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1.
J Ethnopharmacol ; 336: 118723, 2025 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-39181285

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Mountain-cultivated Panax ginseng C.A.Mey. (MCG) with high market price and various properties was valuable special local product in Northeast of Asia. MCG has been historically used to mitigate heart failure (HF) for thousand years, HF is a clinical manifestation of deficiency of "heart-qi" in traditional Chinese medicine. However, there was little report focus on the activities of extracted residue of MCG. AIM OF THE STUDY: A novel glycopeptide (APMCG-1) was isolated from step ethanol precipitations of alkaline protease-assisted extract from MCG residue. MATERIALS AND METHODS: The molecular weight and subunit structure of APMCG-1 were determined by FT-IR, HPLC and GPC technologies, as well as the H9c2 cells, Tg (kdrl:EGFP) zebrafish were performed to evaluated the protective effect of APMCG-1. RESULTS: APMCG-1 was identified as a glycopeptide containing seven monosaccharides and seven amino acids via O-lined bonds. Further, in vitro, APMCG-1 significantly decreased reactive oxygen species production and lactate dehydrogenase contents in palmitic acid (PA)-induced H9c2 cells. APMCG-1 also attenuated endoplasmic reticulum stress and mitochondria-mediated apoptosis in H9c2 cells via the PI3K/AKT signaling pathway. More importantly, APMCG-1 reduced the blood glucose, lipid contents, the levels of heart injury, oxidative stress and inflammation of 5 days post fertilization Tg (kdrl:EGFP) zebrafish with type 2 diabetic symptoms in vivo. CONCLUSIONS: APMCG-1 protects PA-induced H9c2 cells while reducing cardiac dysfunction in zebrafish with type 2 diabetic symptoms. The present study provides a new insight into the development of natural glycopeptides as heart-related drug therapies.


Assuntos
Diabetes Mellitus Tipo 2 , Glicopeptídeos , Insuficiência Cardíaca , Panax , Peixe-Zebra , Animais , Panax/química , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/prevenção & controle , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ratos , Linhagem Celular , Glicopeptídeos/farmacologia , Glicopeptídeos/química , Apoptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Cardiotônicos/farmacologia , Cardiotônicos/química , Cardiotônicos/isolamento & purificação , Cardiotônicos/uso terapêutico , Miócitos Cardíacos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos
2.
Clin Chim Acta ; 564: 119937, 2025 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-39173701

RESUMO

BACKGROUND: End-stage renal disease (ESRD) necessitating hemodialysis pose substantial cardiovascular risks, with cardiovascular disease (CVD) as a leading cause of mortality. Biomarkers like copeptin have emerged as potential indicators of cardiovascular stress and prognosis in CKD populations. OBJECTIVE: This study aimed to assess the prognostic value of copeptin in predicting major adverse cardiovascular events (MACEs) among hemodialysis patients, alongside traditional cardiac biomarkers. METHODS: ESRD patients undergoing maintenance hemodialysis were enrolled. Copeptin levels were measured, and patients were followed for MACEs, defined as cardiovascular deaths, myocardial infarction, stroke, or heart failure-related hospitalizations. Cox proportional-hazards models were used to evaluate the association between copeptin and outcomes, adjusting for relevant covariates. RESULTS: Among 351 patients followed for a median of 22.7 months, elevated copeptin levels were significantly associated with an increased risk of MACEs (HR 1.519, 95 % CI 1.140 to 2.023; p = 0.00425). Copeptin demonstrated predictive capability across multiple statistical tests (Log-rank p = 0.024; Gehan p < 0.001; Tarone-Ware p < 0.001; Peto-Peto p = 0.027), although significance was attenuated in pairwise comparisons post-adjustment for multiple testing. Combining copeptin with NT-proBNP or hs-cTnT further enhanced risk stratification for MACEs. CONCLUSION: Elevated copeptin levels independently predict adverse cardiovascular outcomes in hemodialysis patients. Integrating copeptin with traditional cardiac biomarkers may refine risk stratification and guide personalized therapeutic strategies in this high-risk population.


