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1.
Food Chem ; 306: 125613, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31610331

RESUMO

Reduction of bitter taste in protein hydrolysates is a challenging task. The aim of this study was to apply a simple two-step approach to prepare low bitter hydrolysates and investigate the influence of peptide modifications on taste characteristics. Protein hydrolysates were prepared from porcine muscle and plasma through simultaneous hydrolysis using endo- and exo-peptidases combined with peptide glycation by glucosamine (GlcN). Spectroscopic analysis and quantification of major alpha-dicarbonyl compounds (α-DCs) indicated the relatively low extent of Maillard reaction in GlcN-glycated protein hydrolysates. Thermal degradation of high MW peptides (>10 kDa) might play a major role in Maillard reaction, reflected by the formation of more Maillard reacted peptides (1-5 kDa), especially in plasma samples. Sensory evaluation indicated that glycation by GlcN can alter taste profiles of protein hydrolysates, which may be attributed to the formation of Maillard reacted peptides and peptide modifications revealed by LC-MS/MS analysis.


Assuntos
Exopeptidases/química , Músculo Esquelético/química , Paladar , Animais , Aspergillus oryzae/enzimologia , Exopeptidases/metabolismo , Glucosamina/química , Glucosamina/metabolismo , Glicosilação , Hidrólise , Reação de Maillard , Músculo Esquelético/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Hidrolisados de Proteína/química , Suínos
2.
Food Chem ; 306: 125615, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31622833

RESUMO

Phycocyanin (PC), a plant-based protein with interesting biological activity, is rarely directly applied in the food industry because it has structural and functional limitations. This study combined ultra-high-pressure (UHP) treatment with glycation to improve PC functionality and explored resulting structural changes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, scanning electron microscopy, Fourier-transform infrared spectroscopy, circular dichroism, and UV-visible spectroscopy. The UHP treatment obviously improved the speed and degree of glycation and the composite-modified PC (CM-PC) showed high solubility and good emulsifying and foaming performance. Scanning electron microscopy images showed the CM-PC surface was loose and fluffy. Gel electrophoresis, Fourier-transform infrared spectroscopy, and circular dichroism results demonstrated that the content of α-helix decreased from 78.1% in PC to 26.6% in CM-PC, and hydroxyl groups were introduced. UV-visible spectroscopy showed that the mechanism of composite modification involved stretching of the PC and promotion of binding with sugars.


Assuntos
Ficocianina/química , Spirulina/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Emulsões/metabolismo , Glicosilação , Pressão , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier
3.
J Sci Food Agric ; 100(2): 607-613, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31591730

RESUMO

BACKGROUND: α-Dicarbonyl compounds are widely generated in the Maillard reaction, caramelization and oil oxidation during heat treatment. These compounds can readily react with lysine and arginine residues of a protein, whereas the influence of these compounds on protein structure and quality has seldom been revealed. This study compared influence of glycation by glucose and α-dicarbonyl compounds on amyloid-like aggregation of ß-lactoglobulin (ß-LG), both fibrillation kinetics and conformation of aggregates were studied. RESULTS: Compared with glycation by glucose, the glycation by α-dicarbonyl compounds resulted in faster reduction of free amino group, sulfydryl group, and the relative content of ß-sheet secondary structure, according to the ultraviolet (UV) spectra or circular dichroism (CD) spectra results. Based on the analysis of fibrillation kinetics using thioflavin T (ThT) binding assay, the glycation by α-dicarbonyls were more efficient in suppressing the growth of fibrillar aggregates. In addition, glycation by α-dicarbonyl resulted in amorphous oligomers, which were compared with the amyloid-like aggregates in control and glucose-glycated samples, based on the transmission electron microscopy (TEM) observation. CONCLUSIONS: Glycation by α-dicarbonyl compounds induced larger decline in the ß-sheet structure of ß-LG than glycation by glucose, and thus largely suppressed the amyloid-like aggregation of ß-LG and changed the morphology of aggregates. © 2019 Society of Chemical Industry.


