强阳离子交换色谱分离多肽混合物的条件优化

以酵母全蛋白提取液的胰蛋白酶切产物为研究对象,对多维色谱分离中常用的强阳离子交换色谱的分离条件,包括上样量、盐的种类、调节缓冲液pH值的酸的种类及有机溶剂的比例进行了考察和比较。结果表明,在强阳离子交换色谱分离线性上样量范围内,在氯化铵溶液作为流动相,用磷酸调节流动相缓冲液pH值至2.7,且流动相中乙腈的体积分数为30%条件下进行梯度洗脱时,可获得最佳的分离结果。本结果可为采用二维色谱-质谱策略分析多肽混合物时的第一维强阳离子交换色谱分离条件的选择提供有益的参考。     

反相高效液相色谱双梯度洗脱分离肽混合物及质谱分析

复杂肽段混合物的有效分离是高覆盖率地鉴定蛋白质混合物的前提。“鸟枪法”(Shotgun)蛋白质组学研究策略通常采用蛋白酶切、二维液相色谱-串联质谱分析肽段混合物从而鉴定蛋白质,其中高效率地分离肽段混合物是关键步骤之一。本文通过pH梯度结合有机溶剂梯度的反相高效液相色谱(RP-HPLC)进行一维液相色谱分离,按等时间间隔收集馏分并将一个梯度的前段的一个馏分与后段一个馏分混合,然后进行纳升级液相色谱-质谱联用(nanoRPLC-MS/MS)分析。将该方法应用于酵母蛋白质的分离和鉴定,实验结果为: 与常规的强阳离子色谱-反相液相色谱-质谱分离鉴定方法相比,采用pH梯度结合有机相梯度的RP-HPLC-RPLC-MS分离鉴定方法多鉴定到567个酵母蛋白质(簇,含有3035个唯一肽段);其中鉴定到肽段的pI分布范围为3.42～12.01,相对分子质量范围为587.67～3499.79;蛋白质的pI分布范围为3.82～12.19,相对分子质量范围为3446.55～432905。该结果表明这种方法在复杂体系蛋白质组分离鉴定中具有明显的优势,在蛋白质组学研究中有较好的应用前景。     

强阳离子交换毛细管液相色谱-反相加压毛细管电色谱二维系统的构建及其在中药黄柏提取物分离中的应用

加压毛细管电色谱(pCEC)具有电泳和液相色谱的双重分离机理,其柱效高、选择性强、分辨率高和分离速度快并可进行梯度洗脱。我们在此基础上加入离子交换色谱模式,构建了强阳离子交换-反相加压毛细管液相色谱(micro strong cation exchange liquid chromatography/reversed phase pressurized capillary electrochromatography, μ-SCXLC/RP-pCEC)二维系统,并对中药黄柏的提取物进行了优化分离。第一维μ-SCXLC采用线性盐梯度分离,样品被切割成11个馏分洗脱收集后进入第二维,第二维脱盐后,采用RP-pCEC进行分离分析,梯度洗脱。以中药黄柏提取物为样品,此二维系统的分辨率和峰容量都较一维系统有很大提高,理论峰容量可达900左右,证明构建的二维体系非常适合复杂样品的分离分析。     

全二维微柱液相色谱-质谱鉴定人胃癌组织中的差异蛋白质

构建了以阳离子交换色谱-反相色谱(SCX-RPLC)为分离模式的新型全二维微柱液相色谱-质谱分离平台.采用了醋酸铵缓冲液梯度洗脱,实现了第一维肽段的分步洗脱,洗脱的肽段经富集除盐后通过接口进入反相色谱微柱,通过线性梯度实现第二维进一步分离,最后进入质谱进行检测.采用此平台分析了人胃癌组织与正常组织提取蛋白质信息,其中正常胃组织鉴定蛋白质数为537个,而癌症组织鉴定蛋白质数目为506个.对胃癌和正常组织两种提取蛋白质酶解产物的蛋白质检索结果进行比较分析,将鉴定的蛋白质按照物理性质进行分布,找出正常组织与癌症组织间蛋白质差异,筛选出一种可能发生变异的癌症特有蛋白.     