Assuntos
Doenças Cardiovasculares , Glicopeptídeos , Falência Renal Crônica , Diálise Renal , Humanos , Glicopeptídeos/sangue , Diálise Renal/efeitos adversos , Masculino , Feminino , Pessoa de Meia-Idade , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/diagnóstico , Falência Renal Crônica/terapia , Falência Renal Crônica/sangue , Falência Renal Crônica/complicações , Idoso , Biomarcadores/sangue
3.
J Chromatogr A ; 1735: 465318, 2024 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-39244913

RESUMO

Protein glycosylation, one of the most important biologically relevant post-translational modifications for biomarker discovery, faces analytical challenges due to heterogeneous glycosite, diverse glycans, and mass spectrometry limitations. Glycopeptide enrichment by removing abundant hydrophobic peptides helps overcome some of these obstacles. Hydrophilic interaction liquid chromatography (HILIC), known for its selectivity, glycan separations, intact glycopeptide enrichment, and compatibility with mass spectrometry, has seen recent advancements in stationary phases like Amide-80, glycoHILIC, amino acids or peptides for improved HILIC-based glycopeptide analysis. Utilization of these materials can improve glycopeptide enrichment through solid-phase extraction and separation via high-performance liquid chromatography. Additionally, using glycopeptides themselves to modify HILIC stationary phases holds promise for improving selectivity and sensitivity in glycosylation analysis. Additionally, HILIC has capability to assess the information about glycosites and structural information of glycans. This review summarizes recent breakthroughs in HILIC stationary materials, highlighting their impact on glycopeptide analysis. Ongoing research on advanced materials continues to refine HILIC's performance, solidifying its value as a tool for exploring protein glycosylation.


Assuntos
Glicopeptídeos , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos , Glicopeptídeos/química , Glicopeptídeos/isolamento & purificação , Glicopeptídeos/análise , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Polissacarídeos/análise , Glicosilação , Cromatografia Líquida/métodos , Cromatografia Líquida de Alta Pressão/métodos , Extração em Fase Sólida/métodos , Humanos
4.
Nat Commun ; 15(1): 8055, 2024 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-39277585

RESUMO

Precision mapping of site-specific glycans using mass spectrometry is vital in glycoproteomics. However, the diversity of glycan compositions across species often exceeds database capacity, hindering the identification of rare glycans. Here, we introduce pGlycoNovo, a software within the pGlyco3 software environment, which employs a glycan first-based full-range Y-ion dynamic searching strategy. pGlycoNovo enables de novo identification of intact glycopeptides with rare glycans by considering all possible monosaccharide combinations, expanding the glycan search space to 16~1000 times compared to non-open search methods, while maintaining accuracy, sensitivity and speed. Reanalysis of SARS Covid-2 spike protein glycosylation data revealed 230 additional site-specific N-glycans and 30 previously unreported O-glycans. pGlycoNovo demonstrated high complementarity to six other tools and superior search speed. It enables characterization of site-specific N-glycosylation across five evolutionarily distant species, contributing to a dataset of 32,549 site-specific glycans on 4602 proteins, including 2409 site-specific rare glycans, and uncovering unexpected glycan fragments.


Assuntos
Glicopeptídeos , Polissacarídeos , Software , Glicoproteína da Espícula de Coronavírus , Glicosilação , Polissacarídeos/metabolismo , Polissacarídeos/química , Humanos , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicopeptídeos/análise , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Animais , Proteômica/métodos , COVID-19/virologia
5.
Int J Mol Sci ; 25(17)2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-39273352

RESUMO

Highly purified human menopausal gonadotropin (HP-hMG [Menopur®, Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur® and two of Menogon® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid®) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.


Assuntos
Gonadotropina Coriônica , Hormônio Luteinizante , Espectrometria de Massas em Tandem , Humanos , Gonadotropina Coriônica/urina , Gonadotropina Coriônica/análise , Hormônio Luteinizante/urina , Hormônio Luteinizante/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Polissacarídeos/análise , Polissacarídeos/química , Polissacarídeos/urina , Glicosilação , Feminino , Menotropinas/urina , Menotropinas/análise , Hormônio Foliculoestimulante/urina , Hormônio Foliculoestimulante/análise , Oxirredução , Glicopeptídeos/análise , Glicopeptídeos/química , Glicopeptídeos/urina
6.
BMC Infect Dis ; 24(1): 906, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39223521

