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1.
Proteomics ; 24(5): e2300314, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38433404

RESUMO

A historic challenge for shotgun proteomics has been the requirement for high quality, simple, and nonredundant curated protein sequences in small .fasta text files. Due to the intrinsic informatic challenges and time required to assemble these files, proteomics has struggled to expand beyond the confines of a few model organisms. When considering post-translational modifications that may or may not be present on a specific peptide sequence, these factors inevitably compound. A study on how mangos continue to ripen on the shelf may not be the first thing you'd think of as proof of a scientific discipline shedding historic limitations. However, Bautiste-Valle et al., may be just that. These authors present a quantitative comparison of both peptide and glycopeptide alterations through the complexity of the fruit ripening process and in this we see the present state of a field that no longer needs to wait on genomics to obtain deep mechanistic insights.


Assuntos
Genômica , Glicopeptídeos , Sequência de Aminoácidos , Processamento de Proteína Pós-Traducional , Proteômica
2.
Nat Commun ; 15(1): 2448, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38503734

RESUMO

Deep learning has achieved a notable success in mass spectrometry-based proteomics and is now emerging in glycoproteomics. While various deep learning models can predict fragment mass spectra of peptides with good accuracy, they cannot cope with the non-linear glycan structure in an intact glycopeptide. Herein, we present DeepGlyco, a deep learning-based approach for the prediction of fragment spectra of intact glycopeptides. Our model adopts tree-structured long-short term memory networks to process the glycan moiety and a graph neural network architecture to incorporate potential fragmentation pathways of a specific glycan structure. This feature is beneficial to model explainability and differentiation ability of glycan structural isomers. We further demonstrate that predicted spectral libraries can be used for data-independent acquisition glycoproteomics as a supplement for library completeness. We expect that this work will provide a valuable deep learning resource for glycoproteomics.


Assuntos
Aprendizado Profundo , Espectrometria de Massas em Tandem , Glicopeptídeos/química , Proteômica , Polissacarídeos/química
3.
J Org Chem ; 89(5): 3390-3402, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38377557

RESUMO

The introduction of alkyne moieties into peptides remains in demand as it represents a promising approach for further structural diversification of peptides. Herein, we describe the Pd(II)-catalyzed C(sp3)-H alkynylation of Ala-Asn-embedded di- and tripeptides using Asn as the endogenous lead group. In addition, a key building block for the glycopeptide Tyc4PG-14 and Tyc4PG-15 was produced by our methodology.


Assuntos
Alanina , Alcinos , Glicopeptídeos , Catálise
4.
J Am Chem Soc ; 146(8): 5502-5510, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38359445

RESUMO

Glycoproteins account for numerous biological processes including those associated with diseases and infections. The advancement of glycopeptides has emerged as a promising strategy for unraveling biological pathways and discovering novel medicines. In this arena, a key challenge arises from the absence of efficient synthetic strategies to access glycopeptides and glycoproteins. Here, we present a highly concise approach to bridging saccharides with amino acids and peptides through an amide linkage. Our amide-linked C-glycosyl amino acids and peptides are synthesized through cooperative Ni-catalyzed and photoredox processes. The catalytic process generates a glycosyl radical and an amide carbonyl radical, which subsequently combine to yield the C-glycosyl products. The saccharide reaction partners encompass mono-, di-, and trisaccharides. All 20 natural amino acids, peptides, and their derivatives can efficiently undergo glycosylations with yields ranging from acceptable to high, demonstrating excellent stereoselectivities. As a substantial expansion of applications, we have shown that simple C-glycosyl amino acids can function as versatile building units for constructing C-glycopeptides with intricate spatial complexities.