Assuntos
Amiloide/química , Lactoglobulinas/química , Animais , Bovinos , Dicroísmo Circular , Glucose/química , Glicosilação , Temperatura Alta , Agregados Proteicos , Estrutura Secundária de Proteína
4.
Biochemistry (Mosc) ; 84(11): 1268-1279, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760917

RESUMO

The review describes the use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitors to study the enzyme and to suppress its activity in various cell types. The main problem of selective GAPDH inhibition is a highly conserved nature of the enzyme active site and, especially, Cys150 environment important for the catalytic action of cysteine sulfhydryl group. Numerous attempts to find specific inhibitors of sperm GAPDH and enzymes from Trypanosoma sp. and Mycobacterium tuberculosis that would not inhibit GAPDH of somatic mammalian cells have failed, which has pushed researchers to search for new ways to solve this problem. The sections of the review are devoted to the studies of GAPDH inactivation by reactive oxygen species, glutathione, and glycating agents. The final section discusses possible effects of GAPDH inhibition and inactivation on glycolysis and related metabolic pathways (pentose phosphate pathway, uncoupling of the glycolytic oxidation and phosphorylation, etc.).


Assuntos
Inibidores Enzimáticos/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Inibidores Enzimáticos/metabolismo , Glutationa/química , Glutationa/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Glicosilação , Mycobacterium tuberculosis/enzimologia , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma/enzimologia
5.
Chem Pharm Bull (Tokyo) ; 67(10): 1072-1075, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582627

RESUMO

Shikonin, a natural naphthoquinone, has attracted much attention due to its various biological activities. Two shikonin glucosides, shikonin-1',8-di-O-ß-D-glucopyranoside (1) and shikonin-1'-O-ß-D-glucopyranoside (2), were biosynthesized through in vitro enzymatic glycosylation and their structures were elucidated using spectroscopic techniques. The water-solubility and stability of compounds 1 and 2 were significantly higher than those of the parent compound. Furthermore, compound 2 showed moderate cytotoxicity against six cancer cell lines, with IC50 values ranging from 36.10 to 67.47 µM. This research indicated that in vitro enzymatic glycosylation of shikonin is an effective strategy to improve it water solubility and chemical stability.


Assuntos
Antineoplásicos/metabolismo , Glucosídeos/biossíntese , Glicosiltransferases/metabolismo , Naftoquinonas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glucosídeos/química , Glucosídeos/farmacologia , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Solubilidade , Relação Estrutura-Atividade , Temperatura Ambiente
6.
Se Pu ; 37(8): 845-852, 2019 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642255

RESUMO

The study of protein glycosylation is important to deepening the current understanding of its biological functions as well as to elucidate novel biomarkers of glycosylation. However, glycopeptides must be enriched prior to analysis due to their naturally low abundance. In this study, tryptophan functionalized polymer materials (denoted as Poly-Trp) were synthesized to serve as a novel biomimetic polymers. The resulting materials were characterized by various methods including scanning electron microscopy, thermal analysis, and infrared spectrometry, validating the successful synthesis of Poly-Trp. The retention of glycopeptides on Poly-Trp was investigated revealing that the retention ability decreased as the acetonitrile content of the mobile phase decreased and its acidity increased. Poly-Trp exhibited higher enrichment selectivity for glycopeptides from bovine fetuin than the commercial material ZIC-HILIC, and aminated silica which could defect the interference of 100-fold amount of substance ratios of bovine serum albumin. Poly-Trp is expected to have further applications in the purification of biological samples for the study of glycosylation.


Assuntos
Biomimética , Glicopeptídeos/química , Polímeros/química , Animais , Bovinos , Fetuínas/química , Glicosilação , Dióxido de Silício
7.
Chem Commun (Camb) ; 55(91): 13741-13744, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31661093

RESUMO

Natural glycopeptides have been shown to possess interesting biological activities. In this work, we have developed a general solid-phase approach to C-terminal glycopeptides. Taking advantage of oxime resin ester bond nucleophile susceptibility, we optimised the nucleophilic cleavage step with glycosylamines and demonstrated the generality and scope of this method. In addition, this reaction has high functional group tolerance and can be used for the preparation of longer C-terminal glycopeptides, demonstrated with the synthesis of a glycododecapeptide in one single step. The results pave the way to access efficiently novel medically relevant compounds.


