Biomarker discovery by CE-MS enables sequence analysis via MS/MS with platform-independent separation

CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.     

Recent developments in the determination of urinary cancer biomarkers by capillary electrophoresis

Investigation of effective biomarkers for cancers is currently a popular area of study in clinical and cancer researches, because it can potentially lead to pre-cancer screening or pre-cancer diagnosis and may provide useful information on cancer type and the disease's stage of progression. More and more biochemical or chemical fluid components of the human body such as urine, blood, and cerebrospinal fluid have been considered to contain biomarkers, which are useful in cancer researches, pre-cancer diagnosis, and cancer follow-ups during or after cancer treatment. Several modern analytical techniques, such as gas chromatography (GC), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and other separation techniques as well as hyphenated techniques, have been extensively used in study of cancer biomarkers. Among these techniques, CE is considered to be a highly efficient and practical analytical technique because of the small sample volume requirement and its wide separation versatility, ranging from small inorganic molecules to large biomolecules. This review discusses the latest developments involving biomarkers and their analysis by CE, including a discussion of instrumental conditions, method developments, and data analysis.     

High-throughput screening of genetic mutations/polymorphisms by capillary electrophoresis

Genetic mutations/polymorphisms analyses play a great role in genetic and medical research, and clinical diagnosis. Most conventional methods for genetic assay are based on slab gel electrophoresis that is both labor-intensive and time-consuming. Recently, capillary electrophoresis (CE) has been used for genetic analysis instead of conventional slab gel electrophoresis. This technique can be automated and is characterized by short analysis time, small sample and reagents requirements, and high separation efficiency. CE has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes, and has shown a great potential for genetic mutation/polymorphism screening of large numbers of clinical samples. In this article, an overview of the fundamental aspects of mutation/polymorphism assay methods in combination with CE is given and some key applications are summarized.     

Highly sensitive detection of cancer cells based on the DNA barcode assay and microcapillary electrophoretic analysis

Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (μCE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the μCE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF‐7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the μCE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF‐7 and SW620 cells in a volume of 200 μL, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the μCE analysis.     

Assessing the suitability of capillary electrophoresis‐mass spectrometry for biomarker discovery in plasma‐based metabolomics

The actual utility of capillary electrophoresis‐mass spectrometry (CE‐MS) for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic profiling study for biomarker discovery by CE‐MS was performed, using pooled human plasma samples with spiked biomarkers. Two studies have been carried out in this work. Focus of study I was on comparing two sets of plasma samples, in which one set (class I) was spiked with five isotope‐labeled compounds, whereas another set (class II) was spiked with six different isotope‐labeled compounds. In study II, focus was also on comparing two sets of plasma samples, however, the isotope‐labeled compounds were spiked to both class I and class II samples but with concentrations which differ by a factor two between both classes (with one compound absent in each class). The aim was to determine whether CEMS‐based metabolomics could reveal the spiked biomarkers as the main classifiers, applying two different data analysis software tools (MetaboAnalyst and Matlab). Unsupervised analysis of the recorded metabolic profiles revealed a clear distinction between class I and class II plasma samples in both studies. This classification was mainly attributed to the spiked isotope‐labeled compounds, thereby emphasizing the utility of CE‐MS for biomarker discovery.     

Cover Picture: Electrophoresis 2'2011

Issue no. 2 is a regular issue assembled of 16 solid and original research articles distributed over 3 distinct parts. Part I is on novel trends in fundamentals and methodologies including theoretical models for selectivity of charged solutes in MEKC, system peaks in indirect detection, measuring epimerization constants by MEEKC, bundled CE using micro‐structured fibers, 2‐D separations by coupling CIEF and CEC, high speed DNA CE, MCE of N‐glycans and mucin expression in a microfluidic gradient device. Part II is concerned with detection, sensitivity enhancement, on‐column preconcentration and microdialysis sampling involving the design of continuous full filling CEC‐ESI‐MS using nanoparticles, CE‐fluorescence using tapered optical fiber, CZE separation of pesticide residues in water samples with acid‐assisted on‐column preconcentration and CE‐LIF to detect neurotransmitter amino acids and carbamathione in brain microdialysis samples. Novel methods for the separation and profiling of various proteins and large nucleic fragments are described in 4 consecutive papers grouped in part III. Featured articles include: Theoretical models of separation selectivity for charged compounds in micellar electrokinetic chromatography (( 10.1002/elps.201000405 )) Bundled capillary electrophoresis using microstructured fibres ( 10.1002/elps.201000442 )) Two‐dimensional separation system by on‐line hyphenation of capillary isoelectric focusing with pressurized capillary electrochromatography for peptide and protein mapping ( 10.1002/elps.201000419 )) Microchip electrophoresis of N‐glycans on serpentine separation channels with asymmetrically tapered turns ( 10.1002/elps.201000461 ))     