Assuntos
Amidas , Aminoácidos , Níquel/química , Peptídeos , Carboidratos/química , Glicopeptídeos , Glicoproteínas , Catálise
5.
Anal Chim Acta ; 1294: 342309, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38336411

RESUMO

BACKGROUND: Glycopeptide antibiotics (GPAs) represented by vancomycin (VAN) are clinically used as a first-line treatment for serious infections caused by Gram-positive pathogens. The use and dosing methods of GPAs are rigorously managed for safety considerations, which calls for fast and accurate quantification approaches. RESULT: A new sort of fluorescent probes for GPAs has been proposed, each of which was integrated by a fluorescein-based reporter and a GPAs' recognition peptide D-alanyl-D-alanine (D-Ala-D-Ala). These probes work as dynamic molecular switches, which mainly exist as non-fluorescent spirolactam forms in the absence of GPAs. GPAs binding with the dipeptide regulates the dynamic balance between fluorescence OFF lactam form and fluorescence ON ring-opened form, rendering these probes capable of GPAs detecting. The most promising one P1 exhibits excellent sensitivity and selectivity towards GPAs detection. SIGNIFICANCE: Different to previous developments, P1 consists of a single fluorophore without the need of a fluorescence-quenching group or a secondary dye, which is the smallest fluorescent probe for GPAs up to now. P1 realizes direct VAN quantification from complex biological samples including real serums, dispensing with additional drug extraction. More interestingly, both P1 and P6 can distinguish GPAs with different peptide backbones, which has not been achieved previously.


Assuntos
Antibacterianos , Glicopeptídeos , Fluorescência , Antibacterianos/química , Glicopeptídeos/química , Vancomicina/química , Alanina
6.
Acta Crystallogr D Struct Biol ; 80(Pt 3): 181-193, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38372589

RESUMO

Low-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to the binding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reductases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future antituberculosis drug development.


Assuntos
Actinobacteria , Glicopeptídeos , Inositol , NADH NADPH Oxirredutases , Oxirredutases , Oxirredutases/metabolismo , Compostos de Sulfidrila/química , Cisteína/química , Cisteína/metabolismo , Oxirredução
7.
Int J Cardiol ; 403: 131879, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38369132

RESUMO

BACKGROUND: The rapid and reliable differentiation of myocardial infarction (MI) due to atherothrombosis (T1MI) from MI due to supply-demand mismatch (T2MI) or acute myocardial injury is of major clinical relevance due to very different treatments, but still a major unmet clinical need. This study aimed to investigate whether copeptin, a stress hormone produced in the hypothalamus, helps to differentiate between T1MI versus T2MI or injury. METHODS: In a retrospective analysis, 1271 unselected consecutive patients presenting with symptoms suggestive of MI to the emergency department were evaluated. Patients diagnosed with ST-elevation MI were excluded. All patients with elevated cardiac troponin I (cTnI) concentration possibly indicating MI were classified into T1MI, T2MI, or acute myocardial injury using detailed clinical assessment and coronary imaging. Copeptin plasma concentration was measured in a blinded fashion. A multicenter diagnostic study with central adjudication of the final diagnosis served as external validation cohort (n = 1390). RESULTS: Among 1161 patients, 154 patients had increased cTnI concentration. Of these, 78 patients (51%) were classified as T1MI and 76 (49%) as T2MI or myocardial injury. Patients with T2MI or myocardial injury had significantly higher copeptin plasma concentration between patients versus T1MI (21,4 pmol/l versus 8,1 pmol/l, p = 0,001). A multivariable regression analysis revealed that higher concentrations of copeptin and C-reactive protein, higher heart rate at presentation and lower frequency of smoking remained significantly associated with T2MI and myocardial injury. Findings were largely confirmed in the external validation cohort. CONCLUSION: In patients without ST-segment elevation, copeptin concentration was higher in T2MI and myocardial Injury versus T1MI and may help in their differential diagnosis.