RESUMO

BACKGROUND: Glycopeptides for ampicillin-susceptible Enterococcus faecalis/faecium bacteremia are readily prescribed depending on the severity of the condition. However, there is limited data on the outcomes of glycopeptide use compared to ampicillin-containing regimens for ampicillin-susceptible E. faecalis/faecium bacteremia. From an antibiotic stewardship perspective, it is important to determine whether the use of glycopeptides is associated with improved clinical outcomes in patients with ampicillin-susceptible E. faecalis/faecium bacteremia. METHODS: This retrospective cohort study was conducted at a university-affiliated hospital between January 2010 and September 2019. We collected data from patients with positive blood cultures for Enterococcus species isolates. The clinical data of patients who received ampicillin-containing regimens or glycopeptides as definitive therapy for ampicillin-susceptible E. faecalis/faecium bacteremia were reviewed. Multivariate logistic regression analysis was performed to identify risk factors for 28-day mortality. RESULTS: Ampicillin-susceptible E. faecalis/faecium accounted for 41.2% (557/1,353) of enterococcal bacteremia cases during the study period. A total of 127 patients who received ampicillin-containing regimens (N = 56) or glycopeptides (N = 71) as definitive therapy were included in the analysis. The 28-day mortality rate was higher in patients treated with glycopeptides (19.7%) than in those treated with ampicillin-containing regimens (3.6%) (p = 0.006). However, in the multivariate model, antibiotic choice was not an independent predictor of 28-day mortality (adjusted OR, 3.7; 95% CI, 0.6-23.6). CONCLUSIONS: Glycopeptide use was not associated with improved mortality in patients with ampicillin-susceptible E. faecalis/faecium bacteremia. This study provides insights to reduce the inappropriate use of glycopeptides in ampicillin-susceptible E. faecalis/faecium bacteremia treatment and promote antimicrobial stewardship.


Assuntos
Ampicilina , Antibacterianos , Bacteriemia , Enterococcus faecalis , Glicopeptídeos , Infecções por Bactérias Gram-Positivas , Sulbactam , Humanos , Estudos Retrospectivos , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Bacteriemia/mortalidade , Ampicilina/uso terapêutico , Ampicilina/farmacologia , Masculino , Feminino , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Enterococcus faecalis/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/mortalidade , Idoso , Pessoa de Meia-Idade , Glicopeptídeos/uso terapêutico , Glicopeptídeos/farmacologia , Sulbactam/uso terapêutico , Sulbactam/farmacologia , Resultado do Tratamento , Testes de Sensibilidade Microbiana , Idoso de 80 Anos ou mais
7.
Sci Transl Med ; 16(759): eabo4736, 2024 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-39110780

RESUMO

Gram-positive bacterial infections present a major clinical challenge, with methicillin- and vancomycin-resistant strains continuing to be a cause for concern. In recent years, semisynthetic vancomycin derivatives have been developed to overcome this problem as exemplified by the clinically used telavancin, which exhibits increased antibacterial potency but has also raised toxicity concerns. Thus, glycopeptide antibiotics with enhanced antibacterial activities and improved safety profiles are still necessary. We describe the development of a class of highly potent semisynthetic glycopeptide antibiotics, the guanidino lipoglycopeptides, which contain a positively charged guanidino moiety bearing a variable lipid group. These glycopeptides exhibited enhanced in vitro activity against a panel of Gram-positive bacteria including clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains, showed minimal toxicity toward eukaryotic cells, and had a low propensity for resistance selection. Mechanistically, guanidino lipoglycopeptides engaged with bacterial cell wall precursor lipid II with a higher binding affinity than vancomycin. Binding to both wild-type d-Ala-d-Ala lipid II and the vancomycin-resistant d-Ala-d-Lac variant was confirmed, providing insight into the enhanced activity of guanidino lipoglycopeptides against vancomycin-resistant isolates. The in vivo efficacy of guanidino lipoglycopeptide EVG7 was evaluated in a S. aureus murine thigh infection model and a 7-day sepsis survival study, both of which demonstrated superiority to vancomycin. Moreover, the minimal to mild kidney effects at supratherapeutic doses of EVG7 indicate an improved therapeutic safety profile compared with vancomycin. These findings position guanidino lipoglycopeptides as candidates for further development as antibacterial agents for the treatment of clinically relevant multidrug-resistant Gram-positive infections.