Assuntos
Glicopeptídeos/química , Oximas/química , Antígenos Glicosídicos Associados a Tumores/química , Biomarcadores Tumorais/síntese química , Biomarcadores Tumorais/química , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/química , Glicopeptídeos/síntese química , Glicosilação , Humanos
8.
BMC Bioinformatics ; 20(1): 513, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640540

RESUMO

BACKGROUND: Carbohydrates are a class of large and diverse biomolecules, ranging from a simple monosaccharide to large multi-branching glycan structures. The covalent linkage of a carbohydrate to the nitrogen atom of an asparagine, a process referred to as N-linked glycosylation, plays an important role in the physiology of many living organisms. Most software for glycan modeling on a personal desktop computer requires knowledge of molecular dynamics to interface with specialized programs such as CHARMM or AMBER. There are a number of popular web-based tools that are available for modeling glycans (e.g., GLYCAM-WEB (http:// https://dev.glycam.org/gp/ ) or Glycosciences.db ( http://www.glycosciences.de/ )). However, these web-based tools are generally limited to a few canonical glycan conformations and do not allow the user to incorporate glycan modeling into their protein structure modeling workflow. RESULTS: Here, we present Glycosylator, a Python framework for the identification, modeling and modification of glycans in protein structure that can be used directly in a Python script through its application programming interface (API) or through its graphical user interface (GUI). The GUI provides a straightforward two-dimensional (2D) rendering of a glycoprotein that allows for a quick visual inspection of the glycosylation state of all the sequons on a protein structure. Modeled glycans can be further refined by a genetic algorithm for removing clashes and sampling alternative conformations. Glycosylator can also identify specific three-dimensional (3D) glycans on a protein structure using a library of predefined templates. CONCLUSIONS: Glycosylator was used to generate models of glycosylated protein without steric clashes. Since the molecular topology is based on the CHARMM force field, new complex sugar moieties can be generated without modifying the internals of the code. Glycosylator provides more functionality for analyzing and modeling glycans than any other available software or webserver at present. Glycosylator will be a valuable tool for the glycoinformatics and biomolecular modeling communities.


Assuntos
Glicoproteínas/química , Simulação de Dinâmica Molecular , Polissacarídeos/química , Software , Glicosilação
9.
J Agric Food Chem ; 67(42): 11752-11757, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31554403

RESUMO

Resveratrol (3,5,4'-trihydroxystilbene) is one of the most abundant polyphenols in red grapes, and red wine represents one of the most important dietary sources of this compound. Although its beneficial properties on human health have been widely investigated over the last 30 years, very little is known about its derivatives. Resveratrol can indeed undergo glycosylation, oligomerization and, upon UV-light exposure, it can isomerize from the trans-to the cis-isomer, which can further cyclize to 2,4,6-trihydroxyphenanthrene (THP). Although the effects of THP on human health are not yet known, being a polycyclic aromatic hydrocarbon, it can be potentially harmful. Because no data about THP occurrence in plant food and beverages are available, a simple procedure based on liquid-liquid extraction and gas chromatography-mass spectrometry has been developed and validated for the simultaneous qualitative and quantitative analysis of trans-resveratrol, cis-resveratrol, and THP in red wine, before and after UV-light exposure.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Fenantrenos/química , Resveratrol/química , Vinho/análise , Vinho/efeitos da radiação , Glicosilação , Isomerismo , Raios Ultravioleta
10.
J Agric Food Chem ; 67(42): 11694-11702, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31558015

RESUMO

Sucrose synthase (SUS) plays an important role in carbohydrate metabolism in plants. The SUS genes in licorice remain unknown. To reveal the sucrose metabolic pathway in licorice, all the 12 putative SUS genes of Glycyrrhiza uralensis were systematically identified by genome mining, and two novel SUSs (GuSUS1 and GuSUS2) were isolated and characterized for the first time. Furthermore, we found that the flexible N-terminus was responsible for the low stability of plant SUSs, and deletion of redundant N-terminus improved the stability of GuSUS1 and GuSUS2. The half-life of both GuSUS1 and GuSUS2 mutants was increased by 2-fold. Finally, the GuSUS1 mutant was coupled with UGT73C11 for the glycosylation of glycyrrhetinic acid (GA) with uridine 5'-diphosphate disodium salt hydrate (UDP) in situ recycling, and GA conversion was increased by 7-fold. Our study not only identified the SUS genes in licorice but also provided a stable SUS mutant for the construction of an efficient UDP-recycling system for GA glycosylation.