Assuntos
Infarto Miocárdico de Parede Anterior , Glicopeptídeos , Traumatismos Cardíacos , Infarto do Miocárdio , Humanos , Estudos Retrospectivos , Infarto do Miocárdio/terapia , Infarto Miocárdico de Parede Anterior/complicações , Troponina I , Biomarcadores
8.
Sci Rep ; 14(1): 3716, 2024 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-38355753

RESUMO

Glycoproteins in urine have the potential to provide a rich class of informative molecules for studying human health and disease. Despite this promise, the urine glycoproteome has been largely uncharacterized. Here, we present the analysis of glycoproteins in human urine using LC-MS/MS-based intact glycopeptide analysis, providing both the identification of protein glycosites and characterization of the glycan composition at specific glycosites. Gene enrichment analysis reveals differences in biological processes, cellular components, and molecular functions in the urine glycoproteome versus the urine proteome, as well as differences based on the major glycan class observed on proteins. Meta-heterogeneity of glycosylation is examined on proteins to determine the variation in glycosylation across multiple sites of a given protein with specific examples of individual sites differing from the glycosylation trends in the overall protein. Taken together, this dataset represents a potentially valuable resource as a baseline characterization of glycoproteins in human urine for future urine glycoproteomics studies.


Assuntos
Glicopeptídeos , Espectrometria de Massas em Tandem , Humanos , Glicopeptídeos/química , Cromatografia Líquida , Glicoproteínas/metabolismo , Proteoma/química , Polissacarídeos/química
9.
Rapid Commun Mass Spectrom ; 38(5): e9690, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38355883

RESUMO

RATIONALE: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. METHODS: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. RESULTS: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. CONCLUSION: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.


Assuntos
COVID-19 , Glicopeptídeos , Humanos , Glicopeptídeos/química , Glicoproteína da Espícula de Coronavírus , Espectrometria de Massas em Tandem/métodos , Deutério , SARS-CoV-2 , Asparagina , Glicoproteínas/química , Polissacarídeos , Íons
10.
BMC Pediatr ; 24(1): 140, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402172

RESUMO

BACKGROUND: Using Zonulin and Copeptin as potential obesity markers in children, hasn't yet been focused. AIM: To evaluate the association between serum levels of both Zonulin and Copeptin with the obesity markers, and to assess their role as metabolic disturbance predictors in obese children. METHODS: A case-control study comprised 111 Egyptian children (45 males and 66 females); aged 6-10 years to avoid the effect of puberty (prepubertal). They were classified according to their body mass index (BMI) percentiles into: 72 obese (BMI ≥ 95th ), and 39 control ones (BMI > 15th - <85th ), based on the Egyptian Growth Charts for children and adolescents. Anthropometric parameters and blood pressure were measured, and body composition analysis, lipid profile, Zonulin, and Copeptin levels were assessed. RESULTS: The obese group showed a significantly higher value of Copeptin and a lower value of Zonulin than the control one Also, the obese group showed significant negative correlations between Zonulin and both anthropometric obesity markers and body composition, whereas Copeptin showed significant positive ones. Moreover, significant positive correlations were found between Copeptin and both body weight and fat distribution. Insignificant correlations were observed between both serum Zonulin and Copeptin levels and blood pressure and lipid profile. CONCLUSION: Zonulin and Copeptin cannot be used as metabolic disturbance predictors, among Egyptian children, as they were insignificantly correlated with lipid profile or blood pressure.


Assuntos
Glicopeptídeos , Haptoglobinas , Obesidade Pediátrica , Precursores de Proteínas , Masculino , Feminino , Adolescente , Humanos , Criança , Estudos de Casos e Controles , Índice de Massa Corporal , Lipídeos
11.
Adv Exp Med Biol ; 1443: 23-32, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38409414

