Individual Electrophoretic Mobilities Research Articles

Accurate Identification of Hemoglobin Variants By Top-Down Protein Analysis Using Capillary Electrophoresis-High-Resolution Mass Spectrometry

Introduction Structural characterization of hemoglobin (Hb) variants, particularly the mutant forms of α- and β-subunits, is of significant value in the clinical diagnosis of hemoglobinopathy. The conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of detailed structural characterization when it goes to the analysis of those Hb variants with similar sizes and charge states. High-resolution mass spectrometry (HR-MS) has been a central technology for structural characterization of proteins. As a novel approach for HR-MS protein analysis, top-down workflow analyzes proteins in intact state without prior enzymatic digestion, and it is capable of identifying unique proteoforms. Capillary electrophoresis (CE), as a powerful separation technology for proteins, has demonstrated excellent separation efficiency in the analysis of intact Hb forms and Hb subunits. CE can be coupled with HR-MS to form a CE-HR-MS system. By this means the CE separation is able to enhance the analytical power of HR-MS to allow for superior analytical performance in Hb analysis. In addition, when neutral coating of capillary is employed to suppress electroosmotic flow, CE separates analytes only by their individual electrophoretic mobilities, which enhances separation efficiency by maximizing the electrophoretic difference between the analytes. Thus, we established a neutral-coating CE-HR-MS method for accurate identification of Hb variants with top-down protein analysis strategy. Method An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 CE unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE separation. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis. Results In the neutral-coating CE, since denaturing conditions were used, intact Hb forms were dissociated in background electrolyte, and baseline separation of individual Hb subunits was observed. Hb subunits in ion electropherograms followed the order of α-, β-, δ-, γ(1)-, γ2- subunit. The identification of Hb variants using CE-HR-MS is a two-step process: (1) intact-protein analysis that preliminarily identifies Hb subunits by precursor ions, and (2) top-down analysis that characterizes the primary structures and confirms the identification of Hb subunits by fragments produced from particular precursor ions. In intact-protein analysis, multiple charge states of each Hb subunit were observed in the mass spectra. At each charge state of a Hb subunit, a cluster of isotopic MS peaks were observed, corresponding to the isotopes of the molecule. All the MS peaks in a mass spectrum can be deconvoluted and those resulted from one analyte can be merged to display a single MS peak at its accurate monoisotopic mass, which can be matched to the theoretical mass of a known Hb subunit or variant. In top-down protein analysis, fragments from a precursor ion were acquired after HCD fragmentation. The MS peaks can be deconvoluted and those resulted from one fragment can be merged to display a single MS peak at its monoisotopic mass. The monoisotopic masses of fragments can be used to characterize the primary structure of the analyte by matching them to the theoretical masses of possible fragments from the structure of a known Hb subunit. The CE-HR-MS method was applied to the analysis of normal Hb forms as well as Hb variants from adults and neonates. The structures of Hb subunit variants β-S, β-C, β-E, β-Riyadh, β-Koln, β-New York, β-G-Accra, β-G-Siriraj, β-Khartoum, β-D-Punjab, β-D-Iran, β-Deer Lodge, α-Tarrant, γ1-Kuala Lumpur, and P-Nilotic β-δ hybrid have been successfully identified and characterized with >30% amino acid residue coverage and >95% matched fragment coverage. As an example, the CE-HR-MS result for the identification of a Hb γ1/γ1-Kuala Lumpur is shown, with intact-protein analysis in Figure 1A and top-down analysis in Figure 1B. Conclusion We have utilized the neutral-coating CE-HR-MS method for top-down structural characterization of hemoglobin variants. With the use of CE, only a simple dilute-and-shoot sample preparation procedure is required. Baseline separation of Hb subunits can be achieved to enhance HR-MS data quality. With these advantages, the CE-HR-MS method is compatible with clinical laboratories and can potentially be used in routine clinical testing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dual-opposite injection capillary electrophoresis: Principles and misconceptions.

