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1.
Hata K Morisaka H Hara K Mima J Yumoto N Tatsu Y Furuno M Ishizuka N Ueda M 《Analytical biochemistry》2006,350(2):292-297
Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses. 相似文献
2.
The first practical fluorometric assay of plasma 25-hydroxyvitamin D3 (25-OH-D3) and 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is described. The method uses a highly fluorescent dienophile, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1, 2,4- triazoline-3,5-dione (DMEQ-TAD), to fluorescence-label vitamin D. Vitamin D metabolites were roughly purified with a short cartridge column followed by HPLC, labeled with DMEQ-TAD, and the product was analyzed on HPLC. In the assay of 25-OH-D3 the new fluorometric method was compared with the HPLC-uv method and was confirmed to be as accurate and reliable (CV, 4-5%) as the HPLC-uv method. Plasma 24,25-(OH)2D3 was accurately assayed by the HPLC-FL method, where the standard addition method was successfully used to calculate the overall recovery. 相似文献
3.
Fiot J Baghdikian B Boyer L Mahiou V Azas N Gasquet M Timon-David P Balansard G Ollivier E 《Phytochemical analysis : PCA》2005,16(1):30-33
An efficient system for the analysis of the total alkaloids extracted from leaves of Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae) by HPLC using a reversed-phase column is described. The chromatographic conditions allowed the separation of indole and oxindole alkaloids in leaf extracts, and the quantification of uncarine D in samples collected in Burkina Faso and Mali. The HPLC method described was validated for its specificity, linearity and precision using an internal standard (naphthalene). The concentrations of uncarine D in various extracts were compared with their in vitro anti-plasmodial activity. The anti-proliferative activity on chloroquine-resistant strain (W2) of Plasmodium falciparum was not correlated with the concentration of uncarine D in leaves. 相似文献
4.
Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry(1,2,3). The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein(4). H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection(2), instead of a HPLC/ESI (electrospray ionization)-MS system(5,6). The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called γ-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A(2;) (sPLA(2;)). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation(7,8,9). 相似文献
5.
Bonetto V Kangur L Palumaa P Mutt V Jörnvall H Sillard R 《Protein expression and purification》1999,17(3):387-391
Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k. 相似文献
6.
In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability. 相似文献
7.
A method is described which enables determination of vitamin D3 and its physiologically most important metabolites, i.e. 25-OHD3, 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,25-(OH)2D3 in a plasma sample of about 2 to 4 ml. The whole procedure involves two preparative and one analytical steps: Extraction with methanol/methylene chloride (2:1), chromatographic separation on Lipidex 5000 using a stepwise gradient of n-hexane and chloroform and finally HPLC separation on Zorbax-Sil columns with n-hexane isopropanol mixtures and subsequently reversed phase separation on RP 18-columns and mixtures of methanol and water. Except for 1,25-(OH)2D3 all D compounds were quantified by UV-detection with 1.4 ng of substance being the lowest detectable amount. 1,25-(OH)2D3 was measured by radioimmunoassay. Prior to HPLC analysis the extract was separated into three fractions on Lipidex 5000 which contained 1) vitamin D3, 2) 25-OHD3 and 3) the dihydroxy metabolites. The three fractions were separated by HPLC using different mixtures of isopropanol/n-hexane and methanol/water, respectively. Retention times of the individual D-components longer than 10 min appeared to be essential to separate these compounds from accompanying material. Overall recoveries of the individual metabolites were for vitamin D3 48.9%, for 25-OHD3 54.2%, for 24,25-(OH)2D3 50.9% and for 1,25-(OH)2D3 52.5%. Application of the methods to plasma samples from pigs with pseudovitamin D deficiency rickets, typ I, revealed a reduced concentration of 1,25-(OH)2D3 and 24,25-(OH)2D3 and an elevated level of 25-OHD3 in these animals. The results obtained by this method contributed substantially to a better understanding of the aetiological factors associated with this disease. 相似文献
8.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,668(1):133-139
An analytical method has been developed and validated for the quantitation of CP-88,059 in human serum. The compound and internal standard were extracted from serum by solid-phase extraction with a weak cation-exchange phase. The analytes were resolved from endogenous interferences using narrow-bore (2.1 mm I.D.) C18 reversed-phase HPLC. Column effluent was monitored by UV absorbance detection at 215 nm. The standard curve range was 1 to 250 ng/ml. The accuracy and precision values for the method were within ±10% and ±15%, respectively. A four-fold detectability enhancement was achieved using a 2.1 mm I.D. HPLC column relative to the more common 4.6 mm I.D. column. A performance comparison was made between the 2.1 mm I.D. column used for validation and a 4.6 mm I.D. column with the same stationary phase. 相似文献
9.