Assuntos
Antibacterianos , Lipoglicopeptídeos , Testes de Sensibilidade Microbiana , Animais , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/uso terapêutico , Lipoglicopeptídeos/farmacologia , Lipoglicopeptídeos/uso terapêutico , Camundongos , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Glicopeptídeos/farmacologia , Glicopeptídeos/química , Glicopeptídeos/uso terapêutico , Bactérias Gram-Positivas/efeitos dos fármacos , Feminino
8.
J Med Microbiol ; 73(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39158416

RESUMO

Introduction. Mycobacterium abscessus (MABS) is a pathogenic bacterium that can cause severe lung infections, particularly in individuals with cystic fibrosis. MABS colonies can exhibit either a smooth (S) or rough (R) morphotype, influenced by the presence or absence of glycopeptidolipids (GPLs) on their surface, respectively. Despite the clinical significance of these morphotypes, the relationship between GPL levels, morphotype and the pathogenesis of MABS infections remains poorly understood.Gap statement. The mechanisms and implications of GPL production and morphotypes in clinical MABS infections are unclear. There is a gap in understanding their correlation with infectivity and pathogenicity, particularly in patients with underlying lung disease.Aim. This study aimed to investigate the correlation between MABS morphology, GPL and infectivity by analysing strains from cystic fibrosis patients' sputum samples.Methodology. MABS was isolated from patient sputum samples and categorized by morphotype, GPL profile and replication rate in macrophages. A high-content ex vivo infection model using THP-1 cells assessed the infectivity of both clinical and laboratory strains.Results. Our findings revealed that around 50 % of isolates displayed mixed morphologies. GPL analysis confirmed a consistent relationship between GPL content and morphotype that was only found in smooth isolates. Across morphotype groups, no differences were observed in vitro, yet clinical R strains were observed to replicate at higher levels in the THP-1 infection model. Moreover, the proportion of infected macrophages was notably higher among clinical R strains compared to their S counterparts at 72 h post-infection. Clinical variants also infected THP-1 cells at significantly higher rates compared to laboratory strains, highlighting the limited translatability of lab strain infection data to clinical contexts.Conclusion. Our study confirmed the general correlation between morphotype and GPL levels in smooth strains yet unveiled more variability within morphotype groups than previously recognized, particularly during intracellular infection. As the R morphotype is the highest clinical concern, these findings contribute to the expanding knowledge base surrounding MABS infections, offering insights that can steer diagnostic methodologies and treatment approaches.


Assuntos
Glicolipídeos , Macrófagos , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Mycobacterium abscessus/isolamento & purificação , Mycobacterium abscessus/classificação , Humanos , Macrófagos/microbiologia , Macrófagos/imunologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Glicolipídeos/análise , Células THP-1 , Fibrose Cística/microbiologia , Fibrose Cística/complicações , Escarro/microbiologia , Glicopeptídeos
9.
J Mass Spectrom ; 59(9): e5083, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39162140

RESUMO

Protein glycosylation is the co- and/or post-translational modification of proteins with oligosaccharides (glycans). This process is not template based and can introduce a heterogeneous set of glycan modifications onto substrate proteins. Glycan structures preserve biomolecular information from the cell, with glycoproteins from different cell types and tissues displaying distinct patterns of glycosylation. Several decades of research have revealed that glycan structures also differ between normal physiology and disease. This suggests that the information stored in glycoproteins and glycans can be utilized for disease diagnosis and monitoring. Methods that enable sensitive and site-specific measurement of protein glycosylation in clinical settings, such as nano-flow liquid chromatography tandem mass spectrometry, are therefore essential. The purpose of this perspective is to discuss recent advances in mass spectrometry and the potential of these advances to facilitate the detection and monitoring of disease-specific glycoprotein glycoforms. Glycoproteomics, the system-wide characterization of glycoprotein identity inclusive of site-specific characterization of carbohydrate modifications on proteins, and glycomics, the characterization of glycan structures, will be discussed in this context. Quantitative measurement of glycopeptide markers via parallel reaction monitoring is highlighted. The development of promising glycopeptide markers for autoimmune disease, liver disease, and liver cancer is discussed. Synthetic glycopeptide standards, ambient ionization mass spectrometry, and consideration of glyco-biomarkers in two- and three-dimensional space within tissue will be critical to the advancement of this field. The authors envision a future in which glycoprotein mass spectrometry workflows will be integrated into clinical settings, to aid in the rapid diagnosis and monitoring of disease.