Capillary Electrophoresis Coupled On-Line with Inductively Coupled Plasma Atomic Emission Spectrometry for Elemental Speciation

Capillary electrophoresis (CE) has become a powerful analytical technique for the separation of a variety of analytes ranging from small inorganic ions to large biomolecules such as proteins and nucleic acids. A selective and sensitive detector for CE has been one of the most important and challenging prerequisites for the growth of CE. On-column UV-Vis detectors are commonly used to determine the analytes separated by CE. However, these detectors are often not very selective. Other detection techniques such as mass spectrometry, laser induced fluorescence, amperometry, and inductively coupled plasma spectrometry have been investigated to provide a more sensitive and selective detection for the target analytes. However, relatively few studies have been published on the use of inductively coupled plasma atomic emission spectrometry (ICP-AES) as a means of detection in CE separation.     

CE methods for analysis of isoforms of prostate-specific antigen compatible with online derivatization for LIF detection

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glycosylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17?min, while the shortest one separates 9 isoforms in < 8?min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.     

毛细管电泳技术在重大疾病特异性蛋白质分析中的应用

人类重大疾病特异性蛋白质的识别、鉴定等是目前生命科学亟待解决的问题。毛细管电泳技术用于蛋白质分析,具有装置简单、分析速度快、试剂和样品消耗少以及有多种分离模式和检测手段等特点。本文综述了毛细管电泳技术在人类重大疾病特异性蛋白质分析中的应用,包括肿瘤疾病、神经退行性疾病和输血性传染病等的特异性蛋白质检测。     

毛细管电泳用于药物分析的研究进展

药物分析是毛细管电泳（CE）的重要应用领域,所有CE分离模式与检测方法都在各种药物及其不同形式样品的分离分析中显示出特色和应用能力。该文从药品分析领域中的小分子药物（包括手性药物）及其有关物质、中药与天然产物、体内药物分析、生物制品药物分析等几个方面,综述了近几年CE在这些传统药物分析领域应用的研究进展。限于篇幅,未包括现代药物分析研究比较活跃的理化常数测定、亲和毛细管电泳与结合常数研究（药物与受体间的相互作用等）、临床生物标志物分析、代谢组学和微流控芯片CE分析等方面的内容。根据目前传统药物分析领域的发展,该文关注到近期CE在顺应药物分析的法规需求、电容耦合非接触电导检测（CE-C⁴ D）、改进检测灵敏度与精密度、CE-十二烷基硫酸钠（SDS）毛细管电泳、全柱成像毛细管等电聚焦（icIEF）、抗体分析等方面的新进展。该文结合文献,讨论了目前传统药物分析领域的需求,以及CE在其中的地位、挑战和机遇。对目前CE主要作为互补分析方法在化学药和中药分析中的应用研究提出了一些针对性的建议,期待CE在生物制品分析中的特色和能力得到进一步的发挥,同时提出CE-MS和对CE分析重复性改进等新进展可能对未来CE应用领域的大幅度扩展。该综述主要涉及近3年（2017年1月到2020年2月）及部分2016年的相关文献。     

Determination of small phosphorus-containing compounds by capillary electrophoresis

The review focuses on the analysis of small phosphorus-containing compounds by capillary electrophoresis (CE) with different detection modes including UV absorption, laser-induced fluorescence, conductometry, amperometry, atomic spectrometry, and mass spectrometry. Determinations of phosphates, organophosphate, and chemical warfare agents in environmental samples such as water, soil and grains are emphasized. To achieve better sensitivity, high-resolving power, and reproducibility, the optimum separation conditions for various analytes (samples) are different. We compare the merits and demerits of the different detection modes for the detection of the analytes. Although the present methods are successful in many cases, there is still a need to develop high-throughput CE techniques for screening numerous environmental samples and sample stacking techniques in CE for the analysis of trace analytes.     

