the bioinformatic predictions. The data set is available at the ProteomeXchange consortium via PRIDE repository (PXD026986).

Designing Atomically Dispersed Au on Tensile-Strained Pd for Efficient CO2 Electroreduction to Formate.

Pd is one of the most effective catalysts for the electrochemical reduction of CO2 to formate, a valuable liquid product, at low overpotential. However, the intrinsically high CO affinity of Pd makes the surface vulnerable to CO poisoning, resulting in rapid catalyst deactivation during CO2 electroreduction. Herein, we utilize the interaction between metals and metal-organic frameworks to synthesize atomically dispersed Au on tensile-strained Pd nanoparticles showing significantly improved formate production activity, selectivity, and stability with high CO tolerance. We found that the tensile strain stabilizes all reaction intermediates on the Pd surface, whereas the atomically dispersed Au selectively destabilizes CO* without affecting other adsorbates. As a result, the conventional COOH* versus CO* scaling relation is broken, and our catalyst exhibits 26- and 31-fold enhancement in partial current density and mass activity toward electrocatalytic formate production with over 99% faradaic efficiency, compared to Pd/C at -0.25 V versus RHE.

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.

Critical Role of Ti4+ in Stabilizing High-Voltage Redox Reactions in Li-Rich Layered Material.

Li-rich layered oxide materials are considered promising candidates for high-capacity cathodes for battery applications and improving the reversibility of the anionic redox reaction is the key to exploiting the full capacity of these materials. However, permanent structural change of the electrode occurring upon electrochemical cycling results in capacity and voltage decay. In view of these factors, Ti4+ -substituted Li2 IrO3 (Li2 Ir0.75 Ti0.25 O3 ) is synthesized, which undergoes an oxygen redox reaction with suppressed voltage decay, yielding improved electrochemical performance and good capacity retention. It is shown that the increased bond covalency upon Ti4+ substitution results in structural stability, tuning the phase stability from O3 to O1' upon de-lithiation during charging compared with O3 to T3 and O1 for pristine Li2 IrO3 , thereby facilitating the oxidation of oxygen. This work unravels the role of Ti4+ in stabilizing the cathode framework, providing insight for a fundamental design approach for advanced Li-rich layered oxide battery materials.

Atomic-level tuning of Co-N-C catalyst for high-performance electrochemical H2O2 production.

Despite the growing demand for hydrogen peroxide it is almost exclusively manufactured by the energy-intensive anthraquinone process. Alternatively, H2O2 can be produced electrochemically via the two-electron oxygen reduction reaction, although the performance of the state-of-the-art electrocatalysts is insufficient to meet the demands for industrialization. Interestingly, guided by first-principles calculations, we found that the catalytic properties of the Co-N4 moiety can be tailored by fine-tuning its surrounding atomic configuration to resemble the structure-dependent catalytic properties of metalloenzymes. Using this principle, we designed and synthesized a single-atom electrocatalyst that comprises an optimized Co-N4 moiety incorporated in nitrogen-doped graphene for H2O2 production and exhibits a kinetic current density of 2.8 mA cm-2 (at 0.65 V versus the reversible hydrogen electrode) and a mass activity of 155 A g-1 (at 0.65 V versus the reversible hydrogen electrode) with negligible activity loss over 110 hours.

Controlled syngas production by electrocatalytic CO2 reduction on formulated Au25(SR)18 and PtAu24(SR)18 nanoclusters.

Syngas, a gaseous mixture of CO and H2, is a critical industrial feedstock for producing bulk chemicals and synthetic fuels, and its production via direct CO2 electroreduction in aqueous media constitutes an important step toward carbon-negative technologies. Herein, we report controlled syngas production with various H2/CO ratios via the electrochemical CO2 reduction reaction (CO2RR) on specifically formulated Au25 and PtAu24 nanoclusters (NCs) with core-atom-controlled selectivities. While CO was predominantly produced from the CO2RR on the Au NCs, H2 production was favored on the PtAu24 NCs. Density functional theory calculations of the free energy profiles for the CO2RR and hydrogen evolution reaction (HER) indicated that the reaction energy for the conversion of CO2 to CO was much lower than that for the HER on the Au25 NC. In contrast, the energy profiles calculated for the HER indicated that the PtAu24 NCs have nearly thermoneutral binding properties; thus, H2 production is favored over CO formation. Based on the distinctly different catalytic selectivities of Au25 and PtAu24 NCs, controlled syngas production with H2/CO ratios of 1 to 4 was demonstrated at a constant applied potential by simply mixing the Au25 and PtAu24 NCs based on their intrinsic catalytic activities for the production of CO and H2.