Assuntos
Glicoproteínas , Polissacarídeos , Proteômica , Humanos , Glicoproteínas/análise , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Proteômica/métodos , Polissacarídeos/análise , Polissacarídeos/química , Biomarcadores/análise , Espectrometria de Massas/métodos , Glicômica/métodos , Glicopeptídeos/análise , Glicopeptídeos/química , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem/métodos , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/química , Hepatopatias/diagnóstico , Hepatopatias/metabolismo , Cromatografia Líquida/métodos
10.
J Sep Sci ; 47(16): e2400310, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39164910

RESUMO

In this work, a composite hydrogel material consisting of chitosan-based composite hydrogel was prepared by a simple and rapid synthetic method and will be named three-dimensional (3D)-IL-COF-1@CS hydrogel. Possessing a stable 3D network structure and outstanding hydrophilicity, the novel hydrogel is capable of capturing glycopeptides. The 3D-IL-COF-1@CS hydrogel showed good sensitivity (0.1 fmol/µL) and selectivity (1:2000). In addition, 19 glycopeptides were captured in standard samples. In the analysis of human serum, 148 glycopeptides assigned to 72 glycoproteins were assayed in the serum of normal individuals, and 245 glycopeptides corresponding to 100 glycoproteins were found in the serum of colorectal cancer (CRC) patients. More importantly, several functional programs based on Gene Ontology analysis supported molecular biological processes that may be relevant to the pathogenesis of CRC, including aging, fibrinogen complex, and arylesterase activity. The low cost, simplicity, rapid synthesis, and good enrichment performance have a great future in glycoproteomics analysis and related diseases.


Assuntos
Neoplasias Colorretais , Glicopeptídeos , Interações Hidrofóbicas e Hidrofílicas , Humanos , Neoplasias Colorretais/sangue , Glicopeptídeos/sangue , Glicopeptídeos/química , Hidrogéis/química , Polímeros/química , Quitosana/química
11.
Glycobiology ; 34(9)2024 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-39115362

RESUMO

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.


Assuntos
Colostro , Lactalbumina , Leite , Lactalbumina/metabolismo , Lactalbumina/química , Animais , Glicosilação , Colostro/química , Colostro/metabolismo , Bovinos , Leite/química , Leite/metabolismo , Feminino , Lactação/metabolismo , Amino Açúcares/química , Amino Açúcares/metabolismo , Glicopeptídeos/metabolismo , Glicopeptídeos/química , Glicopeptídeos/análise , Lactose/metabolismo , Lactose/química
12.
Int J Mol Sci ; 25(16)2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39201804

RESUMO

An asymmetric synthesis is a favorable approach for obtaining enantiomerically pure substances, but racemic resolution remains an efficient strategy. This study aims to elucidate the chiral resolution of aromatic amino acids and their elution order using glycopeptides as chiral selectors through molecular docking analysis. Chiral separation experiments were conducted using Vancomycin as a chiral additive in the mobile phase (CMPA) at various concentrations, coupled with an achiral amino column as the stationary phase. The Autodock Vina 1.1.2 software was employed to perform molecular docking simulations between each enantiomer (ligand) and Vancomycin (receptor) to evaluate binding affinities, demonstrate enantiomeric resolution feasibility, and elucidate chiral recognition mechanisms. Utilizing Vancomycin as CMPA at a concentration of 1.5 mM enabled the separation of tryptophan enantiomers with a resolution of 3.98 and tyrosine enantiomers with a resolution of 2.97. However, a poor chiral resolution was observed for phenylalanine and phenylglycine. Molecular docking analysis was employed to elucidate the lack of separation and elution order for tryptophan and tyrosine enantiomers. By calculating the binding energy, docking results were found to be in good agreement with experimental findings, providing insights into the underlying mechanisms governing chiral recognition in this system and the interaction sites. This comprehensive approach clarifies the complex relationship between chiral discrimination and molecular architecture, offering valuable information for creating and improving chiral separation protocols.