Dual-opposite injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. The electroosmotic flow (EOF) is suppressed to allow all ions to reach the detector quickly. Depending on the individual electrophoretic mobilities of the analytes of interest and the effective length that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. This review discusses the principles, applications, and limitations of dual-opposite injection capillary electrophoresis. Common misconceptions regarding DOI-CE are clarified.

A systematic approach for avoiding co-detection of oppositely charged analytes in dual-opposite-injection capillary electrophoresis

Dual-opposite-injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. Depending on the individual electrophoretic mobilities of the analytes of interest and the migration distance that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. One consequence of a variable elution order is that co-detection of oppositely charged analytes becomes possible under certain experimental conditions. While several approaches currently exist to avoid co-detection, they are often time consuming and inefficient. This paper describes a systematic approach to easily avoid co-detection by analyzing the net mobilities of the analytes of interest to aid in choosing appropriate capillary lengths for a particular separation. Simulated electropherograms that highlight various aspects of this new approach are shown. An electropherogram from the literature is replicated and the derived relationship is applied to show the potential advantages of this new approach.

Describing autophagy via analysis of individual organelles by capillary electrophoresis with laser induced fluorescence detection.

Autophagy is a cellular process responsible for the degradation of intracellular cargo. Its dynamic nature and the multiple types of autophagy organelles present at a given time make current measurements, such as those done by Western blotting, insufficient to understand autophagy and its roles in aging and disease. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) has been used previously to count and determine properties of individual organelles, but has never been used on autophagy organelles or for determination of changes of such properties. Here we used autophagy organelles isolated from L6 cells expressing GFP-LC3, which is an autophagy marker, to develop a CE-LIF method for the determination of the number of autophagy organelles, their individual GFP-LC3 fluorescence intensities, and their individual electrophoretic mobilities. These properties were compared under basal and rapamycin-driven autophagy, which showed differences in the number of detected organelles and electrophoretic mobility distributions of autophagy organelles. Vinblastine treatment was also used to halt autophagy and further characterize changes and provide additional insight on the nature of autophagy organelles. This approach revealed dramatic and opposite directions in changes of organelle numbers, GFP-LC3 contents, and electrophoretic mobilities during the duration of the vinblastine treatment. These trends suggested the identity of organelle types being detected. These observations demonstrate that individual organelle analysis by CE-LIF is a powerful technology to investigate the complexity and nature of autophagy, a process that plays critical roles in response to drug treatments, aging, and disease.

Development and characterization of a novel semiautomated arrangement for electrochemically assisted injection in combination with capillary electrophoresis time‐of‐flight mass spectrometry

Electrochemically assisted injection (EAI) is an attractive injection concept for CE that enables the separation of neutral analytes via electrochemical generation of charged species during the injection process. A new semiautomated EAI configuration was developed and applied in conjunction with CE-MS (EAI-CE-MS). The EAI cell arrangement consists of an integrated buffer reservoir for CE separations and a compartment holding screen-printed electrodes. A drop of sample solution (50 μL) was sufficient to cover the three-electrode structures. A piezo motor provided a fast and precise capillary positioning over the screen-printed electrode assembly. Using ferrocene methanol as a model system, the EAI arrangement was characterized regarding coulometric efficiency, precision, and sensitivity of electrospray ionization-time-of-flight-MS. The formation of the cationic oxidation product of ferrocene methanol enhanced the sensitivity of CE-MS determination by two orders of magnitude and the electrochemically formed product showed a migration time corresponding to its individual electrophoretic mobility. Preliminary studies of EAI-CE-MS in the field of the analysis of nitroaromatic compounds were carried out. The formation of corresponding hydroxylamines and amines paved the way for selective and sensitive CE-MS determinations without the need of adding surfactants to the electrophoresis buffer.

