色谱技术在药物-血浆蛋白相互作用研究中的应用进展

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α₁ -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。     

Human complement factor 3 polymorphism determination by capillary electrophoresis of serum

Variability of complement factor 3 (C3) mobility in serum protein electrophoresis was investigated. We found that the migration time of C3 can be reproducibly determined (beween‐run CV=0.76%) using clinical capillary electrophoresis (CE) equipment (the Capillarys? 2 system, Sebia). Moreover, we found a significant difference (p<0.001) in migration times of the major C3 phenotypes FF (fast‐fast), FS (fast‐slow) and SS (slow‐slow). Glycosylation did not significantly affect test results. This is the first report on the migration time of C3 phenotypes on a clinical CE instrument. The presented method allows faster data than agarose‐electrophoresis or genotyping. Moreover, reference ranges for serum C3 concentration depend on C3 phenotype, which allows a better tailored clinical interpretation of C3 concentrations.     

Capillary electrophoresis-based separation techniques for the analysis of proteins

CE offers the advantages of high speed, great efficiency, as well as the requirement of minimum amounts of sample and buffer for the analysis of proteins. In this review, we summarize the CE-based techniques coupled with absorption, LIF, and MS detection systems for the analysis of proteins mostly within the past 5 years. The basic principle of each technique and its advantages and disadvantages for protein analysis are discussed in brief. Advanced CE techniques, including on-column concentration techniques and high-efficiency multidimensional separation techniques, for high-throughput protein profiling of complex biological samples and/or of single cells are emphasized. Although the developed techniques provide improved peak capacity, they have not become practical tools for proteomics, mainly because of poor reproducibility, low-sample lading capacity, and low throughput due to ineffective interfaces between two separation dimensions and that between separation and MS systems. In order to identify the complexities and dynamics of the proteomes expressed by cells, tissues, or organisms, techniques providing improved analytical sensitivity, throughput, and dynamic ranges are still demanded.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.     

Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.     

The analysis of human amniotic fluid using capillary electrophoresis

This study has investigated the composition of amniotic fluid (AF) using capillary electrophoresis (CE). A detailed optimisation investigation was undertaken to obtain the best resolution of the major peaks in amniotic fluid. In the final method, capillary zone electrophoresis (CZE) of AF was performed on a Hewlett Packard3D CE instrument using a fused-silica capillary of 44 cm total length (36 cm to the detector) with in internal diameter of 50 microm. The background electrolyte was 20 mM sodium tetraborate containing 0.8 mM EDTA adjusted to pH 9.0. AF was diluted 1 plus 1 with deionised water prior to hydrodynamic injection for 3 s at 50 mbar. The separation was performed at +22.5 kV and resulted in a current of 65 microA. The capillary temperature was 28 degrees C. Using this CZE method, some eight peaks were consistently resolved in AF samples and several other more transient peaks have been separated from AF in less than 10 min. A scheme for the identification of peaks once they had been separated was also developed. Four peaks have been identified as proteins, i.e., gamma-globulin, alpha1-antitrypsin, transferrin and albumin. Surprisingly, one major peak was shown to be the purine catabolite, xanthine.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.     

Cover Picture: Electrophoresis 21'2010

Issue no. 21 is a regular issue with "Emphasis on Nucleic Acids" and comprises 17 contributions distributed over 4 distinct parts. Part I is on nucleic acids and has 8 research articles on various aspects of nucleic acid research including an automated instrument for human STR identification, a high resolution chip‐CE assay for rapid detection of gene mutations and amplifications, a fluorescence‐based sequence‐specific primer PCR, pulsed field CE for RNA separation, methylated DNA immunoprecipitation sequencing, PCR‐SSCP, DNA methylation, and analysis of SNPs. Part II has 4 research articles dealing with studies on protein and proteomics analysis, e.g., microchip IEF, CE analysis of microheterogeneous glutelin subunits in rice, protein precipitation methods and prefractionation of plasma for proteomic analysis. Part III has 2 contributions that describe the separation of the enantiomers of DL‐isocitric acid by CE and the analysis of trace degradation products of S‐adenosylmethionine, which are decarboxylated diastereomers, by CE. The last 3 articles in this issue (Part IV) are on novel methodologies for facile interfacing of CE with MS, electrokinetic chromatography for the determination of contaminants and impurities of heparin samples, and particle electrophoresis. As such, issue no. 21 is packed with the latest developments and innovations in the field. Featured articles include: An automated instrument for Human STR identification: design, characterization, and experimental validation (doi: 10.1002/elps.201000305 )) Miniature flowing atmospheric‐pressure afterglow ion source for facile interfacing of capillary electrophoresis with mass spectrometry (doi: 10.1002/elps.201000350 )) Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.). (doi: 10.1002/elps.201000333 ))     