Assuntos
Glucosiltransferases/metabolismo , Glycyrrhiza uralensis/enzimologia , Proteínas de Plantas/metabolismo , Difosfato de Uridina/metabolismo , Biocatálise , Glucosiltransferases/química , Glucosiltransferases/genética , Glicosilação , Ácido Glicirretínico/metabolismo , Glycyrrhiza uralensis/química , Glycyrrhiza uralensis/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Difosfato de Uridina/química
11.
J Agric Food Chem ; 67(38): 10774-10781, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31479258

RESUMO

The released milk N-glycome has been found to possess antipathogenic activity. Natively, they are covalently linked onto proteins. Whether the conjugated N-glycans still have antipathogenic properties and how the glycosylation influences the antipathogenic activity of proteins remain unclear. Herein, we compared the quantitative differences of milk protein N-glycosylation and the antilisterial differences of native milk proteins, released N-glycan pools, and deglycosylated proteins between human and bovine milk. N-glycosylation exhibited to be quantitatively species-specific. The entire growth inhibitory activity and the majority of the antiadhesive activity against Listeria monocytogenes of milk whey proteins, although not as high as the released N-glycans, are attributed to N-glycosylation. Moreover, all N-glycan-bearing samples from human milk showed better growth inhibitory activities than those from bovine milk. Generally, N-glycosylation significantly contributes to the antilisterial function of milk proteins and to the functional differences between species. This gives novel insights into the role of these glycoconjugates in nature.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Proteínas do Leite/química , Proteínas do Leite/farmacologia , Leite Humano/química , Animais , Aderência Bacteriana/efeitos dos fármacos , Células CACO-2 , Bovinos , Glicosilação , Humanos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/fisiologia , Polissacarídeos/química , Especificidade da Espécie
12.
Adv Exp Med Biol ; 1148: 1-24, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482492

RESUMO

The use of therapeutic enzymes embraces currently a vast array of applications, abridging from diggestive disorders to cancer therapy, cardiovascular and lysosomal storage diseases. Enzyme drugs bind and act on their targets with great affinity and specificity, converting substrates to desired products in a reduced time frame with minimal side reactions. These characteristics have resulted in the development of a multitude of enzyme biopharmaceuticals for a wide range of human disorders.The advances in genetic engineering and DNA recombination techniques facilitated the production of therapeutical human-like enzymes, using different cells as host organisms. The selection of hosts generally privileges those that secrete the enzyme into the culture medium, as this eases the purification process, and those that are able to express complex glycoproteins, with glycosylation patterns and other post-translational modifications close to human proteins. Moreover, engineering approaches such as pegylation, encapsulation in micro- and nanocarriers, and mutation of amino acid residues of the native enzyme molecule to yield variants with improved therapeutic activity, half-life and/or stability, have been also addressed. Engineered enzyme products have been designed to display enhanced delivery to target sites and reduced adverse side-effects (e.g., immunogenicity) upon continuous drug administration.Irrespectively of the production method, the final formulation of therapeutic enzymes must display high purity and specificity, and they are often marketed as lyophilized pure preparations with biocompatible buffering salts and diluents to prepare the reconstituted aqueous solution before treatment.


Assuntos
Enzimas/biossíntese , Enzimas/isolamento & purificação , Enzimas/farmacologia , Produtos Biológicos , Meios de Cultura , Engenharia Genética , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes
13.
Cell Biochem Biophys ; 77(4): 335-342, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31489526

RESUMO

Alpha-2-macroglobulin (A2M) is a glycosylated broad spectrum inhibitor of numerous proteases, including those involved in blood coagulation. Glycosylation characteristics can affect protein structure and function. This study compares glycosylation characteristics of A2M in newborn umbilical cord (NUCP) and adult pooled plasmas. Peptide N-Glycosidase F treatment was used to evaluate the total N-glycan content of the molecules. Neuraminidase treatment, and affinity for Ricinus Communis Agglutinin I were used to examine terminal sialic acid and galactose content, respectively. Two-dimensional (2D) electrophoresis was used to determine charge-related isoform profiles and fluorophore-assisted carbohydrate electrophoresis (FACE) was used to characterize N-glycan profiles. Results revealed no difference in total N-glycan mass, however, a statistically significant difference was shown in the change in charge associated with sialic acid loss in the NUCP A2M population. 2D electrophoresis indicated a lower pI range for NUCP A2M isoforms. In addition, NUCP A2M displayed a trend toward higher terminal galactose quantities than adult A2M. FACE revealed an increased abundance of more branched, higher molecular weight glycans in NUCP A2M. These differences in glycan branching and charged residues may impact A2M receptor-based clearance and thus could be responsible for the increased A2M concentration seen in NUCP, and newborns.