Bipolar electrode focusing: tuning the electric field gradient

Bipolar electrode (BPE) focusing is a developing technique for enrichment and separation of charged analytes in a microfluidic channel. The technique employs a bipolar electrode that initiates faradaic processes that subsequently lead to formation of an ion depletion zone. The electric field gradient resulting from this depletion zone focuses ions on the basis of their individual electrophoretic mobilities. The nature of the gradient is of primary importance to the performance of the technique. Here, we report dynamic measurements of the electric field gradient showing that it is stable over time and that its axial position in the microchannel is directly correlated to the location of an enriched tracer band. The position of the gradient can be tuned with pressure-driven flow. We also show that a steeper electric field gradient decreases the breadth of the enriched tracer band and therefore enhances the enrichment process. The slope of the gradient can be tuned by altering the buffer concentration: higher concentrations result in a steeper gradient. Coating the channel with the neutral block co-polymer Pluronic also results in enhanced enrichment.

Development of a membrane-less dynamic field gradient focusing device for the separation of low-molecular-weight molecules.

Dynamic field gradient focusing uses an electric field gradient generated by controlling the voltage profile of an electrode array to separate and concentrate charged analytes according to their individual electrophoretic mobilities. This study describes a new instrument in which the electrodes have been placed within the separation channel. The major challenge faced with this device is that when applied voltages to the electrodes are larger than the redox potential of water, electrolysis will occur, producing hydrogen ions (H+) plus oxygen gas on the anodes and hydroxide (OH(-)) plus hydrogen gas on the cathodes. The resulting gas bubbles and pH excursions can cause problems with system performance and reproducibility. An on-column, degassing system that can remove gas bubbles "on-the-fly" is described. In addition, the use of a high capacity, low-conductivity buffer to address the problem of the pH shift that occurs due to the production of H+ on the anodes is illustrated. Finally, the successful separation of three, low-molecular-weight dyes (amaranth, bromophenol blue and methyl red) is described.

Characterization of voltage degradation in dynamic field gradient focusing.

Dynamic field gradient focusing (DFGF) is an equilibrium gradient method that utilizes an electric field gradient to simultaneously separate and concentrate charged analytes based on their individual electrophoretic mobilities. This work describes the use of a 2-D nonlinear, numerical simulation to examine the impact of voltage loss from the electrodes to the separation channel, termed voltage degradation, and distortions in the electric field on the performance of DFGF. One of the design parameters that has a large impact on the degree of voltage degradation is the placement of the electrodes in relation to the separation channel. The simulation shows that a distance of about 3 mm from the electrodes to the separation channel gives the electric field profile with least amount of voltage degradation. The simulation was also used to describe the elution of focused protein peaks. The simulation shows that elution under constant electric field gradient gives better performance than elution through shallowing of the electric field. Qualitative agreement between the numerical simulation and experimental results is shown. The simulation also illustrates that the presence of a defocusing region at the cathodic end of the separation channel causes peak dispersion during elution. The numerical model is then used to design a system that does not suffer from a defocusing region. Peaks eluted under this design experienced no band broadening in our simulations. Preliminary experimental results using the redesigned chamber are shown.

Individual Electrophoretic Mobilities of Liposomes and Acidic Organelles Displaying pH Gradients Across Their Membranes

This report focuses on measuring the individual electrophoretic mobilities of liposomes with different pH gradients across their membrane using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The results from the individual analysis of liposomes show that, using surface electrostatic theories and the electrokinetic theory as the first approximation, zeta potential contributes more significantly to the electrophoretic mobility of liposomes than liposomal size. For liposomes with an outer pH 7.4 (pH(o) 7.4) and a net negative outer surface charge, the most negative electrophoretic mobilities occur when the inner pH (pH(i)) is 6.8; at higher or lower pH(i), the electrophoretic mobilities are less negative. The theories mentioned above cannot explain these pH-induced electrophoretic mobility shifts. The capacity theory, predicting an induced electrical charge on the surface of liposomes, can only explain the results at pH(i) > 6.8. In this report, we hypothesize that there is a flip-flop process of phospholipids, which refers to the exchange of phospholipids between the outer and inner layers of the membrane. This flip-flop is caused by the pH gradient and membrane instability and results in the observed electrophoretic mobility changes when pH(i) is <6.8. Furthermore, it is found that the mobilities of acidic organelles are consistent with the predictions of liposome models we used here.