新型全二维微柱液相色谱分离平台的构建

采用自动进样器和一套二元梯度泵构建了以阳离子交换色谱-反相色谱(SCX-RPLC)为分离模式的新型全二维微柱液相色谱分离平台.在第一维分离中,通过自动进样器将不同浓度的盐溶液以台阶梯度输送至SCX柱上,实现肽段的分级洗脱.洗脱下的肽段经C8预柱富集、除盐后,进入第二维C18 RPLC柱上,通过线性梯度实现进一步的分离.采用该平台分离了5种标准蛋白的酶解产物,系统峰容量可达1 467.结果表明,该平台可以自动完成进样、除盐、分离及检测.     

制药废液中氨基酸的分离与测定

为测定维生素 B12 制药厂排出的废液中氨基酸量,先在所取的废液样品中加入乙酸铅使其中蛋白质及其他大分子有机物产生沉淀并用离心法分离.上层澄清液中的氨基酸通过装有强酸性阳离子交换树脂的交换柱(25 cm×2.5 cm)予以分离,用1 mol·L-1 氨水将氨基酸从柱上洗脱.在所得洗脱液中用气相色谱-质谱及傅立叶变换红外光谱法对氨基酸进行定性及定量测定.     

用阳离子交换树脂和pH梯度洗脱分离头孢菌素混合物

合成了一种聚苯乙烯结构的凝胶型1×2强酸性阳离子交换树脂,结合pH梯度或盐浓度梯度洗脱法分离头孢菌素类抗生素的混合物,并与市售的树脂比较,考察其分离性能。1×2型树脂具有较小的颗粒度(0.2～0.3mm)和较小的交换容量(在60%乙醇溶液中),用pH梯度洗脱法可以将5种头孢菌素按照等电点顺序完全分离,结构稳定的3种头孢菌素的收率均大于95%,1×2型强酸性阳离子交换树脂结合pH梯度洗脱法适用于水溶性头孢菌素类抗生素的制备分离。     

LC-ESI MS/MS测定蜜胺餐具中三聚氰胺的迁移量

建立了液相色谱串联电喷雾正离子源质谱(LC-ESI MS/MS)检测蜜胺餐具中三聚氰胺迁移量的方法.采用强阳离子交换柱,流动相为乙腈-10 mmol/L乙酸铵/乙酸缓冲溶液(pH 4.0)(40:60,体积比),以多反应离子监测对三聚氰胺做定性定量分析.在水、3%乙酸、10%乙醇模拟物中,三聚氰胺在3.00～130.0...     

二维液相色谱-质谱联用分离中药复方葛根芩连汤中的有效成分

建立了二维液相色谱-质谱联用方法分离中药复方葛根芩连汤的成分。以CN柱作第一维色谱柱,水和甲醇梯度洗脱分离;以ODS柱作第二维色谱柱,20 mmol/L乙酸铵缓冲液和乙腈梯度洗脱分离;质谱检测采用电喷雾电离/大气压化学电离（ESI/APCI）复合离子源,正负离子扫描。实验结果表明搭建的二维液相色谱的峰容量显著高于一维色谱,分离效率得到了明显的提高。以第一维色谱的第3个流分为例,对其二维分离进行仔细分析,发现质谱比紫外光谱检测到的组分多,质谱中采用负离子模式比正离子模式检测到的组分多。表明搭建的二维液相色谱-质谱分离平台分离效果好,提高了液相色谱的峰容量和分离效率。该方法操作简便,可作为中药等复杂体系分离分析的有效手段。     

离子交换色谱-荧光检测法测定化妆品中苯海拉明及串联质谱确证

建立了化妆品中苯海拉明的离子交换色谱-荧光检测方法及液相色谱-串联质谱确证方法。样品用含0.2%甲酸的乙腈提取,定量检测时以乙腈-0.05 mol/L KH2PO4溶液(7+3,V/V)为流动相,强阳离子交换色谱柱(250×4.6 mm,5μm)分离,荧光检测激发波长258 nm,发射波长288 nm,外标法定量,苯海拉明检出限为1.5 mg/kg,在0.2~20 mg/L范围内线性关系良好,相关系数为0.9999,方法的回收率在87.6%~106.3%范围内,相对标准偏差低于6.3%。质谱确证时用BEH C18色谱柱分离,乙腈-0.2%甲酸系统梯度洗脱,采用质谱碎片和相对离子丰度进行定性确认。     