One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro‐heterogeneity can result from post‐translational modifications such as sialylation, galactosylation, C‐terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on‐line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post‐translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on‐line or at‐line measurements of these attributes. In this work, we describe the development of an at‐line assay to separate MAb charge variants in near real‐time, which could ultimately be used to implement on‐line quality control strategies for MAb production. The assay consists of a 2D‐HPLC method with sequential in‐line Protein A and WCX‐10 HPLC column steps. To perform the 2D‐HPLC assay at‐line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D‐HPLC for analysis. With this at‐line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:249–255, 2014 相似文献
10.
Rapuri PB Gallagher JC 《The Journal of steroid biochemistry and molecular biology》2004,(1-5):601-604
Vitamin D(2) and D(3) are generally considered equipotent in humans. As Vitamin D(2) supplements are commonly used by elderly in United States, we determined the contribution of 25OHD(2) to the total serum 25OHD levels by HPLC in elderly women who reported taking Vitamin D(2) supplements (n = 56) and also in a group of randomly selected unsupplemented women (n = 60). In addition, we compared the total serum 25OHD measured by HPLC with competitive protein-binding assay (CPBA), a method routinely employed to measure Vitamin D status. A correlation of 0.91 (P < 0.001) was observed between the two methods for the serum total 25OHD measurement. The mean serum 25OHD level in Vitamin D(2) supplemented group was significantly higher than in unsupplemented group measured by HPLC (32 versus 28 ng/ml) and marginally higher measured by CPBA (33 vs. 31 ng/ml). Seventy eight percent of women taking Vitamin D(2) supplements had appreciable amounts of circulating 25OHD(2,) which constituted about 25 percent of their total serum 25OHD. It is also interesting to note that Vitamin D deficiency was less prevalent in elderly women taking Vitamin D(2) supplements (1.8%) compared to women not taking any supplements (12%). 相似文献
11.
This paper describes a method that substantially improves the sensitivity of high-performance liquid chromatography hydrogen exchange-mass spectrometry (HPLC HX MS). The success of this method relies on using a capillary HPLC column (0.1mm IDx5cmL) to increase the sensitivity of electrospray ionization, while keeping analysis times short to minimize hydrogen/deuterium (H/D) exchange. A small, immobilized pepsin column and a capillary C18 trap were included in the capillary HPLC MS system to provide rapid digestion, peptide concentration, and desalting while maintaining slow H/D exchange conditions. To minimize the analysis time, dead volumes and capacities of all components were optimized. Fully deuterated cytochrome c and its fully deuterated peptic peptides were used to evaluate deuterium recovery at amide linkages. The deuterium recovery measured at low flow rates using this system spanned a range of 66-77% (average of 71%), which was similar to the range measured for a much larger system (67-80%, average 75%). Signal levels of most peptides for the downsized system increased by about 100-fold compared with the signal for the larger system. These results greatly strengthen the HPLC HX MS technique for studies where the quantity of protein is small. 相似文献
12.
A new HPLC method was set up for the simultaneous evaluation of the amount of uric acid and NADH produced by incubation of tissue fractions containing xanthine oxidase, from which the activity of both type "O" (oxidase) and type "D" (dehydrogenase) xanthine oxidase can be calculated. After incubation of the enzyme fraction and ethanol extraction, HPLC analysis is directly carried out. Sensitivity of the method is high enough for the evaluation of xanthine oxidase activity at the lowest reported tissue values. The reliability of the method was tested measuring the enzyme activity in rat heart and kidney extracts. 相似文献
13.
Determination of saposin proteins (sphingolipid activator proteins) in human tissues 总被引:2,自引:0,他引:2
Saposins are small glycoproteins which are required for sphingolipid hydrolysis by lysosomal hydrolases. Each saposin (A, B, C, and D) stimulates a different enzymatic activity. A new simple HPLC method to determine the levels of saposins A, C, and D in tissue was developed. Tissues were homogenized in 20 vol of water, boiled, and centrifuged. The supernatant was lyophilized and redissolved in 5 ml of water. A 1.5-ml sample of the solution was applied to a reverse-phase HPLC column (C4 column) and eluted with an acetonitrile gradient. Most contaminants eluted from the column prior to the saposins, which were eluted later as a cluster of peaks. This cluster was collected and then analyzed by another HPLC system equipped with an AX-300 anion-exchange column using a NaCl gradient. Saposins D, A, and C eluted from the AX-300 column separately and in that order. Quantitation of the saposins was made by measuring the sizes of each peak. Standard curves made from pure saposins showed that quantification was linear over a range from 1 to 5 micrograms. Saposin B was measured by its stimulation activity on pure human liver GM1 ganglioside beta-galactosidase. Stimulation was linear up to 80 micrograms of saposin B. Application of this method to analysis of human tissues for their saposin content is presented. 相似文献
14.
