Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: II. Experimental results

Part I of this study investigated the theory and basic characteristics of “serial displacement chromatofocusing” (SDC). In Part II of this study, SDC is applied to two prototype applications which have potential uses in proteomics and related areas involving the analysis of complex analyte mixtures. In the first application, SDC was used as a prefractionation method prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate a human prostate cancer cell lysate. It was observed that the resolution achieved in narrow-pI-range 2D-PAGE was improved when using SDC prefractionation, so that SDC may be useful as a low-cost, high-speed, and highly scalable alternative to electrophoretic prefractionation methods for 2D-PAGE. The second application involves the use of SDC as the first dimension, and reversed-phase chromatography as the second dimension, to produce a novel, fully automated, two-dimensional high-performance liquid chromatography technique. The method was shown to have performance advantages over one-dimensional reversed-phase chromatography for peptide separations.     

High-throughput analytical strategy with combined planar and column liquid chromatography for improvement of the poppy (Papaver somniferum L.) with a high alkaloid content

Summary Poppy capsules with a high alkaloid content are of great importance to the pharmaceutical industry, because approximately 35 000 tons of straw (capsule with short stem), 350 tons of straw concentrate, and 1000 tons of opium are used annually for extraction of morphinane alkaloids. The combined use of four different liquid chromatographic methods is proposed for determination of alkaloid content. In the first, semi-quantitative, method screening is performed by multilayer overpressured-layer chromatography (MLOPLC) in which 142 samples are separated on silica as stationary phase within 5 s per sample. If the morphine content is >1.3% it is then measured quantitatively by NPHPTLC (normal-phase high-performance thin-layer chromatography) combined with densitometric evaluation. A second, quantitative, determination is always performed by means of a rapid RPHPLC (reversed-phase high-performance liquid chromatography) method in which the separation time is less than 4 min per sample. If the difference between the alkaloid content measured by use of the last two methods is >12% a confirmatory RPHPLC method with increased selectivity and analysis time (15 min) must be used. The high throughput strategy presented has been used for analysis of ca. 15 000 samples per year, per genotype. Systematic selection, cross-breeding, and this analytical strategy with combined planar and column liquid chromatographic methods resulted in the discovery of two new candidate cultivars with high morphine (ca. 2%) and thebaine (ca. 1.5%) content. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001.     

Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography

We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.     

Separation of Diethylstilbestrol and Derivatives in Biological Fluids and Tissues by High Pressure Liquid Chromatography

Abstract

A method was developed to identify radiolabeled DES and DES metabolites in biological fluids and tissues. After a rapid initial clean-up step, the biological samples were analyzed with a single-column, reversed-phase, highpressure liquid chromatography system. The parent compound and metabolites were simultaneously resolved and tentatively identified by comparing their retention times to those of known standards. Positive identification of some metabolites was achieved by field-desorption and capillary-column mass-spectrometric analysis. The method was found to be rapid and reproducible, and sample recovery was >80%.     

利用亲水富集策略分析人血浆中N-糖基化蛋白及N-糖类型

蛋白质的N-糖基化是最重要的翻译后修饰之一,许多已知的血浆肿瘤诊断标志物及治疗靶标都是N-糖基化蛋白。针对血浆的糖蛋白质组研究有利于发现新的蛋白标志物。然而,血浆蛋白质浓度分布的动态范围非常宽,且同一位点上的糖链存在微观不均一性,影响了血浆中糖蛋白的鉴定效率。本文利用亲水材料ZIC-HILIC制备亲水富集柱分别对人血浆中的N-糖链和N-糖肽进行富集,并结合碱性反相色谱进行肽段的预分离和高准确度质谱分析,最终在健康人的血浆中鉴定到了299个糖基化蛋白、637个糖基化位点,并识别出31种不同的糖型。在这些鉴定到的糖基化位点中,新发现有107个N-糖基化位点(占总位点数的16.8%)。本方法操作简单,可以有效富集N-糖肽和N-糖,为在血浆中寻找糖蛋白和糖链生物标志物提供了可靠的手段。     

A comprehensive two-dimensional normal-phase × reversed-phase liquid chromatography based on the modification of mobile phases