Assuntos
Aminoácidos Aromáticos , Glicopeptídeos , Simulação de Acoplamento Molecular , Glicopeptídeos/química , Aminoácidos Aromáticos/química , Estereoisomerismo , Vancomicina/química , Cromatografia Líquida de Alta Pressão/métodos , Ligantes
13.
Sci Rep ; 14(1): 20000, 2024 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-39198565

RESUMO

Epithelial ovarian cancer (EOC) is widely recognized as the most lethal gynecological malignancy; however, its early-stage detection remains a considerable clinical challenge. To address this, we have introduced a new method, named Comprehensive Serum Glycopeptide Spectral Analysis (CSGSA), which detects early-stage cancer by combining glycan alterations in serum glycoproteins with tumor markers. We detected 1712 glycopeptides using liquid chromatography-mass spectrometry from the sera obtained from 564 patients with EOC and 1149 controls across 13 institutions. Furthermore, we used a convolutional neural network to analyze the expression patterns of the glycopeptides and tumor markers. Using this approach, we successfully differentiated early-stage EOC (Stage I) from non-EOC, with an area under the curve (AUC) of 0.924 in receiver operating characteristic (ROC) analysis. This method markedly outperforms conventional tumor markers, including cancer antigen 125 (CA125, 0.842) and human epididymis protein 4 (HE4, 0.717). Notably, our method exhibited remarkable efficacy in differentiating early-stage ovarian clear cell carcinoma from endometrioma, achieving a ROC-AUC of 0.808, outperforming CA125 (0.538) and HE4 (0.557). Our study presents a promising breakthrough in the early detection of EOC through the innovative CSGSA method. The integration of glycan alterations with cancer-related tumor markers has demonstrated exceptional diagnostic potential.


Assuntos
Biomarcadores Tumorais , Carcinoma Epitelial do Ovário , Glicopeptídeos , Neoplasias Ovarianas , Humanos , Feminino , Carcinoma Epitelial do Ovário/sangue , Carcinoma Epitelial do Ovário/diagnóstico , Carcinoma Epitelial do Ovário/patologia , Biomarcadores Tumorais/sangue , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Glicopeptídeos/sangue , Pessoa de Meia-Idade , Curva ROC , Antígeno Ca-125/sangue , Estadiamento de Neoplasias , Adulto , Idoso , Cromatografia Líquida/métodos , Detecção Precoce de Câncer/métodos , Estudos de Casos e Controles , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/análise , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/metabolismo
14.
Acta Biochim Biophys Sin (Shanghai) ; 56(8): 1172-1183, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39118567

RESUMO

N-linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, N-glycosylation introduces an additional layer of complexity with tens of thousands of possible structures arising from various dimensions, including different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformations. This results in additional microheterogeneity of the modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio. N-glycosylation regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis. Numerous advanced methods ranging from sample preparation to mass spectrum analysis have been developed to distinguish N-glycan structures. Chemical derivatization of monosaccharides, online liquid chromatography separation and ion mobility spectrometry enable the physical differentiation of samples. Tandem mass spectrometry further analyzes the macro/microheterogeneity of intact N-glycopeptides through the analysis of fragment ions. Moreover, the development of search engines and AI-based software has enhanced our understanding of the dissociation patterns of intact N-glycopeptides and the clinical significance of differentially expressed intact N-glycopeptides. With the help of these modern methods, structure-specific N-glycoproteomics has become an important tool with extensive applications in the biomedical field.


