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1.
J Chromatogr A ; : 460934, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32029268

RESUMO

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.

2.
Analyst ; 145(2): 582-587, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31769453

RESUMO

A new quantitative analysis method was proposed, aiming at resolving the difficulty encountered in accurately determining the trace content of a free acid in lithium-ion battery electrolytes in the past 30 years. The presented method overcame the three restrictive factors of lithium-ion battery electrolytes, namely, poor thermal stability, the formation of hydrofluoric acid with water and difficulty in the accurate determination of trace free acids. The free acid in lithium-ion battery electrolytes was directly titrated with ethoxide ions generated through the electrolyzation of a 0.50 mol L-1 LiCl ethanol solution. The content of free acid was obtained according to the Faraday's law, and the whole determination process could be completed in 5 minutes. The relative standard deviation was below 2.0% when the content of free acid was 2.0 µg and above. The detection limit was 1.0 µg and the recovery rate was 99.5%-102.5%. In this method, free acid determination was not affected by temperature change and the existence of a small amount of water. Thus, this study provides a simple, fast and accurate analytical method with a lower detection limit for free acid in the lithium-ion battery industry and comprehensively improves the quality of lithium-ion battery electrolytes and the performance of lithium-ion battery products.

3.
J Chromatogr A ; 1569: 8-16, 2018 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-30041872

RESUMO

The pretreatment of low-abundance glycans/glycopeptides prior to mass spectrometry (MS) analysis is of great significance for glycosylation identification of proteins. Herein, a simple one-step purification and enrichment procedure of glycans and glycopeptides was achieved using cellulose microspheres-filled pipet tips (CM-PTs). CM-PTs were fabricated by conveniently filling cellulose microspheres synthesized by sol-gel transition method into 100 µL pipet tips. The whole enrichment and purification process were completed within two minutes by pipetting up and down for dozens of times (30-40), followed by the direct analysis of MALDI TOF-MS. Upon the assessment of model glycoproteins, including human immunoglobulin G (IgG) and horse radish peroxidase (HRP), the CM-PT method exhibited good selectivity (IgG/BSA = 1:100), high sensitivity (1 ng µL-1 of IgG), large binding capacity (125 mg g-1, IgG/cellulose microspheres) and excellent reproducibility in glycopeptide enrichment. The capability for purifying glycans of CM-PTs was also investigated, presenting high binding capacity (8 mg g-1, maltoheptaose/cellulose microspheres) and good recoveries (81.3-89.8%). The developed in-pipet-tip system shows a great potential in efficient and high-throughput analysis of protein glycosylation.


Assuntos
Celulose/química , Técnicas de Química Analítica/instrumentação , Glicopeptídeos/isolamento & purificação , Microesferas , Polissacarídeos/isolamento & purificação , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Imunoglobulina G/química , Espectrometria de Massas , Reprodutibilidade dos Testes
4.
J Chromatogr A ; 1535: 114-122, 2018 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-29329887

RESUMO

Robust, efficient identification and accurate quantification of N-glycans are of great significance in N-glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N-glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N-glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N-glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N-glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis.


Assuntos
Aminoquinolinas/química , Carbamatos/química , Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Polissacarídeos/análise , Glicômica , Glicosilação , Humanos , Indicadores e Reagentes , Micro-Ondas , Reprodutibilidade dos Testes
5.
Anal Chim Acta ; 1002: 50-61, 2018 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-29306413

RESUMO

Quantitative analysis of glycans is an emerging field in glycomic research. Herein we present a rapid and effective dual-labeling strategy, in the combination of isotopic derivatization of N-glycosylamine-based glycans by d0/d5-benzoyl chloride and methylamidation of sialic acids, to relatively quantify both neutral and sialylated N-glycans simultaneously by MALDI-MS. The derivatization efficiencies were increased by microwave-accelerated deglycosylation which not only largely reduce the time of glycoprotein deglycosylation but also inhibit the hydrolysis of intermediate glycosylamines produced by PNGase F digestion. Three model glycoproteins, including RNase B, bovine fetuin and IgG from human serum, were applied to validate this technique. Results showed that the glycans from microgram level of glycoprotein can be successfully quantified with high reproducibility and the whole time of analytical procedure was shortened to 4 h. Furthermore, this proposed method was applied for the comparative analysis of N-glycans from serum of healthy donors and multiple myeloma patients. It was found that five N-glycans may be as the potential biomarkers for rapid detection of early multiple myeloma, indicating the feasibility of this strategy in monitoring subtle quantitative differences of serum glycomics.


Assuntos
Benzoatos/química , Deutério/química , Glicômica/métodos , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Glicosilação , Humanos , Marcação por Isótopo , Polissacarídeos/sangue , Processamento de Proteína Pós-Traducional
6.
Anal Bioanal Chem ; 410(1): 79-89, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29071364

RESUMO

Derivatization of carbohydrates with aminooxy agents to form oximes can be used for qualitative and quantitative analysis of carbohydrates; however, the formation of isomeric products limits its application. A new reductive oxyamination procedure developed for the analysis of monosaccharides with a novel fluorescent O-substituted aminooxy reagent, 4-((aminooxy)methyl)-6-chloro-7-hydroxycoumarin (AOCC), is reported. In this procedure, monosaccharides undergo an oxime formation reaction with AOCC and are then readily reduced with 2-picoline-borane, followed by analysis with high-performance liquid chromatography with fluorescence detection. Good separation of five monosaccharide derivatives was achieved within 40 min with acetonitrile-water-tetrahydrofuran as the mobile phase. The detection limits were on the order of femtomoles. The linear range was 0.2-4000 nM, with a good correlation coefficient (R ≥ 0.9985). Furthermore, the method was applied for analysis of real samples, such as bovine milk powder, without complicated and tedious sample treatment. This reductive oxyamination method circumvents the problem caused by oxime isomers and can be used for the highly sensitive and selective analysis of monosaccharides with high accuracy, providing an effective and promising method for the analysis of carbonyls with aminooxy agents.

7.
Talanta ; 178: 834-841, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29136902

RESUMO

A nitrone formation method using a novel fluorescent reagent 4-hydroxylaminopropyl-7-methoxylcoumarin (HAMC) has been developed and applied for the determination of furfurals in foods using high performance liquid chromatography with fluorescence detection (HPLC-FLD). Furfurals samples were derivatized by HAMC in aqueous condition at room temperature in less than 30min and were subjected to direct HPLC-FLD analysis without extra purification or extraction. Compared with other conventional methods, nitrone formation method produced single chromatographic peak for each analyte, leading to simpler chromatograms and enhancing the overall sensitivity. Low detection limits at sub-nM level (0.08-0.1nM) and high repeatabilities (intra-day RSD ≤ 4.2% and inter-day RSD ≤ 6.7%) were achieved. The linear range of the calibration curve was 0.2-4000nM with good correlation coefficients (R ≥ 0.9989). This method was successfully applied for furfurals determination in food samples, including honey and coffee. The accuracy was satisfactory with recoveries ranging from 89.7% to 106.7% Above all, this pre-column derivatization method is simple, reliable and highly sensitive, providing a promising way for future studies of aldehydes from various sources.


Assuntos
Análise de Alimentos/métodos , Furaldeído/análise , Furaldeído/química , Óxidos de Nitrogênio/química , Cromatografia Líquida de Alta Pressão , Limite de Detecção , Espectrometria de Fluorescência
8.
Anal Chim Acta ; 987: 47-55, 2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-28916039

RESUMO

A pre-column fluorescent derivatization method based on nitrone formation has been applied to determine furfurals (e.g. furfural (F), 5-methyfurfural (5-MF) and 5-hydroxymethylfurfural (5-HMF)) in food samples for the first time. An N-substituted hydroxylamine reagent 4-((hydroxyamino)butyl)-7-hydroxycoumarin (HAHC) was used to react with the aldehyde group of furfurals to form stable nitrone derivatives with high fluorescence intensities. The reactions proceeded under mild conditions in 30 min with high derivatization yields (>93%). A baseline-separation of three furfurals derivatives was subsequently achieved within 25 min on a reversed-phase column. The detection limits were at the low femtomol level (S/N = 3, 20 µL per injection). The linear range of the calibration curve was 0.4-4000 nM with good correlation coefficients (R2 ≥ 0.9991). The proposed method was further applied for food sample analysis, such as bovine milk powder and raisin. Satisfactory recoveries were obtained in the range of 94.7%-103.5%. Above all, this pre-column derivatization method is simple, fast and highly sensitive, providing an effective and promising way for future studies of aldehydes in different matrices.


Assuntos
Frutas/química , Furaldeído/análise , Leite/química , Vitis/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão
9.
J Org Chem ; 82(19): 10388-10397, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28886243

RESUMO

The first chiral Brønsted acid-catalyzed asymmetric cascade allylboration/oxo-Michael reaction between o-formyl chalcones and allylboronate has been successfully discovered, which afforded chiral 1,3-disubstituted 1,3-dihydroisobenzofurans with a yield, diastereoselective ratio (dr) and enantioselective excess (ee) up to 94%, 2.5:1, and 98%, respectively. In addition, 2,3-dienylboronic pinacol ester was also applied into this cascade reaction with good catalytic results.

10.
Anal Bioanal Chem ; 409(16): 4027-4036, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28417175

RESUMO

A rapid and sensitive N-glycan profiling strategy for MALDI-MS incorporating the use of deglycosylation with microwave assistance and the co-derivatization of glycosylamine labeling with tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu) and methylamidation has been developed in this work. Notably, highly efficient release and tagging of N-glycans from ribonuclease B was achieved in less than 90 min, providing up to 35-fold enhancement of MALDI-MS sensitivity with comparison to underivatized N-glycans. After further validation with other two standard glycoproteins (ovalbumin and bovine fetuin), the proposed strategy was applied to human serum for preliminary pathological analysis of N-glycans between healthy and lung cancer individuals. As a result, significant differences (T test p value <0.01) of 6 glycan structures were determined from 54 detected N-glycan structures with only 50 nL of loading amount and further confirmed through PCA and ROC (AUC) analyses between two sample sets. Subsequently, the trend of each lung cancer stage and controls in expression of the selected glycans was implemented with T test and box-plots. Accordingly, these structures can be used as potential lung cancer glycan-based biomarkers and for further definition of cancer progression highlighting the ability of proposed method to rapidly and efficiently analyze N-glycome present in human serum. Graphical abstract MALDI-TOF MS analysis of N-glycans by microwave-assisted deglycosylation and glycosylamine derivatization.


Assuntos
Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esterificação , Glicoproteínas/química , Glicosilação , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/química , Manose/análise , Micro-Ondas , Ácido N-Acetilneuramínico/análise , Compostos Organofosforados/química , Polissacarídeos/sangue , Succinimidas/química
11.
Anal Chem ; 86(15): 7913-9, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25022802

RESUMO

The analysis of isomeric glycans is a challenging task. In this work, a new strategy was developed for isomer-specific glycan profiling using nanoLC-MS with PGC as the stationary phase. Native glycans were derivatized in the presence of methylamine and trispyrrolidinophosphonium hexafluorophosphate and reduced by the ammonia-borane complex. Methylamidation stabilized the retention time and peak width and improved the detection sensitivity of sialylated glycans to 2-80-fold in comparison to previous ESI-MS methods using the positive-ion mode. Up to 19 tetrasialylated glycan species were identified in the derivatized human serum sample, which were difficult to detect in the sample without derivatization. Furthermore, due to high detection sensitivity and chromatographic resolution, more isomeric glycans could be identified from the model glycoprotein Fetuin and the human serum sample. As a result, up to seven isomers were observed for the disialylated biantennary glycan released from Fetuin, and three of them were identified for the first time in this study. Using the developed analytical strategy, a total of 293 glycan species were obtained from the human serum sample, representing an increase of over 100 peaks in comparison to the underivatized sample. The strategy greatly facilitates the profiling of isomeric glycans and the analysis of trace-level samples.


Assuntos
Amidas/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Ácido N-Acetilneuramínico/química , Polissacarídeos/química , Isomerismo , Limite de Detecção , Nanotecnologia , Reprodutibilidade dos Testes
12.
J Mass Spectrom ; 46(6): 546-52, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21630382

RESUMO

Neutral hydroxymethylene HCOH is an important intermediate in several chemical reactions; however, it is difficult to observe due to its high reactivity. In this work, neutral hydroxymethylene and formaldehyde were generated by charge exchange neutralization of their respective ionic counterparts and then were reionized and detected as positive-ion recovery signals in neutralization-reionization mass spectrometry in a magnetic sector instrument of BEE geometry. The reionized species were characterized by their subsequent collision-induced dissociation mass spectra. The transient hydroxymethylene neutral was observed to isomerize to formaldehyde with an experimental time span exceeding 13.9 µs. The vertical neutralization energy of the HCOH(+•) ion has also been assayed using charge transfer reactions between the fast ions and stationary target gases of differing ionization energy. The measured values match the result of ab initio calculations at the QCISD/6-311 + G(d,p) and CCSD(T)/6-311 + + G(3df,2p) levels of theory. Neutral hydroxymethylene was also produced by proton transfer from CH(2) OH(+) to a strong base such as pyridine, confirmed by appropriate isotopic labeling. There is a kinetic isotope effect (KIE) for H(+) versus D(+) transfer from the C atom of the hydroxymethyl cation of ∼3, consistent with a primary KIE of a nearly thermoneutral reaction.


Assuntos
Formaldeído/química , Metanol/química , Espectrometria de Massas em Tandem/métodos , Cátions/química , Isótopos , Cinética , Modelos Moleculares , Picolinas/química , Piridinas/química
13.
J Am Soc Mass Spectrom ; 22(1): 75-80, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21472546

RESUMO

In high-energy (keV) CID experiments, oxygen has the unique ability to enhance specific ion fragmentation pathways that lie within a relatively narrow band of activation energy. It has been previously proposed that this oxygen-enhanced dissociation phenomenon is due to the participation of the O(2) B(3)Σ(u)(+) - X(3)Σ(g)(-) (Schumann-Runge) system in the collision complex. During the collision, oxygen is first excited to its [Formula: see text] state before it returns this energy to the projectile ion. This energy drives the nonstatistical dissociation of the projectile provided there is an energetically accessible pathway in resonance with the absorbed radiation. To probe the validity of this hypothesis, a modified VG-ZAB mass spectrometer was used to observe the photon emissions from keV collisions of a selection of projectile ions with O(2) target gas. By studying the resulting collision-induced emission (CIE) spectra, a second potential mechanism came to light, one that involves the near-isoenergetic O(2) (+.) A (2)Π(u)→X (2) Π(g) state transition.

14.
J Phys Chem A ; 112(34): 7761-7, 2008 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-18680262

RESUMO

Collision-induced emission spectra from 190-1020 nm were obtained for N 2 (+*)/Ar collisions at laboratory frame collision energies from 2 to 8 keV. The relative emission intensities of N 2 (+*), N (+), and N (*) are independent of the ion translational energy within the studied energy range, supporting the curve-crossing mechanism for collisional excitation. The role of the target gas in keV N 2 (+*)/He and N 2 (+*)/Ar collisions was investigated by both emission spectroscopy and theoretical calculations. Adiabatic potential energy surfaces calculated at the CISD/6-311+G(2df) level of theory for N 2 (+*)/He and N 2 (+*)/Ar collisions indicate that excitation to the A (2)Pi u and B (2)Sigma u (+) states involves curve-crossing first to the C (2)Sigma u (+) state, and the difference in N 2 (+*) emission intensities from the two systems can be accounted for by the slope difference at the crossing points based on the Landau-Zener curve-crossing model.

15.
J Chromatogr A ; 1021(1-2): 209-13, 2003 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-14735990

RESUMO

A novel method based on capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF) was developed for the determination of abscisic acid (ABA), which is an essential phytohormone during plant growth and development. ABA was labeled with 8-aminopyrene-1,3,6-trisulfonate via reductive amination in presence of acetic acid and sodium cyanoborohydride. The derivatization yield was maximized by optimizing several derivatization parameters including derivatization reagent concentration, reaction temperature and time. The conjugate was separated and quantitated by CE-LIF. The linearity of ABA was determined in the range from 0.1 to 10 micromol l(-1) with a correlation of 0.9979. The derivatization limit of detection for ABA was found to be 56 fmol (corresponding to the concentration of 2.8 x 10(-8) mol l(-1)). The detection limit for ABA was 5.5 amol for an injection volume of 5 nl. As a preliminary application, the proposed method was successfully applied to determining trace amount of ABA in the crude extracts of tobacco without extra purification and enrichment procedure and showed a better selectivity and sensitivity than those conventional methods used in determination of ABA.


Assuntos
Ácido Abscísico/análise , Eletroforese Capilar/métodos , Espectrometria de Fluorescência/métodos , Lasers , Sensibilidade e Especificidade
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