Chaisuwan P Kongprasertsak T Sangcakul A Smith NW Nachapricha D Wilairat P Uraisin K 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(23):2185-2188
A simple CE-C(4)D method has been developed for the determination of glucosamine by direct injection of human serum and pharmaceutical samples. Glucosamine was electrokinetically injected and analysed in its protonated form using 20mM MES/His (pH 6) as background electrolyte in order to separate it from the matrix and to provide a better response to the C(4)D detector. Separation of glucosamine in human serum and pharmaceutical samples was performed in 3 min without the need for protein precipitation or matrix removal. Good precision in terms of %RSD for the migration time and peak area were less than 1.91% (n = 10). The conductivity signal was linear with glucosamine concentration in the range 0.10-2.50mg/mL, with a detection limit of 0.03 mg/mL. Recoveries of glucosamine in serum and pharmaceutical samples were 86.5-104.78%. The method was successfully applied for the determination of the glucosamine content in pharmaceutical formulations and validated with high performance liquid chromatography (HPLC). Good agreements were observed between the developed method, label values and the HPLC method. Glucosamine could be detected in spiked serum sample by direct injection. This was not possible by HPLC due to co-eluting interferences. 相似文献
15.
Y Huang H Matsunaga A Toriba T Santa T Fukushima K Imai 《Analytical biochemistry》1999,270(2):257-267
A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals. 相似文献
16.
A significant chromatographic isotope effect is reported for 1,25-dihydroxyvitamin D3 in a wide variety of HPLC separation systems. The effect is also observed for 24,25-dihydroxyvitamin D3. Retention times differ from less than 1% up to 4% depending on the separation system and the degree and position of tritium substitution. Such an effect must be corrected for whenever both labeled and unlabeled vitamin D metabolites are used in HPLC cochromatography or assay recovery studies. 相似文献
17.
Ranasinghe JG Sakakibara Y Harada M Nishiyama K Liu MC Suiko M 《Bioscience, biotechnology, and biochemistry》1999,63(1):229-231
A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions. 相似文献
18.
《Chirality》2017,29(6):294-303
(+)‐R ,R ‐D‐84 ((+)‐R ,R ‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol) is a promising pharmacological tool for the dopamine transporter (DAT), due to its high affinity and selectivity for this target. In this study, an analytical method to ascertain the enantiomeric purity of this compound was established. For this purpose, a high‐performance liquid chromatographic (HPLC) method, based on a cellulose derived chiral stationary phase (CSP) was developed. The method was characterized concerning its specificity, linearity, and range. It was shown that the method is suitable to determine an enantiomeric excess of up to 99.8%. With only a few adjustments, this analytical CSP‐HPLC method is also well suited to separate (+)‐R ,R ‐D‐84 from its enantiomer in a semipreparative scale. 相似文献
19.
D、L型氨基糖经荧光手性试剂 (S) ( ) 2 叔丁基 2 甲基 苯并 1 ,3 二氧杂环戊烷 4 甲酸 [(S) TBMB甲酸 ]标记、完全乙酰基化得到非对映的氨基糖完全乙酰基化N (S) TBMB羧酸衍生物。其1 HNMR图谱 ,特别是强的叔丁基及甲基信号峰被用于分析氨基糖的D、L构型。此外 ,还利用反相HPLC及同样的荧光标识方法创立了简便的高灵敏度氨基糖D、L构型分析方法。其全部分析操作时间在 2h内 ,检测极限为 0 .2 pmol 相似文献
20.
H Tsurui Y Kumazawa R Sanokawa K Watanabe A Wada T Shirai 《Nucleic acids symposium series》1991,(25):149-150
A novel method for the purification of a specific tRNA using solid phase DNA probe is developed. With this method, the probe DNA immobilized on HPLC gel hybridized with target tRNA within a minute at room temperature. The hybridizing capacity of the solid phase probe was about 20 O.D. per gram dry gel when yeast phenylalanine tRNA was used. The specificity of this method was extremely high and the recovery rate was about 90%. 相似文献