Assuntos
Envelhecimento , Polissacarídeos/análise , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Adulto , Eletroforese em Gel Bidimensional , Glicosilação , Humanos , Recém-Nascido , alfa 2-Macroglobulinas Associadas à Gravidez/análise , Isoformas de Proteínas/metabolismo , Cordão Umbilical/metabolismo
14.
J Agric Food Chem ; 67(38): 10744-10755, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31525900

RESUMO

We previously reported that ß-glucosidase BGL1 at low concentration (15 µg mL-1) from Coprinopsis cinerea exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 µg mL-1) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 µg mL-1) showed transglycosylation but BGL1 at high concentration (200 µg mL-1) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a ß(1 → 3) or ß(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-ß-d-gentiobiosyl-d-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.


Assuntos
Agaricales/enzimologia , Dissacarídeos/metabolismo , Proteínas Fúngicas/química , Oligossacarídeos/metabolismo , beta-Glucosidase/química , Agaricales/química , Agaricales/genética , Sequência de Aminoácidos , Dissacarídeos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Hidrólise , Cinética , Estrutura Molecular , Oligossacarídeos/química , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
15.
J Agric Food Chem ; 67(41): 11307-11311, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31514500

RESUMO

Current reports increasingly associate dietary "advanced glycation end products" ("AGEs") resulting from the Maillard reaction (glycation) between reducing sugars and amino compounds in foods with pathophysiological consequences, such as chronic inflammation, atherosclerosis, and metabolic syndrome. Heated foods are therefore suggested to pose a potential risk for human health. However, studies in this field are very often based on questionable quantitative data and inadequate structural characterization. To improve the situation, the present perspective suggests quality criteria for future studies and the assessment of the currently available literature.


Assuntos
Alimentos/efeitos adversos , Produtos Finais de Glicação Avançada/metabolismo , Animais , Culinária , Dieta , Produtos Finais de Glicação Avançada/efeitos adversos , Produtos Finais de Glicação Avançada/química , Glicosilação , Saúde , Humanos , Reação de Maillard
16.
J Agric Food Chem ; 67(38): 10702-10712, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31490688

RESUMO

Human milk oligosaccharides are complex carbohydrates with multibiofunctional health benefits to newborns. Human milk free oligosaccharides (HMOs) are well characterized. However, changes in the N/O-glycome during lactation are poorly reported. Herein, we qualitatively and quantitatively investigated N/O-glycome profiles and their alteration in human milk at different lactation stages. N-Glycans were mainly fucosylated and nonsialylated, nonfucosylated throughout lactation. O-Glycans mainly consisted of sialylated and nonsialylated, nonfucosylated in colostrum and transitional milk, and fucosylated and nonfucosylated, nonsialylated in mature milk. Fucosylated and sialylated N-glycans gradually decreased and increased, respectively, as lactation progressed; O-glycans showed the reverse. Interestingly, changes in HMO abundance decreased during lactation, complementing HMG N/O-glycome changes. In conclusion, temporal HMG glycosylation changes provide the groundwork for developing infant formula that is closer to breast milk at different lactation stages.


Assuntos
Glicoproteínas/química , Lactação , Leite Humano/química , Adulto , Colostro/química , Feminino , Glicoproteínas/metabolismo , Glicosilação , Humanos , Espectrometria de Massas , Leite Humano/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
17.
Nat Methods ; 16(9): 902-910, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31384044

RESUMO

We report a liquid chromatography coupled to tandem mass spectrometry O-glycoproteomics strategy using data-independent acquisition (DIA) mode for direct analysis of O-glycoproteins. This approach enables characterization of glycopeptides and structures of O-glycans on a proteome-wide scale with quantification of stoichiometries (though it does not allow for direct unambiguous glycosite identification). The method relies on a spectral library of O-glycopeptides; the Glyco-DIA library contains sublibraries obtained from human cell lines and human serum, and it currently covers 2,076 O-glycoproteins (11,452 unique glycopeptide sequences) and the 5 most common core1 O-glycan structures. Applying the Glyco-DIA library to human serum without enrichment for glycopeptides enabled us to identify and quantify 269 distinct glycopeptide sequences bearing up to 5 different core1 O-glycans from 159 glycoproteins in a SingleShot analysis.


Assuntos
Biologia Computacional/métodos , Simulação por Computador , Glicopeptídeos/análise , Glicoproteínas/análise , Polissacarídeos/análise , Proteoma/análise , Proteômica/métodos , Glicosilação , Humanos
18.
J Agric Food Chem ; 67(33): 9411-9422, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31393126

RESUMO

Royal jelly (RJ) is secreted by young worker bees, and it plays key roles in the development and physiological function in honeybees and can improve human health. Although there have been analyses on the glycosylation modification of RJ proteins, none of these methods have been conducted on a site-specific analysis of glycosylation from these glycoproteins. Here, a combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity of RJ glycoproteins. First, global characterization of the N-glycome of RJ was performed using a direct infusion ion trap-sequential mass spectrometry (IT-MSn) method. Second, tryptic glycopeptides were enriched and separated by hydrophilic interaction liquid chromatography-ion trap-sequential mass spectrometry (HILIC-IT-MSn). A total of 50 N-glycopeptides and 30 N-glycans have been site-specific glycosylation profiled in major royal jelly protein 1 (MRJP1) and MRJP2 of RJ for the first time. Eighteen of the identified N-glycans have been structurally characterized by IT-MSn, including oligosaccharide composition, sequence, branching, and linkage. Two N-glycosylation sites (N177 and N394), 3 sites (N145, N178, and N92), and 1 site of N183 were identified in MRJP1, MRJP2, and MRJP3, respectively. There were 18, 17, and 2 N-glycans attached to MRJP1, MRJP2, and MRJP3, respectively. The diversity of N-glycans attached to each single glycosylation site of these glycoproteins confirmed that MRJP1 and MRJP2 heterogeneity was mostly associated with their glycoform populations. Understanding the properties of the site-specific glycosylation heterogeneity of the RJ glycoproteins can be potentially useful for producing a glycoprotein with desirable pharmacokinetic and biological activity.


Assuntos
Ácidos Graxos/química , Glicoproteínas/química , Proteínas de Insetos/química , Motivos de Aminoácidos , Animais , Abelhas , Cromatografia Líquida , Glicômica , Glicosilação , Espectrometria de Massas em Tandem
19.
J Agric Food Chem ; 67(37): 10543-10551, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31464438

RESUMO

Adulteration of meat and meat products causes concerns to consumers. It is necessary to develop novel robust and sensitive methods that can authenticate the origin of meat by qualitative and quantitative means to minimize the drawbacks of the existing methods. This study has shown that the protein N-glycosylation profiles of different meats are species specific and thus can be used for meat authentication. Based on the N-glycan pattern, the investigated five meat species (beef, chicken, pork, duck, and mutton) can be distinguished by principal component analysis, and partial least square regression was performed to build a calibration and validation model for the prediction of adulteration ratio. Using this method, beef samples adulterated with a lower-value duck meat could be detected down to the addition ratio as low as 2.2%. The most distinguishing N-glycans from beef and duck were elucidated for the detailed structures.


Assuntos
Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Glicômica/métodos , Polissacarídeos/química , Animais , Bovinos , Galinhas , Análise Discriminante , Patos , Glicosilação , Carne , Análise de Componente Principal , Carne Vermelha , Suínos
20.
Artigo em Inglês | MEDLINE | ID: mdl-31454680

RESUMO

Glycosylation is one of the most common post-translational protein modifications and is closely associated with muscle atrophy. This study aims to investigate the changes in glycan profiles in the fast-twitch extensor digitorum longus (EDL) muscles of Daurian ground squirrels (Spermophilus dauricus) during hibernation as well as the correlation between protein glycosylation and muscle atrophy prevention in hibernating animals. The results showed that there was no significant change in the muscle-to-body mass ratio, muscle fiber cross-sectional area (CSA), fiber distribution and ultrastructures in the EDL muscles of ground squirrels during hibernation. Alterations of six glycans comprising sialic acid α2-3 galactose (Sia2-3Gal) and Fucα1-2Galß1-4Glc(NAc) in the EDL muscles were observed. In addition, the observed downregulation of sialyltransferase (ST3Gals) mRNA levels and upregulation of fucosyltransferase (FUT1 and FUT2) mRNA levels during hibernation and the subsequent restoration to normal levels during periodic interbout arousal were consistent with the changes in sialic acid and fucose modifications. Our results indicate that changes in ST3Gals and FUTs in the EDL muscles of Daurian ground squirrels during hibernation can alter sialylation and fucosylation of muscle glycoproteins, which may protect the skeletal muscles of hibernating Daurian ground squirrels from disuse atrophy.


Assuntos
Músculo Esquelético/metabolismo , Animais , Fucose/metabolismo , Glicosilação , Hibernação/fisiologia , Ácido N-Acetilneuramínico/metabolismo , Sciuridae
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