Analysis of mitochondria isolated from single cells

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

Direct Sampling from Muscle Cross Sections for Electrophoretic Analysis of Individual Mitochondria

Muscle is a highly heterogeneous tissue. Practical approaches to sample selectively small regions of muscle cross sections would help to effectively utilize analytical techniques on muscle studies while taking into account tissue heterogeneity. In this report, semimembranosus muscle tissue cross sections were directly sampled and analyzed by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Prior to CE-LIF analysis, a small region in the muscle cross section was stained with 10-nonyl acridine orange (NAO) which is a mitochondrion-selective fluorescent probe known to form a stable complex with cardiolipin, a phospholipid found only in mitochondria. By micromanipulation, the injection end of the capillary was brought into contact with the tissue exhibiting fluorescently labeled mitochondria. Sampling from a region similar in size to the cross section of a single fiber was carried out by applying 11 kPa of negative pressure for 3 s. When an electric field of -200V/cm was applied, fluorescently labeled mitochondria electromigrated and were individually detected by postcolumn LIF detection. For each sample, the electropherogram displays a migration time window with a collection of narrow peaks. The collection of individual peak measurements is represented as a distribution of individual intensities related to cardiolipin content of mitochondria and a distribution of individual electrophoretic mobilities. Positioning the capillary injection end was sufficiently spatially accurate to deplete mitochondria in the sampled region upon repetitive injections. Treatment of a muscle cross section with a protease (trypsin) prior to mitochondria sampling resulted in a higher number of detected mitochondria, suggesting that one of the effects of this enzyme is a partial digestion of the muscles myofibrils, which eases the release of interfibrillar mitochondria entangled within these fibers. The protease treatment also resulted in changes to the electrophoretic mobility distribution of individual mitochondria, which may imply that partial digestion of proteins bound to the mitochondria contributes to the alteration in the electrophoretic mobility of mitochondria. The ability to sample a region as small as a single muscle fiber cross section and its direct CE-LIF analysis opens exciting possibilities for the direct analysis of muscle biopsies and mapping the mitochondrial electrophoretic properties in highly heterogeneous tissues.

Analysis of individual acidic organelles by capillary electrophoresis with laser-induced fluorescence detection facilitated by the endocytosis of fluorescently labeled microspheres.

Submicrometer-sized fluorescent microspheres were loaded into the acidic organelles of NS-1 mouse myeloma cells via endocytosis. Confocal microscopy imaging showed that microspheres colocalized nearly perfectly with LysoTracker Red, a probe that stains acidic organelles. Unlike LysoTracker dyes that seem to leak from acidic organelles upon cell disruption, microspheres are retained within these organelles, facilitating their analysis following isolation. Using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF), the electrophoretic mobilities of acidic organelles were individually calculated and fluorescence intensities individually measured. When cells were incubated for sufficient time to allow for endocytosis (48 h) with 3.9 x 10(3) microspheres/cell, replicate CE-LIF analyses of the corresponding isolated fraction indicated a dramatic increase in the number of detected events (n = 1990 +/- 234) and in the overall fluorescence intensity of the individual events (0.38 +/- 0.01 RFU; average +/- SD; n = 3) over the corresponding <10-min incubations (n = 60; 0.21 RFU, respectively). In addition, a treatment with 4-fold increase in microsphere density (1.6 x 10(4) microspheres/cell), increased the number of detected individual events (n = 3427 +/- 101) and altered only slightly the fluorescence intensity and electrophoretic mobility distributions. The individual electrophoretic mobility values ranged from -1.45 x 10(-)(4) to -3.0 x 10(-)(4) cm(2) V(-)(1) s(-)(1) while the individual fluorescence values ranged from 0.1 V to over 8 V, demonstrating the benefit of detecting organelles individually rather than averaging their properties over single cells or bulk homogenates.

High-throughput single-molecule DNA screening based on electrophoresis.

In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Many protein and DNA assays relevant to disease diagnosis are based on such separations. However, standard protocols are not only slow (minutes to hours) but also insensitive (many molecules in a detectable band). We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement only requires a few milliseconds to complete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. We developed three different procedures to measure the individual molecular mobilities. The results correlate well with capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis.