Attomole protein analysis by CIEF with LIF detection

We have coupled CIEF with an LIF detector that is based on a post‐column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the ε‐amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503‐labeled β‐lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.     

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 sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells

Capillary sodium dodecyl sulfate (SDS)-DALT electrophoresis (SDS-DALT-CE) refers to CE separation of proteins based on their size; DALT is the abbreviation for Dalton, the unit used to describe molecular weight. In this work, seven proteins from 18 to 116 kDa were denatured by SDS, labeled by 3-(2-furoyl) quinoline-2-carboxaldehyde, separated by SDS-DALT-CE in polyethylene oxide sieving matrix, and detected by laser-induced fluorescence (LIF) in a sheath flow cuvette. This method was combined with detergent differential fractionation, which is a protein fractionation method using a series of detergent-containing buffers to sequentially extract protein fractions from cells, to analyze the proteins in HT29 human colon adenocarcinoma cells. In addition, on-column labeling was demonstrated for protein analysis by SDS-DALT-CE with LIF, and applied to analysis of proteins in a single HT29 cancer cell. Most proteins had molecular masses from 10 to 120 kDa. Similar protein profiles were obtained for single cells and protein extract of a large cell population.     

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.     

Cover Picture: Electrophoresis 14'2011

Issue no. 14 is a regular issue consisting of 17 contributions distributed over 3 distinct parts. Part I has 7 articles describing studies on proteins and proteomics including measuring protein mobility using laser Doppler electrophoresis, high sensitivity protein analysis by FESI‐CE‐MALDI‐MS, protocol for SDS‐PAGE separation of myosin heavy chain isoforms, proteomics analysis of a spring wheat cultivar in response to prolonged cold stress, analysis of changes in the 20S proteasome, treatment of acute lymphoblastic leukemia with L‐asparaginase, and improved method for immunostaining of mucin. Part II is on nucleic acids and has 4 contributions on multiplex PCR‐CE analysis, copy number variation, ethnic difference, SNPs, CE‐SSCP, allelic ladder and SNaPshot technique. Part III has 6 articles on various fundamentals and methodologies, including nanofluidics, nanopore sensing, sequential injection setup for CIEF combined with MS detection, determination of imatinib mesylate in chronic myeloid leukemia patients by CE, UV detection of plasma malondialdehyde using CE‐FASI, enantioseparation of basic drugs by CE using clarithromycin lactobionate chiral selector and CE determination of bioactive constituents in Herba Houttuyniae. Featured articles include: Advances in the measurement of protein mobility using laser Doppler electrophoresis – the diffusion barrier technique (( 10.1002/elps.201100108 )) Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure (( 10.1002/elps.201000608 )) Strategy for high‐fidelity multiplex DNA copy number assay system using capillary electrophoresis devices (( 10.1002/elps.201100093 )) Electrokinetic particle translocation through a nanopore containing a floating electrode (( 10.1002/elps.201100050 )) Sequential injection setup for capillary isoelectric focusing combined with MS detection (( 10.1002/elps.201100012 ))     

A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

Here, a CIEF‐LIF method for multiple protein kinase simultaneous analysis and inhibitors throughput screening with fast rate and low cost is presented. Comparing with CZE, CIEF‐LIF exhibited great focusing ability and high separation efficiency for substrate and phosphorylated peptides, and is applicable for multiple kinases simultaneous analysis regardless of their substrate peptides compositions and charge statuses. Thus, highly sensitive analysis for cyclic adenosine 3’, 5’‐monophosphate‐dependent protein kinase (PKA) and cyclin‐dependent kinase 1 (CDK1) was achieved in CIEF‐LIF analysis with detection sensitivity up to 1.25 mU/μL and 0.4 mU/μL, respectively, two magnitudes higher than that of CZE and comparable with that in nanomaterials or green fluorescent protein‐based kinase assay. Moreover, the inhibition effect of inhibitors on multiple kinases could be simultaneously readout in a single electrophoretic run, with half maximal inhibitory concentration of H‐89 for PKA and Ro‐3306 for CDK1 calculated as 37.0 and 35.9 nM, respectively, consistent with literatures reported. The CIEF‐LIF also exhibited strong anti‐interference ability in human breast cancer cell lysates analysis and simulators such as forskolin and 3‐isobutyl‐1‐methylxantine assessment. Therefore, CIEF‐LIF is desirable for future biological application and clinical diagnostics and drug discovery.     