Purification of the glycoprotein glucose oxidase from Penicillium amagasakiense by high-performance liquid chromatography

Fast protein liquid chromatography (FPLC) in combination with ion-exchange chromatography on a Mono Q column was used to purify glucose oxidase from Penicillium amagasakiense to homogeneity. Purification was performed with a mixed pH and salt gradient, with 20 mM phosphate buffer (pH 8.5) as starting buffer (A) and 50 mM acetate buffer (pH 3.6) with 0.1 M NaCl as elution buffer (B). Elution conditions were optimized to permit the simultaneous purification and separation of the glucose oxidase isoforms. Three peaks, each consisting of 1-2 isoforms and exhibiting a homogeneous titration curve profile, were resolved with a very flat linear gradient of 5.0-5.1% B in 40 ml. Three more peaks, each consisting of several isoforms, were eluted at 10%, 30% and 100% B. Optimization of the elution conditions and separation of the glucose oxidase isoforms was only possible because of the rapidity of each purification step and the high resolution provided by FPLC and Mono Q.     

Monolithic capillary columns synthesized from a single phosphate-containing dimethacrylate monomer for cation-exchange chromatography of peptides and proteins

Monoliths containing phosphoric acid functional groups were synthesized from only one monomer, bis[2-(methacryloyloxy)ethyl] phosphate (BMEP), in 75-μm i.d. UV transparent fused-silica capillaries by photo-initiated polymerization for cation exchange chromatography of peptides and proteins. Various synthetic conditions, including porogen solvents, monomer concentration, and polymerization time, were studied. The hydrophobicities of the resulting monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. These monoliths exhibited low hydrophobicities and relatively low porosities due to their highly cross-linked structures. A dynamic binding capacity (lysozyme) of 73 mg/mL of column volume was measured using the best performing monolith. Synthetic peptides were eluted in approximately 30 min without addition of acetonitrile to the mobile phase, yielding a peak capacity of 28. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. The effects of separation conditions, i.e., mobile phase pH and salt gradient rate, were studied. Good run-to-run reproducibility was achieved with a relative standard deviation (RSD) less than 1.5% for retention times of proteins. The column-to-column retention time reproducibility for peptides was less than 3.5% RSD. A monolithic column was used to follow the deamidation of ribonuclease A. The kinetics of deamidation were founded to be first order with a half life of 195 h. A cytochrome C digest was also separated using a linear gradient of sodium chloride.     

Ligand exchange chromatography for analysis and preparative separation of tritium-labelled amino acids

Racemic tritium-labelled amino acids were separated into optical isomers by chromatography on a chiral polyacrylamide sorbent filled with copper ions. The polyacrylamide sorbent is synthesized by Mannich's reaction through the action of formaldehyde and L-phenylalanine upon polyacrylamide Biogel P-4 in an alkali phosphate buffer. Tritiumlabelled amino acids are eluted by a weak alkali solution of ammonium carbonate. Data are presented on the ligand exchange chromatography of amino acids depending on the degree to which the sorbent is filled with copper ions and on the eluent concentration. Conditions are suggested for the quantitative separation of amino acid racemates. Amino acids are isolated from the eluent on short columns filled with sulfonated cation exchanger in the H⁺ form. HPLC on modified silica gel sorbents is also used for the analysis of tritium-labelled optically active amino acids. Amino acids are eluted by a weakly acidic water-methanol solution containing ammonium acetate. UV and scintillation flow type detectors are used.     

Analysis of synthetic peptides by capillary electrophoresis: effect of organic solvent modifiers and variable electrical potentials on separation efficiencies

In this study, procedures based on volatile ammonium acetate buffer electrolytes of high pH value containing different organic solvent modifiers have been developed to achieve very high efficiency separations of histidine-containing synthetic peptides by high-performance capillary electrophoresis (HPCE) employing untreated fused silica capillaries. Different organic solvents, including acetonitrile, methanol and ethanol, at high volume fractions were used to modify the composition of the background buffer electrolyte. With the peptides investigated, it was found that methanol had the greatest effect in terms of enhancement of separation efficiency, as determined from the evaluation of theoretical plate numbers, N, of these HPCE systems. On the other hand, separation selectivities, e.g. the alpha(ij) values, did not change significantly as the volume fraction, psi, of the organic solvents was increased up to psi = 60% (v/v). Under these conditions, very rapid, e.g. 1-2 min, separation times could be still achieved. Compared to the effect of carrying out the separation of these peptides at constant voltage, a dramatic increase in the separation efficiency was also achieved by applying a linear voltage gradient during the HPCE experiment. Under optimal conditions of organic solvent composition and linear voltage gradient ramps, very high peak efficiencies for the studied set of synthetic peptides with N values of approximately 2-3 million theoretical plates per meter could be routinely obtained with fast analysis times. Moreover, these buffer electrolyte conditions are compatible with direct interfacing of the HPCE effluent to electrospray ionisation and ion trap mass spectrometers, thus expanding the analytical capabilities of these HPCE systems.     