Quality Indicators for Evaluating the Health Care of Patients with Rheumatoid Arthritis: a Korean Expert Consensus.

BACKGROUND: There is increasing interest in the quality of health care and considerable efforts are being made to improve it. Rheumatoid arthritis (RA) is a disease that can result in favorable outcomes when appropriate diagnosis and treatment are provided. However, several studies have shown that RA is often managed inappropriately. Therefore, the Korean College of Rheumatology aimed to develop quality indicators (QIs) to evaluate and improve the health care of patients with RA. METHODS: Preliminary QIs were derived based on the existing guidelines and QIs for RA. The final QIs were determined through two separate consensus meetings of experts. The consensus was achieved through a panel of experts who voted using the modified Delphi method. RESULTS: Fourteen final QIs were selected among 70 preliminary QIs. These included early referral to and regular follow-up with a rheumatologist, radiographs of the hands and feet, early initiation and maintenance of disease-modifying anti-rheumatic drug (DMARD) therapy, periodic assessment of disease activity, screening for drug safety and comorbidities, including viral hepatitis and tuberculosis before biologic DMARD therapy, periodic laboratory testing, supplementation with folic acid, assessment of the risk for cervical spine instability before general anesthesia, patient education, and specialized nurse. CONCLUSION: These QIs can be used to assess and improve the quality of health care for patients with RA.

Atomically Precise Gold Nanoclusters as Model Catalysts for Identifying Active Sites for Electroreduction of CO2.

Accurate identification of active sites is critical for elucidating catalytic reaction mechanisms and developing highly efficient and selective electrocatalysts. Herein, we report the atomic-level identification of active sites using atomically well-defined gold nanoclusters (Au NCs) Au25 , Au38 , and Au144 as model catalysts in the electrochemical CO2 reduction reaction (CO2 RR). The studied Au NCs exhibited remarkably high CO2 RR activity, which increased with increasing NC size. Electrochemical and X-ray photoelectron spectroscopy analyses revealed that the Au NCs were activated by removing one thiolate group from each staple motif at the beginning of CO2 RR. In addition, density functional theory calculations revealed higher charge densities and upshifts of d-states for dethiolated Au sites. The structure-activity properties of the studied Au NCs confirmed that dethiolated Au sites were the active sites and that CO2 RR activity was determined by the number of active sites on the cluster surface.

Bioinformatic Prediction of Gene Ontology Terms of Uncharacterized Proteins from Chromosome 11.

In chromosome 11, 71 out of its 1254 proteins remain functionally uncharacterized on the basis of their existence evidence (uPE1s) following the latest version of neXtProt (release 2020-01-17). Because in vivo and in vitro experimental strategies are often time-consuming and labor-intensive, there is a need for a bioinformatics tool to predict the function annotation. Here, we used I-TASSER/COFACTOR provided on the neXtProt web site, which predicts gene ontology (GO) terms based on the 3D structure of the protein. I-TASSER/COFACTOR predicted 2413 GO terms with a benchmark dataset of the 22 proteins belonging to PE1 of chromosome 11. In this study, we developed a filtering algorithm in order to select specific GO terms using the GO map generated by I-TASSER/COFACTOR. As a result, 187 specific GO terms showed a higher average precision-recall score at the least cellular component term compared to 2413 predicted GO terms. Next, we applied 65 proteins belonging to uPE1s of chromosome 11, and then 409 out of 6684 GO terms survived, where 103 and 142 GO terms of molecular function and biological process, respectively, were included. Representatively, the cellular component GO terms of CCDC90B, C11orf52, and the SMAP were predicted and validated using the overexpression system into 293T cells and immunofluorescence staining. We will further study their biological and molecular functions toward the goal of the neXt-CP50 project as a part of C-HPP. We shared all results and programs in Github (https://github.com/heeyounh/I-TASSER-COFACTOR-filtering.git).

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.