Cover Picture: Electrophoresis 6/2008

Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting‐edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two‐dimensional electrophoresis. In addition, issue no. 6 features a series of 7 important papers on "Microfluidics and Miniaturization" dealing with LCD‐based optoelectronic tweezers, cell sorting, single cell clone analysis and cultivation, integrated ITP stacking and gel electrophoresis, floating injection, electrokinetic flows on thermosensitive surfaces and flow velocity measurement in CE chip instruments.     

A PDMS sheath flow cuvette for high‐sensitivity LIF measurements in CE

A simple method for producing a sheath flow cuvette in PDMS suitable for post‐column detection in CE is described. Two types of cuvette were investigated. In the first, the sheath flow channel had a round cross‐section of approximately 635 μm diameter, whereas the second cuvette had a 300×300 μm² square channel. Both cuvettes produced laminar flows that ensheathed the separation capillary's effluent allowing sensitive fluorescence measurements. The elasticity of the PDMS allowed the 300×300 μm² square sheath flow channel to expand uniformly and accommodate the larger 330–340 μm od round separation capillary, producing a self‐aligning cuvette with robust mechanical properties. With this cuvette, linear calibrations of over five orders of magnitude and 15–30 zmol fluorescein detection limits were obtained for 12 and 50 μm id capillaries.     

Cover Picture: Electrophoresis 11/2008

Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting‐edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two‐dimensional electrophoresis. Issue 11 also offers a series of 9 papers on bioanalysis related to proteomics, protein analysis, genes, DNA sequencing, SNP, genotyping and SSCP.     

CE-based analysis of hemoglobin and its applications in clinical analysis

This review focuses on the developments and trends in CE including CIEF, CZE, MEKC, two-dimensional conjunction of CIEF-capillary gel electrophoresis, and MEKC-CZE on microfluidic devices coupled to different detection approaches, such as UV absorbance, LIF, MS, and chemiluminescence etc. for performing analysis of hemoglobin (Hb), also with an emphasis on its applications in clinical analysis. Analysis of human Hb is of important clinical sense for numerous hemoglobinopathies associated with the congenital defects and abnormal contents of Hb. The diversiform modes render CE a comprehensive primary clinical tool for Hb analysis, which is rapid, sensitive, high-resolution, and not labor-intensive.     

Cover Picture ‐ Electrophoresis 3/2008

Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting‐edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two‐dimensional electrophoresis.     

Fiber optic illumination of a poly(dimethylsiloxane) sheath flow cuvette for diode laser induced fluorescence detection in capillary electrophoresis

A Tee configuration sheath flow cuvette with square cross‐section channels has been produced in PDMS for CE detection. The output of a 1.4 W laser diode operating at 450 nm was focused onto the 300 μm core of a 370 μm od fiber optic whose end was inserted into one arm of the Tee for LIF. The optimal configuration had the fiber optic positioned 500 μm downstream from the intersection and the end of the 35 cm 50 μm id 365 μm od capillary just outside the intersection and in the leg of the Tee, resulting in a 90° configuration. Detection limits of 50 and 3 pM and linear calibrations of at least three orders of magnitude were obtained for Lucifer Yellow and fluorescein, respectively.     

Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Cover Picture: Electrophoresis 3'2010

Issue no. 3 is a regular issue with Emphasis on “Proteins and Proteomics”. The first part has 8 articles on proteins and proteomics covering various topics, e.g. preparative divergent flow IEF, multichannel gel electrophoresis, capillary gel electrophoresis, nanoparticle‐based CE of proteins, 2‐DE in a radial gel format, depletion of high abundance proteins, and proteomic investigation of fetal brain and lentil seed. The remaining 10 articles are concerned with nucleic acids, gene expression, methodologies and application. Featured articles include: Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples (( 10.1002/elps.200900484 )) SDS‐PAGE and two‐dimensional maps in a radial gel format (( 10.1002/elps.200900526 )) Analysis of Effect of Electrolyte Types on Electrokinetic Energy Conversion in Nanoscale Capillaries (( 10.1002/elps.200900409 )) A simple method to determine the surface charge in microfluidic channels (( 10.1002/elps.200900603 ))     

Cover Picture: Electrophoresis 20'2010

Issue no. 20 is a regular issue comprising 17 contributions distributed over 7 distinct parts. Part I has 3 research articles on particle analysis. Part II has 2 research articles dealing with hyphenated techniques such as CITP‐CZE and CE‐ESI‐MS. Part III reports a variety of methodologies and systems for proteins and proteomics that are described in 4 research articles. Gel DNA: theory and visualization are treated in 2 research articles making up part IV. Part V has 2 contributions that describe CE systems for water analysis. The following Part VI is on affinity and Immunoaffinity CE and has 2 research articles. The last 2 articles in this issue (Part VII) are on MEEKC and free flow electrophoresis. Featured articles include: Dielectrophoretically patterned carbon nanotubes to sort microparticles ((doi: 10.1002/elps.201000104 )) Analysis of 5‐methyltetrahydrofolate in human blood, serum and urine by on‐line coupling of capillary isotachophoresis and zone electrophoresis ((doi: 10.1002/elps.201000193 ))     

Coupling of capillary electrophoresis with electrospray ionization multiplexing ion mobility spectrometry

In this work, ion mobility spectrometry (IMS) function as a detector and another dimension of separation was coupled with CE to achieve two‐dimensional separation. To improve the performance of hyphenated CE‐IMS instrument, electrospray ionization correlation ion mobility spectrometry is evaluated and compared with traditional signal averaging data acquisition method using tetraalkylammonium bromide compounds. The effect of various parameters on the separation including sample introduction, sheath fluid of CE and drift gas, data acquisition method of IMS were investigated. The experimental result shows that the optimal conditions are as follows: hydrodynamic sample injection method, the electrophoresis voltage is 10 kilo volts, 5 mmol/L ammonium acetate buffer solution containing 80% acetonitrile as both the background electrolyte and the electrospray ionization sheath fluid, the ESI liquid flow rate is 4.5 μL/min, the drift voltage is 10.5 kilo volts, the drift gas temperature is 383 K and the drift gas flow rate is 300 mL/min. Under the above conditions, the mixture standards of seven tetraalkylammoniums can be completely separated within 10 min both by CE and IMS. The linear range was 5–250 μg/mL, with LOD of 0.152, 0.204, 0.277, 0.382, 0.466, 0.623 and 0.892 μg/mL, respectively. Compared with traditional capillary electrophoresis detection methods, the developed CE‐ESI‐IMS method not only provide two sets of qualitative parameters including electrophoresis migration time and ion drift time, ion mobility spectrometer can also provide an additional dimension of separation and could apply to the detection ultra‐violet transparent compounds or none fluorescent compounds.     

Cover Picture ‐ Electrophoresis 2/2008

Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting‐edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two‐dimensional electrophoresis. In addition Issue 2 offers two Fast Track articles describing particularly important investigations in electrophoresis: Continuum transport model of Ogston sieving in patterned nanofilter arrays for separation of rod‐like biomolecules Zi Rui Li, Gui Rong Liu, Yu Zong Chen, Jian‐Sheng Wang, Hansen Bow, Yuan Cheng, Jongyoon Han A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene Shanan S. Tobe, Adrian M. T. Linacre     

Cover Picture: Electrophoresis 15/2008

Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting‐edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two‐dimensional electrophoresis. Selected topics of issue 15 are: The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus‐1 gp41 ectodomain by electrophoresis Metabolic fingerprinting of schistosoma mansoni infection in mice urine with capillary electrophoresis Supercritical fluid extraction as an on‐line clean‐up technique for determination of riboflavin vitamins in food samples by capillary electrophoresis with fluorimetric detection A two‐step electro‐dialysis method for DNA purification from polluted metallic environmental samples.