毛细管电泳-激光诱导荧光检测法分析胃癌组织及癌旁正常组织蛋白质的差异性

胃癌是临床常见的恶性肿瘤之一。近年来寻找肿瘤相关特异蛋白质是蛋白质组学研究的热点。本文通过考察毛细管动态涂层方法、筛分介质聚环氧乙烷(PEO)的浓度、缓冲液的pH值、分离电压、温度及荧光染料对分离效果的影响,建立了毛细管电泳-激光诱导荧光法分离胃癌组织及癌旁正常组织蛋白质的方法;通过分离检测,获得两者的蛋白质指纹图谱。经分析,两者的指纹图谱相似度达到0.8以上,差异蛋白质分子质量集中在50000~100000 Da之间,提示某些小分子蛋白质可能是和肿瘤发生相关的特异蛋白质,从而缩小了特异性分子标记物的筛选范围。病理组织学分型及蛋白质电泳峰数目的统计结果验证了该方法的可靠性。该方法具有临床应用的潜力。     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

Determination of pharmaceutical and personal care products in wastewater by capillary electrophoresis with UV detection

Capillary electrophoresis (CE) offers a fast and cost-effective alternative analytical technique to LC-MS/MS for separation and quantitation of many PPCP compounds in wastewater. In this study, we have developed a method that can simultaneously analyze eight different PPCP compounds in untreated wastewater (ibuprofen, triclosan, carbamazepine, caffeine, acetaminophen, sulfamethoxazole, trimethoprim, and lincomycin), using capillary electrophoresis with UV detection (CE-UV). The method detection limit (MDL) ranged from 1.6 to 68.7 ppb through solid phase extraction. The standard limit of quantification (LOQ) ranged from 0.63 to 7.72 ppm. Factors affecting separation and quantification of PPCPs, such as pH, electrophoretic potential, buffer strength, buffer type, and additives, were investigated and optimized. Water samples from two different wastewater treatment plants were collected and analyzed. The results obtained were comparable with those of LC-MS/MS. The technique developed in this study provides a low cost, simple, fast, and relatively sensitive method for determination of various PPCPs in wastewater samples for PPCP screening.     

亲水作用色谱/质谱联用方法用于膀胱癌患者血清代谢组学研究

膀胱癌是泌尿系统最常见的恶性肿瘤之一,具有高发病率、高复发率和高进展率的特点.本研究应用69个极性代谢物标样选择合适的分离系统,建立了两性离子亲水作用色谱/质谱联用的代谢组学分析方法.本方法线性范围较宽,检出限低于ng/mL数量级.将本方法用于血清代谢组学分析,85%以上代谢物峰面积的RSD<30%.对64例膀胱癌患者和32例正常人的血清进行代谢组学研究,发现溶血磷脂酰胆碱、游离脂肪酸、氨基酸、胆汁酸、有机酸、核苷等在患病组和正常组中存在显著差异.经筛选和验证,甘磷酸胆碱、胱氨酸、十二碳烯酸、二十碳烯酸和鹅去氧胆酸5种代谢物可以作为区分膀胱癌和正常人的潜在标志物.本研究结果表明,基于亲水作用色谱/质谱联用的代谢组学方法是发现癌症诊断潜在生物标志物的有效手段.     

Integrated dielectrophoretic and impedimetric biosensor provides a template for universal biomarker sensing in clinical samples

The detection and quantification of nucleic acid and proteomic biomarkers in bodily fluids is a critical part of many medical screening and diagnoses. However, majority of the current detection platforms are not ideal for routine, rapid, and low-cost testing in point-of-care settings. To address this issue, we developed a concept for a disposable universal point-of-care biosensor that can detect and quantify nucleic acid and proteomic biomarkers in diluted serum samples. The central tenet of sensing is the use of dielectrophoresis, electrothermal effects, and thermophoresis to selectively and rapidly isolate the biomarkers of interest in electrodes and then quantify using electrical impedance. When the sensor was applied to quantify microRNA and antigen biomarker molecules directly in diluted serum samples, it produced a LOD values in the fM range and sensitivity values from 10¹² to 10¹⁵ Ω/M with a 30 min assay time and assay cost of less than $50 per assay.