Assuntos
Glicoproteínas , Proteômica , Proteômica/métodos , Humanos , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Polissacarídeos/química , Polissacarídeos/metabolismo , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Glicopeptídeos/química , Glicopeptídeos/análise , Glicopeptídeos/metabolismo , Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodos , Animais
15.
MAbs ; 16(1): 2395503, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39192481

RESUMO

Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos , Espectrometria de Massas em Tandem , Glicosilação , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Polissacarídeos/química , Polissacarídeos/análise , Humanos , Processamento de Proteína Pós-Traducional , Glicopeptídeos/química , Glicopeptídeos/análise , Glicopeptídeos/metabolismo
16.
Org Lett ; 26(34): 7128-7133, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39155450

RESUMO

Using l-methionine (Met) as the endogenous directing group, we developed Pd-catalyzed ß-C(sp3)-H glycosylation of peptides with 1-iodoglycals. A wide range of tri- to hexapeptides containing the Ala-Met motifs underwent Ala C-H glycosylation under the standard conditions to give the glycopeptides smoothly. 15 proteinogenic amino acids (with easily removable protecting groups) were well tolerated. Control experiments indicated that Met acted as a N,S-bidentate directing group and exhibited an effect superior to other amino acid residues such as l-aspartic acid (Asp), l-asparagine (Asn), and S-protected l-cysteine (Cys). In addition, further transformation by HFIP-promoted 1,4-elimination furnished another type of glycopeptide with the 1,3-diene motif, which provides a handle for further derivatization.


Assuntos
Metionina , Metionina/química , Glicosilação , Estrutura Molecular , Catálise , Peptídeos/química , Glicopeptídeos/química , Paládio/química
17.
Elife ; 122024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-39207917

RESUMO

Mycobacterium tuberculosis's (Mtb) autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA sequencing performed under various stress conditions, we identified that genes belonging to Mtb sulfur metabolism pathways are significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role of non-canonical L-cysteine synthases, CysM and CysK2. While transcriptome signatures of RvΔcysM and RvΔcysK2 appear similar under regular growth conditions, we observed unique transcriptional signatures when subjected to oxidative stress. We followed pool size and labelling (34S) of key downstream metabolites, viz. mycothiol and ergothioneine, to monitor L-cysteine biosynthesis and utilization. This revealed the significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant during infection. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection.


Assuntos
Vias Biossintéticas , Cisteína Sintase , Cisteína , Inositol , Mycobacterium tuberculosis , Estresse Oxidativo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Cisteína/metabolismo , Cisteína/biossíntese , Cisteína Sintase/metabolismo , Cisteína Sintase/genética , Vias Biossintéticas/genética , Inositol/metabolismo , Inositol/biossíntese , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ergotioneína/biossíntese , Ergotioneína/metabolismo , Regulação Bacteriana da Expressão Gênica , Camundongos , Glicopeptídeos/metabolismo , Glicopeptídeos/biossíntese , Tuberculose/microbiologia
18.
Clin Neurol Neurosurg ; 244: 108432, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38986366

RESUMO

OBJECTIVE: Traumatic brain injury (TBI) necessitates reliable biomarkers to improve patient care. This study explored copeptin as a potential biomarker in TBI and its relation to vasopressin (ADH) in such patients. METHODS: A cross-sectional study was conducted on 50 TBI patients. Exclusion criteria included specific medical conditions and recent traumatic events. Copeptin and ADH testing were performed within 30 days post-trauma. Patient data, Glasgow Coma Scale (GCS) scores, imaging results, and the need for surgical intervention were obtained from medical charts. RESULTS: Copeptin levels negatively correlated with GCS scores (ρ = - 0.313, p = 0.027), indicating a potential association with trauma severity. Copeptin levels (mean: 3.22 pmol/L, median 2.027 pmol/L, SD = 3.15) tended to be lower than those found in the normal population, suggesting possible neuroendocrine dysfunction post-TBI. ADH levels (mean: 67.93 pmol/L, median 56.474 pmol/L SD = 47.67) were higher than the normal range and associated with the need for surgery (p = 0.048). Surprisingly, copeptin and ADH levels negatively correlated (r = - 0.491; p < 0.001), potentially due to differences in degradation processes and physiological variations in TBI patients. CONCLUSION: Copeptin shows potential as a predictive biomarker for assessing TBI severity and predicting patient outcome. However, its complex relationship with ADH in TBI requires further investigation. Careful interpretation is needed due to potential variations in excretion dynamics and metabolism. Larger studies on TBI patient cohorts are essential to validate copeptin as a reliable biomarker and improve patient care in TBI.