Optimization of capillary electrophoresis conditions for coupling to a mass spectrometer via a sheathless interface

When optimizing a capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) system, consideration has to be given not only to the separation but also to the electrospray stability. Methods developed for CE/UV analysis of drugs and peptides were considered and modified to be suitable for a CE/MS system with a robust sheathless interface. Different concentrations of the organic modifiers acetonitrile, methanol and 2-propanol were used in the separation buffer. The type and concentrations of these modifiers were also compared with reference to electrospray stability, sensitivity and time of analysis. In addition, different ionic strengths in the buffers were evaluated with reference to electrospray stability. The repeatability was used for the estimation of electrospray stability. The degree to which these parameters influenced the separation and the ESI stability was studied using a nine-peptide standard mixture and the antibiotic drugs bacampicillin and ampicillin as test substances. The analysis time and resolution were used as measures of the efficiency of the separation. A time-of-flight MS analyzer was used since it has the potential advantages of becoming a better fit for integration of CE with MS owing to the speed and sensitivity of this mass analyzer. The detection limit, i.e. 1 microM, for bacampicillin was comparable to what could be achieved with CE/MS on a quadrupole instrument using selected ion monitoring and sheath flow ESI.     

Use of Multiplexed CE for Pharmaceutical Analysis

This report describes the evaluation of a multiplexed, 96-capillary array instrument for analytical performance with a view to implementation for routine, high-throughput use. The need for higher throughput analytical capability for today’s busy pharmaceutical laboratory continues to rise. The use of multiplexed capillary electrophoresis is discussed, and the 96 multiplexed capillary array instrument with UV absorbance detection was used to perform high throughput pharmaceutical analysis. Pharmaceutical product assay, pK _a and log P determinations were achieved. Reproducibility of relative migration time and relative peak area between capillaries was found to be acceptable. The multiplexed instrument offered equivalent performance for tablet assay to a conventional CE instrument. Using the multiplexed CE, pK _a values approaching those of the literature values were obtained for a range of drugs. A microemulsion electrokinetic chromatography (MEEKC) method was successfully used without resulting in excessive operating current. The use of multiplexed CE for high throughput analysis has been shown to be a highly viable alternative to HPLC and other conventional analytical techniques.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

Determination of 5-methyl-cytosine and cytosine in tumor DNA of cancer patients

The determination of the relative methylation in DNA tumor samples, in order to evaluate the activity of some anti-cancer drugs, has become a very important issue in the clinical field. Capillary electrophoresis (CE) applications in this area have been done previously but no good separation for model samples or tumor samples has been reported. In this work, the CE conditions have been optimized in order to obtain baseline separation and efficient peaks for cytosine and 5-methylcytosine in both, standard mixtures and actual tumor samples; other bases (adenine, uracil, guanine, and thymine) have also been integrated in the optimization studies. More efficient peaks and shorter analysis time compared with the already reported conditions have been obtained employing a fused-silica capillary (75 microm inner diameter) of 44.5 cm effective length, 20 mM carbonate buffer (pH 9.6) plus 80 mM sodium dodecyl sulfate, a separation voltage of 20 kV, and detection at 223 nm.     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

Automated detection and removal of capillary electrophoresis artifacts due to spectral overlap

While DNA detection using capillary electrophoresis has enabled improvements in both resolution and throughput, the use of CE – particularly with multiple dye channels – can introduce artifacts that can complicate analyses. Undetected pull‐up artifacts can pose a challenge to investigators, especially in low‐level samples, while partial pull‐up peaks can distort peak height balance within a locus and impact the downstream likelihood ratio. Current methods for addressing pull‐up are typically manually implemented. This study presents an effective alternative: a series of mathematical models, created using symbolic regression achieved through genetic programming. The models estimate the amount of pull‐up expected in a peak from a true allele for a given dye‐dye relationship and instrument type. This leads to the removal of artifactual pull‐up peaks and peak height corrections when pull‐up is present within true alleles. When models are used in conjunction with a dynamic threshold, pull‐up peaks were automatically detected and removed with an accuracy rate of 96.1%. The removal of partial pull‐up from true allele peaks led to a more accurate heterozygote balance for the affected locus. These models have been optimized for use with any analytical threshold and can be implemented by any lab using a 3100 or 3500 instrument series.     