A comprehensive orthogonal two-dimensional liquid chromatography (2D-LC) based on the modification of mobile phases was developed with a sample loop–valve interface. To improve the compatibility of mobile phases and analysis speed, some special solvents were chosen as the mobile phases, and the column temperature was elevated to decrease the viscosity of mobile phases of reversed-phase liquid chromatography (RPLC). Based on this principle, the first dimension was normal-phase liquid chromatography (NPLC) with a SiO₂ column, and the second dimension was reversed-phase liquid chromatography containing two tandem C18 columns. 1,4-Dioxane was used in the NPLC mobile phase, and isopropyl alcohol was employed in the RPLC mobile phase. Moreover, the elevated column temperature enabled the reduction of the backpressure and using tandem C18 columns to improve the resolving power in RPLC. The new comprehensive 2D-LC system and applied strategy offered a novel idea for construction of 2D-LC system. A traditional Chinese medicine, Zhengtian pill, was used as the test sample to evaluate the constructed 2D-LC system. 876 peaks were detected, and the peak capacity reached 1740.     

Liquid chromatographic characterization of the deoxyribonucleoside-5’-phosphates and deoxyribonucleoside-3’,5’-bisphosphates obtained by32P-postlabeling of DNA

Summary The use of high-performance liquid chromatography (HPLC) for the characterization of the adducts formed by the³²P-postlabeling of DNA is described. Adducts formed from the reaction of DNA with small molecules are characterized as deoxyribonucleoside-5′-monophosphates. Adducts formed from the reaction of DNA with larger molecules are characterized as 3′,5′-bisphosphates. In either case, good separations are obtained for adducts, other than methyl, by conventional reversed-phase chromatography using a C₁₈ column. Ion-exchange chromatography can be used in selected circumstances where reversed-phase techniques are not successful. Using sample concentration techniques, sensitivities are achievable which make this technique applicable toin vivo situations.     

Evaluation of sample fractionation using micro-scale liquid-phase isoelectric focusing on mass spectrometric identification and quantitation of proteins in a SILAC experiment

Mass spectrometric methods based on stable isotopes have shown great promise for identification and quantitation of complex mixtures. Stable isotope labelling by amino acids in cell culture (SILAC) is a straightforward and accurate procedure for quantitation of proteins from cell lines, that are cultured in media containing the natural amino acid or its isotopically labelled analogue, giving rise to either 'light' or 'heavy' proteins. The two cell populations are pooled and treated as a single sample, which allows the use of various protein purification methods without introducing errors into the quantitative analysis. The quantitation of the proteins is based on the intensities of the light and heavy peptides. The increased number of peptides in a quantitative experiment arising from peptide pairs implies that prefractionation is critical prior to liquid chromatography/mass spectrometric (LC/MS) analysis to minimise signal suppression effects and errors in measurements of the intensity ratios. In this study, the effect of a prefractionation step on identification and quantitation of proteins in a SILAC experiment was evaluated. We show that micro-scale liquid-phase isoelectric focusing in the Micro Rotofor separates proteins into well-defined fractions and reduces the sample complexity. Furthermore, the fractionation enhanced the number of identified proteins and improved their quantitation.     

Quantitation of Branched Chain α-Keto Acids in Sheep Plasma Using Reversed-Phase High Performance Liquid Chromatography and Quinoxalinol Derivatization

Abstract

The concentration of branched chain α-keto acids in sheep plasma was determined by reversed-phase high performance liquid chromatography using precolumn quinoxalinol formation and ion exchange chromatography for sample preparation. The clear resolution, high degree of precision and accuracy, and relatively simple sample cleanup and derivatization procedures render this technique suitable for the routine analysis of physiological fluids.     

Prefractionation techniques in proteome analysis: the mining tools of the third millennium

The present review deals with prefractionation protocols used in proteomic investigation in preparation for mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) map analysis. Briefly, reported methods focus on cell organelle differential centrifugation and on chromatographic approaches, to continue in extenso with a panoply of electrophoretic methods. In the case of chromatography, procedures useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins, revealed several hundreds of new species, previously undetected in unfractionated samples. Novel chromatographic prefractionation methods are also discussed such as a multistaged fractionation column, consisting in a set of immobilized chemistries, serially connected in a stack format (an assembly of seven blocks), each capable of harvesting a given protein population. Such a method significantly simplifies the complexity of treated samples while concentrating species, all resulting in a larger number of visible proteins by MS or 2-DE. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separation are multichamber apparatus, such as the multicompartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads. Multicup device electrophoresis and several others, exploiting the conventional technique of carrier ampholyte focusing, are reviewed. This review also reports approaches for sample treatments in order to detect low-abundance species. Among others, a special emphasis is made on the reduction of concentration difference between proteins constituting a sample. This latter consists in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of 20 amino acids, resulting in millions of different structures. When these beads are impregnated with complex proteomes (e.g., human sera) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility to evidence low-abundance species. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".     

Preparative Reversed-Phase Liquid Chromatographic Isolation of Azadirachtin from Neem Kernels (1)

Abstract

Azadirachtin is a tetranortriterpenoid insect feeding deterrent and growth regulator present in the kernels of the neem tree, Azadirachta indica A. Juss. A procedure was developed whereby 8.7 g of >90% pure azadirachtin was obtained from 48.2 kg of neem kernels by the use of open-column reversed-phase liquid chromatography on Phase-bonded C-18 Hi-Flosil and high-performance liquid chromatography on μ Bondapak C-18 for monitoring (217 nm) the purification.     

Reversed-phase high-performance liquid chromatography prefractionation prior to two-dimensional difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat

Two-dimensional difference gel electrophoresis (2-D DIGE), in combination with mass spectrometry, is a highly effective method for the rapid and reproducible detection of differentially expressed proteins. This approach, however, has the unfortunate drawback that it preferentially displays rather abundantly expressed proteins. Nevertheless, comparison of the protein expression levels of the striate cortex of adult cats and 30-day-old kittens, resulted in the identification of several proteins related to postnatal brain development and possibly age-dependent plasticity as well (Van den Bergh et al., J. Neurochem. 2003, in press). The goal of the present study was the selective enrichment and identification of less abundant proteins within the same paradigm. Hereto, we performed a reversed-phase chromatography prefractionation of our tissue lysate to separate the proteins in four fractions based on their hydrophobicity prior to 2-D DIGE analysis. This approach not only confirmed the differential expression levels of a number of proteins from the previous study, but also identified three additional proteins preferentially expressed in kitten visual cortex and five additional proteins with higher expression levels in adult cat visual cortex. These spots were not visible on the total tissue lysate protein maps, indicating that the high-performance liquid chromatography (HPLC) prefractionation enabled us to visualize additional, less abundant proteins.     

Dilute-and-shoot approach for determination of several biomarkers and pharmaceuticals in wastewater using nanoflow liquid chromatography – Orbitrap mass spectrometry

A new method for quantitative analysis of several biomarkers and pharmaceutical compounds in wastewater has been developed employing nanoflow liquid chromatography with Orbitrap mass spectrometry. An easy dilute-and-shoot approach has been used for sample preparation with a dilution factor of 5. Improved retention of ionic and highly polar compounds has been achieved by the addition of tetrabutylammonium bromide as an ion pair reagent into the final diluted sample. The new nanoflow liquid chromatography method has demonstrated low matrix effects (70%–111%), high sensitivity in terms of limits of quantification (0.005 to 0.3 μg/L), low injection volume (70 nl) and solvent consumption, and the ability to analyze diverse polar and ionic analytes within one run using a single reversed-phase nanoflow liquid chromatography column. Wastewater samples (n = 116) from the wastewater treatment plants of different cities in Latvia were analyzed using the developed method. The observed concentrations of biomarkers were in line with the literature data.     

Reversal of the enantiomeric elution order of some aromatic amino acids using reversed-phase chromatographic supports coated with the teicoplanin chiral selector

In this paper, two chiral stationary phases were prepared by coating the surface of both C8 and C18 high-performance liquid chromatography (HPLC) supports with the teicoplanin chiral selector. The hydrophobic C11 acyl side chain, attached to the d-glucosamine group of teicoplanin, served as anchor moiety for the immobilization of the chiral selector on the apolar support material. The retention and enantioselectivity of these coated stationary phases were studied using some aromatic amino acids as probe solutes and an aqueous solution as mobile phase. It was found that the enantiomer elution order on the modified C8 and C18 stationary phases was reversed (l > d) relatively to that classically observed with a teicoplanin covalently immobilized on a silica support (d > l). Such a dynamic coating on the reversed-phase supports was found to be of interest since the apparent enantioselectivity was not significantly changed by the use during an extended period of time or following a long-term storage of the columns.     