Assuntos
Biomarcadores , Lesões Encefálicas Traumáticas , Glicopeptídeos , Humanos , Lesões Encefálicas Traumáticas/sangue , Lesões Encefálicas Traumáticas/diagnóstico , Glicopeptídeos/sangue , Biomarcadores/sangue , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Estudos Transversais , Idoso , Escala de Coma de Glasgow , Adulto Jovem , Vasopressinas/sangue , Adolescente
19.
ACS Biomater Sci Eng ; 10(8): 5168-5180, 2024 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-39016069

RESUMO

The radiation-induced skin injury (RISI) remains a great challenge for clinical wound management and care after radiotherapy, as patients will suffer from the acute radiation injury and long-term chronic inflammatory damage during the treatment. The excessive ROS in the early acute stage and prolonged inflammatory response in the late healing process always hinder therapeutic efficiency. Herein, we developed an extracellular matrix (ECM)-mimetic multifunctional glycopeptide hydrogel (oCP@As) to promote and accelerate RISI repair via a dual-modulation strategy in different healing stages. The oCP@As hydrogel not only can form an ECM-like nanofiber structure through the Schiff base reaction but also exhibits ROS scavenging and DNA double-strand break repair abilities, which can effectively reduce the acute radiation damage. Meanwhile, the introduction of oxidized chondroitin sulfate, which is the ECM polysaccharide-like component, enables regulation of the inflammatory response by adsorption of inflammatory factors, accelerating the repair of chronic inflammatory injury. The animal experiments demonstrated that oCP@As can significantly weaken RISI symptoms, promote epidermal tissue regeneration and angiogenesis, and reduce pro-inflammatory cytokine expression. Therefore, this multifunctional glycopeptide hydrogel dressing can effectively attenuate RISI symptoms and promote RISI healing, showing great potential for clinical applications in radiotherapy protection and repair.


Assuntos
Glicopeptídeos , Hidrogéis , Lesões por Radiação , Pele , Cicatrização , Animais , Hidrogéis/química , Hidrogéis/farmacologia , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Glicopeptídeos/uso terapêutico , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Pele/patologia , Lesões por Radiação/tratamento farmacológico , Lesões por Radiação/prevenção & controle , Cicatrização/efeitos dos fármacos , Camundongos , Humanos , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Masculino
20.
Anal Methods ; 16(30): 5304-5310, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39028021

RESUMO

Protein glycosylation plays a crucial role in various biological processes and is related to various diseases. Highly specific enrichment of glycopeptides before mass spectrometry detection is crucial for comprehensive glycoproteomic analysis. However, it still remains a great challenge due to the absence of affinity materials with excellent enrichment efficiency. In this work, a triazine structure linked by a -NH- bond of two-dimensional (2-D) covalent organic framework (COF) nanosheets was synthesized as an affinity adsorbent for the selective capture of glycopeptides. In particular, by introducing hydrophilic monomers via a bottom-up approach, the 2-D COF (denoted as NENP-1) nanosheets were provided with abundant amino groups and inherent hydrophilicity. Owing to the specific surface area and excessive accessible sites for hydrophilicity, the resulting NENP-1 nanosheets exhibited an outstanding glycopeptide enrichment efficiency from standard samples with a superior detection sensitivity (1 × 10-10 M), good enrichment selectivity (1 : 800, HRP tryptic digest to BSA protein), excellent binding capacity (100 mg g-1), great reusability, and recovery (60.2%). Furthermore, using the NENP-1 nanosheet adsorbent, twenty-four endogenous glycopeptides in the serum of patients with gastric cancer were successfully identified by LC-MS/MS technology, which illustrates a promising prospective of hydrophilic COF nanosheets in glycoproteomics research.


Assuntos
Glicopeptídeos , Interações Hidrofóbicas e Hidrofílicas , Nanoestruturas , Triazinas , Glicopeptídeos/química , Glicopeptídeos/sangue , Glicopeptídeos/análise , Humanos , Nanoestruturas/química , Triazinas/química , Glicosilação , Estruturas Metalorgânicas/química , Espectrometria de Massas em Tandem/métodos
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