High-throughput capillary electrophoresis analysis of biopharmaceuticals utilizing sequential injections

The complexity of biotherapeutic products implies an ever-increasing list of product quality attributes that need to be monitored and characterized. In addition, the growing interest in implementing process analytical technology in biopharmaceutical production has further increased the testing burden, together with the need for rapid testing that can facilitate real-time or near-real-time decision-making. Capillary electrophoresis (CE) has made a place in biopharmaceutical analysis but is regarded as a low-throughput method, with the instrument dead time constituting more than 80% of the total time of analysis. In this study, the dead time of CE was utilized to analyse 3 mAb samples in a single-CE run. This approach resulted in an up to 77% reduction in the total analysis time and increased the productivity by up to 300%, compared to traditional single CE-ultraviolet runs, without compromising resolution or relative peak areas. Additionally, good method reproducibility was observed. The compatibility of the method has been demonstrated with protein A eluate and cation exchange chromatography fractions. We, thus, propose that sequential injections can be applied for fast and robust CE analysis of biopharmaceuticals.     

Surface sampling capillary electrophoresis–mass spectrometry for a direct chemical characterization of tissue and blood samples

Capillary electrophoresis (CE) is a powerful separation tool for non-targeted analysis of chemically complex samples, such as blood, urine, and tissue. However, traditionally CE requires samples in solution for analysis, which limits information on analyte distribution and heterogeneity in tissue. The recent development of surface sampling CE–mass spectrometry (SS-CE–MS) brings these advantages of CE to solid samples and enables chemical mapping directly from the tissue surface without laborious sample preparation. Here, we describe developments of SS-CE–MS to increase reproducibility and stability for metabolite, lipid, and protein extraction from tissue sections and dried blood spots. Additionally, we report the first electrokinetic sequential sample injection for high throughput analysis. We foresee that the wide molecular coverage from a distinct tissue region in combination with higher throughput will provide novel information on biological function and dysfunction.     

Matrix-assisted laser desorption/ionization time-of-flight based wheat gliadin protein peaks are useful molecular markers for wheat genetic study

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation has been used to analyze wheat seed gliadins as an alternative to other established methods, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), etc. The MALDI-TOF approach has shown to have many advantages such as high resolution, cost effectiveness and high throughput. MALDI-TOF-based gliadin profiles have been used for fast wheat cultivar identification. However, the genetic information represented by individual gliadin peaks has not been utilized. In this study a wheat doubled haploid population with a genetic linkage map of good coverage was used to assay individual gliadin peaks from MALDI-TOF profiles as molecular markers. Eight segregating peaks in the population were scored as polymorphic across the population. The 1 to 1 segregating ratios validated the scoring of the peaks and all peaks were mapped to the expected chromosomes or linkage groups on the available linkage map: 1 peak on chromosome 1A, 1 on 6A, 4 on 6B and 2 on 6D.     

Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

 Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection     

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.     

Attempt to run urinary protein electrophoresis using capillary technique

The study of urinary protein has a predominant place in the diagnosis of kidney disease. The most common technique is agarose gel electrophoresis (AGE). For several years, the technique of choice applied to the analysis of serum proteins has been CE, a system that uses capillary fused silica, subjected to high voltage to separate and measure serum proteins. The purpose of this paper was to perform capillary electrophoresis on urinary proteins which, at present, are not interpretable due to the many nonspecific peaks visible when using gel electrophoresis. In order to carry out our research, we used a capillary V8 analyzer together with an agarose gel system from the same company. AGE was taken as the reference method, for which urine was used without any pretreatment. For the V8 system, urine was subjected to purification on granular‐activated carbon and then inserted into the V8 analyzer, selecting a program suitable for liquids with low protein content. We examined 19 urine samples collected over 24 hrs from both hospitalized and external patients with different types of proteinuria plus a serum diluted 1/61 considered as a control to recognize the bands. Both methods showed the same protein fractions and classified the proteinuria in a similar way.