复杂样品痕量极性小分子化合物的样品前处理及分析方法研究进展

核苷、胺、氨基酸等极性小分子化合物是生物、食品、环境等领域重要的研究对象,但各种复杂基体中痕量极性小分子的分离分析需要高效的前处理介质和技术以及快速灵敏的分析方法。本文综述了硅胶材料、有机聚合物、炭材料和硼酸材料等样品前处理分离介质及反相液相色谱、亲水作用色谱等分析方法在复杂样品痕量极性小分子化合物分析中的应用,并展望了其发展趋势。     

Analysis of doping control samples using supercritical fluid chromatography-tandem mass spectrometry: Ready for routine use

Supercritical fluid chromatography is proving to be a good separation and sample preparation tool for various analytical applications and, as such, has gained the attention of the anti-doping community. Here, the applicability of supercritical fluid chromatography hyphenated to tandem mass spectrometry for routine doping control analysis was tested. A multi-analyte method was developed to cover 197 drugs and metabolites that are prohibited in sport. More than 1000 samples were analyzed by applying a “dilute and inject” approach after hydrolysis of glucuronide metabolites. Additionally, a comparison with routinely used liquid chromatography-mass spectrometry was performed with 250 of the 1000 samples and a number of past positive anti-doping samples. It revealed some features where supercritical fluid chromatography-tandem mass spectrometry was found to be complementary or advantageous to liquid chromatography-mass spectrometry for anti-doping purposes, such as better retention of analytes that are poorly retained in reversed-phase liquid chromatography. Our results suggest that supercritical fluid chromatography-tandem mass spectrometry is sensitive (limit of detection <50% relevant minimum required performance level required by the World Anti-Doping Agency for anti-doping analysis), reproducible, robust, precise (analytes of interest area coefficient of variation <5%; retention time difference coefficient of variation <1%) and complementary to existing techniques currently used for routine analysis in the World Anti-Doping Agency accredited laboratories.     

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity > 95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.     

Free flow electrophoresis coupled with liquid chromatography-mass spectrometry for a proteomic study of the human cell line (K562/CR3)

The requirement for prefractionation in proteomic analysis is linked to the challenge of performing such an analysis on complex biological samples and identifying low level components in the presence of numerous abundant housekeeping and structural proteins. The employment of a preliminary fractionation step results in a reduction of complexity in an individual fraction and permits more complete liquid chromatography/mass spectrometry (LC/MS) analysis. Free flow electrophoresis (FFE), a solution-based preparative isoelectric focusing technique, fractionates and enriches protein fractions according to their charge differences and is orthogonal in selectivity to the popular reversed phase high performance liquid chromatography (HPLC) fractionation step. In this paper, we explored the advantages of a combination of FFE and liquid chromatography/mass spectrometry to extend the dynamic range of a proteomic analysis of a complex cell lysate. In this study, the whole cell lysate of a chronic myelogeneous leukemia cell line, K562/CR3, was prefractionated by FFE into 96 fractions spanning pH 3-12. Of these, 35 fractions were digested with trypsin and then analyzed by LC/MS. Depending on the algorithm used for peptide assignment from MS/MS data, at least 319 proteins were identified through database searches. The results also suggested that pI could serve as an additional criterion besides peptide fragmentation pattern for protein identification, although in some cases, a pI shift might indicate post-translational modification. In summary, this study demonstrated that free flow electrophoresis provided a useful prefractionation step for proteomic analysis and when combined with LC/MS allowed the identification of significant number of low level proteins in complex samples.     

Rapid analysis of peptide toxins in cyanobacteria

A quick and easy-to-perform method for routine analysis of cyanobacterial (blue-green algal) peptide toxins is proposed. The toxins are analysed by means of high-performance liquid chromatography using a recently developed internal surface reversed-phase column. The sample clean-up work is minimized and the total analysis time is thus shortened considerably compared to previously described methods.