RESUMO
Two pectin-polysaccharides, ASP-B2 (Mw, 5.32 kDa) and B3 (Mw, 30.51 kDa), were obtained from Acanthopanax senticosus leaves. An HILIC-UPLC-ESI--HCD-MS/MS technique was introduced for structural elucidations of skeletal structure of pectin-polysaccharides in addition to empolying SEC-MALLS, GC-MS, UPLC-ESI+-MS, methylation analysis, FT-IR and NMR methods. Skeletal structure of ASP-B2 was characterized by combining HG smooth region and RG hairy region with a 2:1 chain length ratio, wheres ASP-B3 had a distinguishing 3:1 chain length ratio (HG:RG). HG were defined as repeated â4GalA1â, and RG were featured by repeated â4GalA1 â 2Rhap1 â with branched points at C-3, C-4 and C-3,4 of rhamnosyl. Side chains mainly consisted of galactans, arabinans and arabinogalactans. Both showed a random coil conformation and no triple-helix conformation. ASP-B2 and B3 possess stronger DPPH and hydroxyl radical scavenging activities, with a relatively low reducing power. Thus, two polysaccharides can be further explored as novel natural antioxidant.
Assuntos
Antioxidantes/farmacologia , Eleutherococcus/química , Folhas de Planta/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Vermelho Congo/química , Metilação , Monossacarídeos/análise , Pectinas/química , Polissacarídeos/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier , Espectrometria de Massas em TandemRESUMO
Royal Jelly (RJ) constitutes one of the most popular beehive products and for this reason the use of inexpensive sweeteners during its production remains an important quality issue. In the present study we report results of metabolic profiling of RJ samples obtained after the application of artificial bee-feeding using different feeding protocols. The hydrophilic content of RJ samples was assessed by applying (HILIC)UPLC-MS/MS. In total 96 crude RJ samples were analysed with the developed method. Multivariate statistical analysis revealed clear differentiation between the RJ samples obtained from control (non-fed) bees and samples obtained after feeding. In total 27 out of 57 detected molecules were statistically found to be significantly altered in the different comparisons. Among them some amino acids (e.g. tryptophan, lysine), amino acid derivatives (pyroglutamic acid), amines (cadaverine, TMAO, etc.), carbohydrates and vitamins were found as potential markers. The results of the study could be further used for the development of an LC-MS based analytical tool for RJ quality control assessment.
Assuntos
Abelhas , Dieta , Ácidos Graxos/química , Aminas/análise , Aminoácidos/análise , Animais , Carboidratos/análise , Cromatografia Líquida , Espectrometria de Massas em Tandem , Vitaminas/análiseRESUMO
Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine-d3 (Cr-d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC-UPLC-MS/MS, we measured cCr and Cr-d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.
Assuntos
Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Creatina/deficiência , Creatinina/análogos & derivados , Fibroblastos/metabolismo , Imidazolidinas/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/tratamento farmacológico , Fosfocreatina/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Encefalopatias Metabólicas Congênitas/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Creatina/metabolismo , Creatinina/farmacocinética , Creatinina/uso terapêutico , Humanos , Imidazolidinas/análise , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Fosfocreatina/análise , Fosfocreatina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Cultura Primária de Células , Espectrometria de Massas em Tandem/métodosRESUMO
Auricularia auricular-judae polysaccharides (AAPs) have been accepted as one important biological constituent. Quality analysis of A. auricular-judae is generally difficult due to technical issues in separating and detecting AAPs. Here, we describe a stepped chromatographic and mass spectrometric approach for discrimination and characterization of AAPs. HPSEC-MALLS-RID and GC-MS techniques have earlier been shown to be ineffective in evaluating wild and cultured AAPs based on molecular weight distributions and monosaccharide compositions. Nevertheless, direct ESI--MS oligosaccharide fingerprints coupled with PCA have the remarkable ability to discriminate wild and cultured ones. HILIC-UPLC-ESI--MS has indicated that wild and cultured AAPs have distinct peak number and intensities in mild acid hydrolysates. HILIC-UPLC-ESI--HCD-MS/MS was further applied in deducing interglycosidic linkages and sequence of monosaccharide residues in oligosaccharides. We demonstrate that linear GlcAwâ¯ââ¯Hexmâ¯ââ¯GlcAxâ¯ââ¯Hexnâ¯ââ¯GlcAyâ¯ââ¯Hexpâ¯ââ¯GlcAzâ¯ââ¯Hexqâ¯ââ¯(w, x, y, zâ¯=â¯0, 1; m, n, p, qâ¯≥â¯0; Hex1â¯ââ¯6Hex, GlcA1â¯ââ¯4Hex, Hex1â¯ââ¯4GlcA) was constructed as the general skeleton of AAPs based on C- and B-type ions as well as cross-ring cleavage A-type series ions. The established workflow has been confirmed to be a feasible approach for comprehensive characterization of polysaccharides in foods and plants.