RESUMO

Protein glycosylation is a post-translational modification involving the addition of carbohydrates to proteins and plays a crucial role in protein folding and various biological processes such as cell recognition, differentiation, and immune response. The vast array of natural sugars available allows the generation of plenty of unique glycan structures in proteins, adding complexity to the regulation and biological functions of glycans. The diversity is further increased by enzymatic site preferences and stereochemical conjugation, leading to an immense amount of different glycan structures. Understanding glycosylation heterogeneity is vital for unraveling the impact of glycans on different biological functions. Evaluating site occupancies and structural heterogeneity aids in comprehending glycan-related alterations in biological processes. Several software tools are available for large-scale glycoproteomics studies; however, integrating identification and quantitative data to assess heterogeneity complexity often requires extensive manual data processing. To address this challenge, we present a python script that automates the integration of Byonic and MaxQuant outputs for glycoproteomic data analysis. The script enables the calculation of site occupancy percentages by glycans and facilitates the comparison of glycan structures and site occupancies between two groups. This automated tool offers researchers a means to organize and interpret their high-throughput quantitative glycoproteomic data effectively.


Assuntos
Glicopeptídeos , Espectrometria de Massas em Tandem , Software , Glicosilação , Polissacarídeos/química
12.
Biomater Sci ; 12(6): 1490-1501, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38329387

RESUMO

Cross-presentation, exogenous antigen presentation onto major histocompatibility complex class I molecules on antigen presenting cells, is crucially important for inducing antigen-specific cellular immune responses for cancer immunotherapy and for the treatment of infectious diseases. One strategy to induce cross-presentation is cytosolic delivery of an exogenous antigen using fusogenic or endosomolytic molecule-introduced nanocarriers. Earlier, we reported liposomes modified with pH-responsive polymers to achieve cytosolic delivery of an antigen. Polyglycidol-based or polysaccharide-based pH-responsive polymers can provide liposomes with delivery performance of antigenic proteins into cytosol via membrane fusion with endosomes responding to acidic pH, leading to induction of cross-presentation. Mannose residue was introduced to pH-responsive polysaccharides to increase uptake selectivity to antigen presenting cells and to improve cross-presentation efficiency. However, direct introduction of mannose residue into pH-responsive polysaccharides suppressed cytoplasmic delivery performance of liposomes. To avoid such interference, for this study, mannose-containing glycans were incorporated separately into pH-responsive polysaccharide-modified liposomes. Soybean agglutinin-derived glycopeptide was used as a ligand for lectins on antigen presenting cells. Incorporation of glycopeptide significantly increased the cellular uptake of liposomes by dendritic cell lines and increased cross-presentation efficiency. Liposomes incorporated both glycopeptide and pH-responsive polysaccharides exhibited strong adjuvant effects in vitro and induced the increase of dendritic cells, M1 macrophages, and effector T cells in the spleen. Subcutaneous administration of these liposomes induced antigen-specific cellular immunity, resulting in strong therapeutic effects in tumor-bearing mice. These results suggest that separate incorporation of glycopeptides and pH-responsive polysaccharides into antigen-loaded liposomes is an effective strategy to produce liposome-based nanovaccines to achieve antigen cross-presentation and induction of cellular immunity towards cancer immunotherapy.


Assuntos
Lipossomos , Neoplasias , Animais , Camundongos , Lipossomos/química , Apresentação de Antígeno , Apresentação Cruzada , Glicopeptídeos/farmacologia , Manose/farmacologia , Antígenos/química , Neoplasias/terapia , Polímeros/química , Concentração de Íons de Hidrogênio , Polissacarídeos/química , Células Dendríticas , Camundongos Endogâmicos C57BL
13.
Hipertens Riesgo Vasc ; 41(1): 35-39, 2024.
Artigo em Espanhol | MEDLINE | ID: mdl-38388322

RESUMO

Preeclampsia represents a specific complication of pregnancy hypertension, which appears de novo after the 20th week of gestation, accompanied by proteinuria and/or maternal or utero-placental organ dysfunction. Despite an uncertain etiopathogenesis, impaired vascular remodeling of the spiral artery and placental ischemia is the most widespread hypothesis. The finding of elevated levels of copeptin in women with preeclampsia compared to normal pregnant women has valued the involvement of arginine vasopressin in the etiopathogenesis of this complication. In this paper, its usefulness as a marker of preeclampsia is considered through the review of the main studies carried out with this molecule.