Separation of pyrimidine deoxyribooligonucleotides according to length and composition using thin-layer chromatography on deae-cellulose.

A procedure has been developed for the fractionation of pyrimidine deoxyribo-oligonucleotides, PynPn+1, according to length and composition using ascending thin-layer chromatography on DEAE-cellulose. The separation of oligonucleotides according to length (n=1-7) into individual isopliths was carried out using a linear 0-0.35 M sodium chloride gradient in 0.01 M sodium acetate buffer of pH 5.1-5.3 and 5 M urea. Then the oligonucleotides of individual isopliths were separated according to composition using the same gradient of sodium chloride in 0.01 M sodium acetate or sodium citrate buffer of pH 3.2. The procedure can be used for rapid fractionation (2-3 h) and serial determination of the frequencies of pyrimidine sequences differing in length and composition in DNAs of various origin. The procedure can be used for both preparative and analytical applications. It was employed to study the distribution of 5-methylcytosine in pyrimidine isopliths of some DNAs.     

Determination of uranium in technological waters by ion-pair liquid chromatography

U(VI) can be efficiently determined in the range 0.3-1OmM after its separation from Th(IV), Zr(IV), Al(III), Fe(III), lanthanides and other ions by ion-pair liquid chromatography on a 3 x 150 mm glass column packed with Separon SGX C18 modified with sorbed ammonium dodecyl sulphate. Traces of uranium can be preconcentrated directly on the analytical column from acidified water solutions and separated from Th, Zr, Al, Fe, lanthanides and other elements, with an enrichment factor of $ 100 and recovery of 98 +/- 8%, by isocratic or pH or concentration gradient elution with ammonium 2-hydroxy-2-methylpropionate or ammonium citrate solution. Post-column derivatization with 25muM Arsenazo III in 0.1M formate buffer at pH 2.7 is used for detection and quantification.     

Chiral CE of aromatic amino acids by ligand-exchange with zinc(II)-L-lysine complex

A novel method of chiral ligand-exchange CE was developed with either L- or D-lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4) x 7H(2)O and 6 mM L-Lys at pH 7.6, unlabeled D,L-tryptophan, D,L-phenylalanine, and D,L-tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2-4 mM and running buffer pH 7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L-Lys to D-Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L-isomers eluted before or after D-isomers in excessive D- or L-Lys, respectively.     

Analysis of neuropeptide Y and its metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger

A novel restricted access cation exchanger with sulphonic acid groups at the internal surface was proven to be highly suitable in the sample clean up of peptides on-line coupled to HPLC-electrospray ionization (ESI)-MS. Neuropeptide Y (NPY) and several of its fragments in plasma were subjected to the sample clean-up procedure. The peptides were eluted by a step gradient from the restricted access column, applying 10 mM phosphate buffer pH 3.5 from 5 to 20% (v/v) of acetonitrile with 1 M NaCl and transferred to a Micra ODS II column (33x4.6 mm). The separation of the peptides and their fragments was performed by a linear gradient from 20 to 60% (v/v) acetonitrile in water with 0.1% formic acid and 0.01% trifluoroacetic acid in 4 min at a flow-rate of 0.75 ml/min. An integrated and completely automated system composed of sample clean up-HPLC-ESI-MS was used to analyze real life samples. The sample volumes ranged between 20 and 100 microl. Peaks due to the fragments NPY 1-36, 3-36 and 13-36 in porcine plasma were identified by ESI-MS. The limit of detection was in the 5 nmol/ml range. The total analysis required 21 min and allowed the direct injection of plasma.     

Separation of proteins by hydrophobic interaction chromatography at low salt concentration

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.