Assuntos
Basidiomycota/química , Carpóforos/química , Polissacarídeos Fúngicos/química , Basidiomycota/crescimento & desenvolvimento , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Polissacarídeos Fúngicos/isolamento & purificação , Hidrólise , Espectrometria de Massas , Metilação , Peso MolecularRESUMO
Most human proteins are glycosylated. Attachment of complex oligosaccharides to the polypeptide part of these proteins is an integral part of their structure and function and plays a central role in many complex disorders. One approach towards deciphering this human glycan code is to study natural variation in experimentally well characterized samples and cohorts. High-throughput capable large-scale methods that allow for the comprehensive determination of blood circulating proteins and their glycans have been recently developed, but so far, no study has investigated the link between both traits. Here we map for the first time the blood plasma proteome to its matching N-glycome by correlating the levels of 1116 blood circulating proteins with 113 N-glycan traits, determined in 344 samples from individuals of Arab, South-Asian, and Filipino descent, and then replicate our findings in 46 subjects of European ancestry. We report protein-specific N-glycosylation patterns, including a correlation of core fucosylated structures with immunoglobulin G (IgG) levels, and of trisialylated, trigalactosylated, and triantennary structures with heparin cofactor 2 (SERPIND2). Our study reveals a detailed picture of protein N-glycosylation and suggests new avenues for the investigation of its role and function in the associated complex disorders.
RESUMO
Guizhi Fuling capsule (GFC), developed from the traditional Chinese prescription of Guizhi Fuling Wan, has been commonly used for the treatment of primary dysmenorrhea (PD). However, the intervention effective mechanism in vivo has not been well elucidated. In this study, an integrated plasma metabonomic strategy based on RP-UPLC-MS coupled with HILIC-UPLC-MS technique has been developed to investigate the global therapeutic effects and intervention mechanisms of GFC on dysmenorrhea rats induced by oxytocin. The 20 potential biomarkers were identified and primarily related to sphingolipid metabolism, steroid hormone biosynthesis, glycerophospholipid metabolism, amino acid metabolism, lipid metabolism and energy metabolism. The results showed that the GFC has therapeutic effects on rats with dysmenorrhea via the regulation of multiple metabolic pathways. Some new potential biomarkers associated with primary dysmenorrhea such as phenylalanine, tryptophan, taurine, carnitine, betaine, creatine and creatinine have been discovered in this study for the first time. This study provides a metabonomic platform based on RP-UPLC-MS complementary to HILIC-UPLC-MS technique to investigate both nonpolar and polar compounds, so as to get a more comprehensive metabolite information to yield insight into the pathophysiology of PD and assessing the efficacy of GFC on PD rats.
Assuntos
Cromatografia de Fase Reversa/métodos , Medicamentos de Ervas Chinesas/farmacologia , Dismenorreia/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica/métodos , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores/sangue , Biomarcadores/metabolismo , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodos , Ratos , Ratos Wistar , Reprodutibilidade dos TestesRESUMO
Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dissacarídeos/análise , Glicosaminoglicanos/química , Heparitina Sulfato/análise , Procainamida/química , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodosRESUMO
Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.
Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Anticorpos Monoclonais/química , Glicoproteínas/química , Polissacarídeos/química , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Corantes Fluorescentes/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
N-linked glycosylation of the fragment crystallizable (Fc)-region of immunoglobulin G (IgG) is known to have a large influence on the activity of the antibody, an effect reported to be IgG subclass specific. This situation applies both to humans and mice. The mouse is often used as experimental animal model to study the effects of Fc-glycosylation on IgG effector functions, and results are not uncommonly translated back to the human situation. However, while human IgG Fc-glycosylation has been extensively characterized in both health and disease, this is not the case for mice. To characterize the glycosylation profile of murine IgG-Fc and in addition evaluate the systematic glycosylation differences between mouse strains, sexes, and IgG subclasses, we used nanoliquid chromatography mass spectrometry (nanoLC-MS(/MS)) to look at the subclass-specific IgG Fc-glycopeptides of male and female mice from the strains BALB/c, C57BL/6, CD-1, and Swiss Webster. The structural analysis revealed the presence of predominantly fucosylated, diantennary glycans, with varying amounts of galactosylation and α2,6-sialylation. In addition, we report glycosylation features not previously reported in an Fc-specific way on murine IgG, including monoantennary, hybrid, and high mannose structures, as well as diantennary structures without a core fucose, with a bisecting N-acetylglucosamine, or with α1,3-galactosylation. Pronounced differences were detected between strains and the IgG subclasses within each strain. Especially the large spread in galactosylation and sialylation levels found between both strains and subclasses may vastly influence IgG effector functions. Mouse strain-based and subclass-specific glycosylation differences should be taken into account when designing and interpreting immunological and glycobiological mouse studies involving IgG effector functions.
RESUMO
Glycosylation of membrane proteins plays a crucial role in various physiological events, including intercellular recognition and intermolecular interactions on the cell surface (Gornik et al., Biochim Biophys Acta 1820:1318-1326, 2012). To study composition and function of N-glycans on membrane proteins one has to have an efficient and reproducible analytical method, which includes protein extraction and analysis of glycans. In this chapter we provide an analytical approach that includes cloud-point extraction (CPE) of total membrane proteins with the non-ionic detergent Triton X-114 and subsequent analysis of their N-glycans using hydrophilic interaction liquid chromatography (HILIC)-UPLC/HPLC. The protocol presented here can be used for parallel analysis of both membrane and intracellular proteins.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Proteínas de Membrana/análise , Polissacarídeos/análise , Animais , Fracionamento Químico/métodos , Precipitação Química , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/isolamento & purificação , Octoxinol , Polietilenoglicóis/química , Polissacarídeos/isolamento & purificaçãoRESUMO
Extraction of N-glycans from intact tissue presents a unique set of challenges which makes it a relatively laborious and time-consuming process in comparison to other sample types, such as plasma. Here we present an approach designed for the extraction, purification, and labeling of free N-glycans from brain tissue. Using this method, up to 16 samples can be processed at once which translates to an output of 48 samples per week when rounds of extraction are staggered. Moreover, although intended for brain tissue, the method could easily be adapted to other tissue types as well. The protocol involves several stages. First, the tissue is homogenized and total proteins are isolated using chloroform-methanol extraction. The proteins are then deglycosylated using the Peptide N-Glycosidase F (PNGase F) enzyme in a reaction lasting two days. The released N-glycans are subsequently cleaned up from the reaction mixture using a centrifugal filter device and dried overnight. Next, the N-glycans are resuspended, labeled with 2-aminobenzamide (2-AB) and once again cleaned up using a filter plate. The purified N-glycans are released from the filter using ultrapure water and are then ready for analysis by for hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC).
Assuntos
Química Encefálica , Cromatografia Líquida de Alta Pressão/métodos , Glicômica/métodos , Glicoproteínas/química , Polissacarídeos/análise , Animais , Corantes Fluorescentes/química , Glicoproteínas/isolamento & purificação , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos/isolamento & purificação , Coloração e Rotulagem/métodos , ortoaminobenzoatos/químicaRESUMO
Root exudation has importance in soil chemical ecology influencing rhizosphere microbiota. Prior studies reported root exudates from host and nonhost plants stimulated resting spore germination of Spongospora subterranea, the powdery scab pathogen of potato, but the identities of stimulatory compounds were unknown. This study showed that potato root exudates stimulated S. subterranea resting spore germination, releasing more zoospores at an earlier time than the control. We detected 24 low molecular weight organic compounds within potato root exudates and identified specific amino acids, sugars, organic acids, and other compounds that were stimulatory to S. subterranea resting spore germination. Given that several stimulatory compounds are commonly found in exudates of diverse plant species, we support observations of nonhost-specific stimulation. We provide knowledge of S. subterranea resting spore biology and chemical ecology that may be useful in formulating new disease management strategies.
Assuntos
Exsudatos de Plantas/farmacologia , Raízes de Plantas/metabolismo , Plasmodioforídeos/patogenicidade , Solanum tuberosum/metabolismo , Esporos de Protozoários/efeitos dos fármacos , Cromatografia Líquida/métodos , Interações Hospedeiro-Patógeno , Espectrometria de Massas/métodos , Metaboloma , Exsudatos de Plantas/química , Exsudatos de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plasmodioforídeos/efeitos dos fármacos , Plasmodioforídeos/fisiologia , Solanum tuberosum/microbiologia , Esporos de Protozoários/patogenicidade , Esporos de Protozoários/fisiologiaRESUMO
An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOF-MS) method using hydrophilic interaction liquid chromatography was developed and validated for simultaneous quantification of 18 free amino acids in urine with a total acquisition time including the column re-equilibration of less than 18 min per sample. This method involves simple sample preparation steps which consisted of 15 times dilution with acetonitrile to give a final composition of 25 % aqueous and 75 % acetonitrile without the need of any derivatization. The dynamic range for our calibration curve is approximately two orders of magnitude (120-fold from the lowest calibration curve point) with good linearity (r (2) ≥ 0.995 for all amino acids). Good separation of all amino acids as well as good intra- and inter-day accuracy (<15 %) and precision (<15 %) were observed using three quality control samples at a concentration of low, medium and high range of the calibration curve. The limits of detection (LOD) and lower limit of quantification of our method were ranging from approximately 1-300 nM and 0.01-0.5 µM, respectively. The stability of amino acids in the prepared urine samples was found to be stable for 72 h at 4 °C, after one freeze thaw cycle and for up to 4 weeks at -80 °C. We have applied this method to quantify the content of 18 free amino acids in 646 urine samples from a dietary intervention study. We were able to quantify all 18 free amino acids in these urine samples, if they were present at a level above the LOD. We found our method to be reproducible (accuracy and precision were typically <10 % for QCL, QCM and QCH) and the relatively high sample throughput nature of this method potentially makes it a suitable alternative for the analysis of urine samples in clinical setting.
Assuntos
Aminoácidos/química , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Aminoácidos/urina , Humanos , Limite de DetecçãoRESUMO
To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.
Assuntos
Anticorpos Monoclonais/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Ácidos Siálicos/análise , Animais , Células CHO , Cricetinae , Cricetulus , Glicosilação , Humanos , Proteínas Recombinantes/químicaRESUMO
Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.
Assuntos
Anticorpos Monoclonais/química , Fragmentos Fc das Imunoglobulinas/química , Animais , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Glicosilação , Espectrometria de MassasRESUMO
In this study, a rapid and sensitive hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS) method was developed for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma. All samples were simply precipitated using acetonitrile and separated on a UPLC-HILIC column under gradient elution with a mobile phase consisting of acetonitrile and water with the addition of 10mm ammonium acetate and 0.1% formic acid at 0.4 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte, and the intra- and inter-day precisions were <12.8%. The analytical runtime within 4.0 min per sample made this method suitable for high throughput determination. The validated method was successfully applied to a clinical pharmacokinetic study of bambuterol in eight healthy volunteers. Furthermore, the effects of the chromatographic conditions on the retention of the analytes on HILIC were investigated, and the benefits of HILIC were evaluated by comparing with a C18 column. The results indicated that liquid-liquid partition and the electrostatic interactions played an important role in the retention of the analytes on HILIC in this study. And HILIC offered particular advantages over RPLC approach in the aspects of the peak symmetry, the column efficiency, and the column pressure.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Terbutalina/análogos & derivados , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Lineares , Pressão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Temperatura , Terbutalina/sangue , Terbutalina/química , Terbutalina/metabolismo , Terbutalina/farmacocinéticaRESUMO
The IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)-UPLC, reversed phase (RP)-UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC-UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques.
Assuntos
Fracionamento Químico/métodos , Saúde , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Polissacarídeos/metabolismo , Animais , Gatos , Bovinos , Cães , Glicosilação , Cavalos , Humanos , Oligossacarídeos/metabolismoRESUMO
In this study, a rapid, simple and sensitive analytical method was developed for the quantitative determination of 20 nucleosides and nucleobases in functional foods at trace levels using hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HILIC-UPLC-TQ-MS/MS) in multiple-reaction monitoring (MRM) mode. Under optimised chromatographic conditions, good separation of 20 target compounds was achieved using a Waters Acquity UPLC BEH Amide column and gradient elution in 11min. The limits of detection (LODs) and quantification (LOQs) were between 0.02-42.54ng/mL and 0.05-98.18ng/mL for the 20 analytes, respectively. This is the first report about simultaneous analysis of nucleosides and nucleobases in functional foods using this method, which afforded good linearity, precision, repeatability and accuracy. The method developed was successfully applied to quantify target compounds in batches of ginkgo seeds. The method potentially could be used to determine polar and trace-level nucleosides and nucleobases in ginkgo seeds.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ginkgo biloba/química , Nucleosídeos/química , Extratos Vegetais/química , Purinas/química , Sementes/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/instrumentaçãoRESUMO
BACKGROUND: Oral cancer is 1 of the 6 most common human cancers, with an annual incidence of >300,000 cases worldwide. This study aimed to investigate potential biomarkers in human saliva to facilitate the early diagnosis of oral squamous cell carcinoma (OSCC). METHODS: Unstimulated whole saliva obtained from OSCC patients (n=30) and apparently healthy individuals (n=30) were assayed with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) in hydrophilic interaction chromatography mode. The data were analyzed using a nonparametric Mann-Whitney U test, logistic regression, and the receiver operating characteristic (ROC) to evaluate the predictive power of each of 4 biomarkers, or combinations of biomarkers, for OSCC screening. RESULTS: Four potential salivary biomarkers demonstrated significant differences (P<0.05) in concentrations between patients at stages I-II and the healthy individuals. The area under the curve (AUC) values in control vs OSCC I-II mode based on choline, betaine, pipecolinic acid, and l-carnitine were 0.926, 0.759, 0.994, and 0.708, respectively. Four salivary biomarkers in combination yielded satisfactory accuracy (0.997), sensitivity (100%), and specificity (96.7%) in distinguishing OSCC I-II from control. CONCLUSIONS: Salivary metabolite biomarkers for the early diagnosis of OSCC were verified in this study. The proposed approach is expected to be applied as a potential technique of preclinical screening of OSCC.