Assuntos
Glicopeptídeos , Pré-Eclâmpsia , Feminino , Gravidez , Humanos , Arginina Vasopressina , Placenta , Vasopressinas , Arginina
14.
J Control Release ; 367: 540-556, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38301927

RESUMO

Cancer presents a high mortality rate due to ineffective treatments and tumour relapse with progression. Cancer vaccines hold tremendous potential due to their capability to eradicate tumour and prevent relapse. In this study, we present a novel glycovaccine for precise targeting and immunotherapy of aggressive solid tumours that overexpress CD44 standard isoform (CD44s) carrying immature Tn and sialyl-Tn (sTn) O-glycans. We describe an enzymatic method and an enrichment strategy to generate libraries of well-characterized cancer-specific CD44s-Tn and/or sTn glycoproteoforms, which mimic the heterogeneity found in tumours. We conjugated CD44-Tn-derived glycopeptides with carrier proteins making them more immunogenic, with further demonstration of the importance of this conjugation to overcome the glycopeptides' intrinsic toxicity. We have optimized the glycopeptide-protein maleimide-thiol conjugation chemistry to avoid undesirable cross-linking between carrier proteins and CD44s glycopeptides. The resulting glycovaccines candidates were well-tolerated in vivo, inducing both humoral and cellular immunity, including immunological memory. The generated antibodies exhibited specific reactivity against synthetic CD44s-Tn glycopeptides, CD44s-Tn glycoengineered cells, and human tumours. In summary, we present a promising prototype of a cancer glycovaccine for future therapeutical pre-clinical efficacy validation.


Assuntos
Vacinas Anticâncer , Neoplasias , Humanos , Vacinas Combinadas , Antígenos Glicosídicos Associados a Tumores/química , Glicoconjugados , Neoplasias/terapia , Imunoterapia , Glicopeptídeos/química , Proteínas de Transporte , Recidiva , Receptores de Hialuronatos
15.
Anal Bioanal Chem ; 416(8): 1867-1881, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38349535

RESUMO

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.


Assuntos
Formiatos , Glicopeptídeos , Metanol , Humanos , Glicopeptídeos/química , 2-Propanol , Cromatografia Líquida/métodos , Solventes , Imunoglobulina G/química , Interações Hidrofóbicas e Hidrofílicas , Extração em Fase Sólida/métodos , Acetonitrilas , Acetatos
16.
Methods Mol Biol ; 2763: 187-199, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38347411

RESUMO

Mucins are sugar-rich glycoproteins. Glycoprotein sugar moieties are structurally diverse, making it difficult to obtain naturally pure glycoproteins. Chemical synthesis is a powerful tool for obtaining target or designed compounds. Automated peptide synthesizers are commercially available, and many use the solid-phase peptide synthesis (SPPS) method. In addition, some of these synthesizers apply microwave irradiation to obtain higher reaction yields, thereby enabling the synthesis of 40 to 50 amino acid residual glycopeptides. Theoretically, glycopeptides can be synthesized using methods similar to those used for peptide synthesis, but glycosylated amino acid synthons are less stable than amino acid synthons and are also very expensive. Therefore, they are not suitable for use in large excess amounts. Many of oligosaccharide-linked amino acid synthons are not commercially available, so they must be specially prepared, and they also require careful handling that demands specific organic synthesis experience and techniques. However, monosaccharide-linked amino acid synthons are commercially available and are relatively easy to handle. Here, as an entry into glycopeptide synthesis, we describe a typical glycopeptide synthesis procedure for a 27 amino acid residual MUC1 repeating unit with monosaccharides.


Assuntos
Glicopeptídeos , Mucinas , Mucinas/química , Glicopeptídeos/química , Mucina-1 , Carboidratos/química , Glicoproteínas , Técnicas de Química Sintética , Açúcares , Aminoácidos/química
17.
Methods Mol Biol ; 2763: 321-327, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38347421

RESUMO

O-Linked glycans potentially play a functional role in cellular recognition events. Recent structural analyses suggest that O-glycosylation can be a specific signal for a lectin receptor which recognizes both the O-glycan and the adjacent polypeptide region. Further, certain antibodies specifically bind to the O-glycosylated peptide. There is growing interest in the mechanism by which O-glycans on proteins are specifically recognized by lectins and antibodies. The recognition system may be common to many O-glycosylated proteins; however, there is limited 3D structural information on the dual recognition of glycan and protein. This chapter describes a solution NMR analysis of the interaction between MUC1 O-glycopeptide and anti-MUC1 antibody MY.1E12.


Assuntos
Glicopeptídeos , Mucina-1 , Glicopeptídeos/química , Anticorpos , Peptídeos , Lectinas , Polissacarídeos/química
18.
Methods Mol Biol ; 2763: 373-379, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38347427

RESUMO

Advances in computer performance and computational simulations allow increasing sophistication in applications in biological systems. Molecular dynamics (MD) simulations are especially suitable for studying conformation, dynamics, and interaction of flexible biomolecules such as free glycans and glycopeptides. Computer simulations are best performed when the scope and limitations in performance have been thoroughly assessed. Proper outputs are obtained only under suitable parameter settings, and results need to be properly validated. In this chapter, we will introduce an example of molecular dynamics simulations of MUC1 O-glycopeptide and its docking to anti-MUC1 antibody Fv fragment.


Assuntos
Simulação de Dinâmica Molecular , Mucina-1 , Anticorpos Monoclonais , Glicopeptídeos/química , Conformação Molecular , Simulação de Acoplamento Molecular
19.
Methods Mol Biol ; 2762: 267-280, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38315371

RESUMO

Glycosylation of proteins is an important post-translational modification that plays a role in a wide range of biological processes, including immune response, intercellular signaling, inflammation, and host-pathogen interaction. Abnormal protein glycosylation has been correlated with various diseases. However, the study of protein glycosylation remains challenging due to its low abundance, microheterogeneity of glycosylation sites, and low ionization efficiency. During the past decade, several methods for enrichment and for isolation of glycopeptides from biological samples have been developed and successfully employed in glycoproteomics research. In this chapter, we discuss the sample preparation protocol and the strategies for effectively isolating and enriching glycopeptides from biological samples, using PolyHYDROXYETHYL A as a hydrophilic interaction liquid chromatography (HILIC) enrichment technique.


Assuntos
Glicopeptídeos , Processamento de Proteína Pós-Traducional , Glicopeptídeos/análise , Cromatografia Líquida/métodos , Glicosilação , Interações Hidrofóbicas e Hidrofílicas
20.
Methods Mol Biol ; 2762: 281-290, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38315372

RESUMO

Glycosylation refers to the biological processes that covalently attach carbohydrates to the peptide backbone after the synthesis of proteins. As one of the most common post-translational modifications (PTMs), glycosylation can greatly affect proteins' features and functions. Moreover, aberrant glycosylation has been linked to various diseases. There are two major types of glycosylation, known as N-linked and O-linked glycosylation. Here, we focus on O-linked glycosylation and thoroughly describe a bottom-up strategy to perform O-linked glycoproteomics studies. The experimental section involves enzymatic digestions using trypsin and O-glycoprotease at 37 °C. The prepared samples containing O-glycopeptides are analyzed using nanoHPLC coupled with tandem mass spectrometry (MS) for accurate identification and quantification.


Assuntos
Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Glicosilação , Peptídeos/metabolismo , Glicopeptídeos/química
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