Zaifang Zhu

Protein separation by capillary gel electrophoresis: A review

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.

Coupling sodium dodecyl sulfate-capillary polyacrylamide gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry via a poly(tetrafluoroethylene) membrane.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS-capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time, and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI-TOF-MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE-resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE-separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes.

Miniaturized Electroosmotic Pump Capable of Generating Pressures of More than 1200 Bar

The pressure output of a pump cannot be increased simply by connecting several of them in series. This barrier is eliminated with the micropump developed in this work. The pump is actually an assembly of a number of fundamental pump units connected in series. The maximum pressure output of this pump assembly is directly proportional to the number of serially connected pump units. Theoretically, one can always enhance the pressure output by adding more pump units in the assembly, but in reality the upper pressure is constrained by the microtees or microunions joining the pump components. With commercially available microtees and microunions, pressures of more than 1200 bar have been achieved. We have recently experimented using open capillaries to build this pump, but many capillaries have to be utilized in parallel to produce an adequate flow to drive HPLC separations. In this paper, we synthesize polymer monoliths inside 75 μm i.d. capillaries, use these monoliths to assemble miniaturized pumps, characterize the performance of these pumps, and employ these pumps for HPLC separations of intact proteins. By tuning the experimental parameters for monolith preparations, we obtain both negatively and positively charged submicrometer capillary channels conveniently. Each monolith in a 75 μm i.d. capillary is equivalent to several thousands of open capillaries.

Stacking open-capillary electroosmotic pumps in series to boost the pumping pressure to drive high-performance liquid chromatographic separations.

Numerous micropumps have been developed, but few of them can produce adequate flow rate and pressure for high-performance liquid chromatography (HPLC) applications. We have recently developed an innovative hybrid electroosmotic pump (EOP) to solve this problem. The basic unit of a hybrid pump consists of a +EOP (the pumping element is positively charged) and a -EOP (the pumping element is negatively charged). The outlet of the +EOP is then joined with the inlet of the -EOP, forming a basic pump unit, while the anode of a positive high voltage (HV) power supply is placed at the joint. The inlet and outlet of this pump unit are electrically grounded. With this configuration, we can stack many of such pump units in series to boost the pumping power. In this work, we describe in details how an open-capillary hybrid EOP is constructed and characterize this pump systematically. We also show that a hybrid EOP with ten serially stacked pump units can deliver a maximum pressure of 21.5 MPa (∼3100 psi). We further demonstrate the feasibility of using this hybrid EOP to drive eluents for HPLC separations of proteins and peptides.

Integrated Bare Narrow Capillary–Hydrodynamic Chromatographic System for Free‐Solution DNA Separation at the Single‐Molecule Level

We report an integrated bare narrow-capillary–hydrodynamic chromatographic system (BaNC-HDC) for rapid, high-resolution, and repeatable separations of a wide size range of DNA at the single-molecule level in free solution. DNA separation is a common task in molecular biological research. Traditionally, DNA molecules are separated using slab-gel electrophoresis,[1] including pulsed field gel electrophoresis (PFGE).[2] To improve resolution, reduce running time, and increase throughput, capillary gel electrophoresis (CGE) and later capillary array electrophoresis (CAE) have been developed, and high resolutions have been achieved.[3] However, both CGE and CAE require viscous polymer sieving matrices, which can be difficult to work with, especially when narrow capillaries are employed. Researchers have experimented with separating DNA in free solutions, but DNA cannot be easily resolved in these media because all DNA molecules have similar mass-to-charge ratios (m/z), and, as a result, similar electrophoretic mobilities. In 1992, Noolandi[4] proposed an approach to solve this problem by attaching a monodisperse entity to each DNA fragment, generating varying m/z values for the DNA to be separated. The idea was experimentally validated in the late 1990s[5] and termed end-labeled free-solution electrophoresis (ELFSE). This method has proven to be effective for separating DNA fragments shorter than a few hundreds of base pairs.[6] Other gel-free approaches for DNA separation include radial migration,[7] liquid chromatography,[8] entropic trapping,[9] and DNA prism.[10] These approaches overcome the problems brought by viscous gels and offer promising alternatives for resolving DNA, but their resolutions are not competitive compared to that of gel electrophoresis. Recently, we have developed a new technique, called BaNC-HDC,[11] for free-solution DNA separations. When DNA molecules are transported inside a narrow capillary under pressure-driven conditions, the DNA molecules move as particles.[12] Larger DNA fragments have greater effective diameters and cannot go as close to the capillary wall (the slow-moving region) as smaller fragments can and, therefore, they move faster. On the basis of this principle, samples of DNA fragments with a wide size range have been separated with resolutions comparable to gel electrophoresis.[11] The minimal waste generation and low operation costs make BaNC-HDC an attractive alternative to gel-based techniques, particularly to PFGE for separating large DNA fragments. Herein, we integrate a high-pressure electroosmotic pump (EOP) and a microfabricated chip-injector with BaNC-HDC; the integrated system enables us to inject samples at low-picoliter (pL) volumes reliably, elute analytes at hundreds of pLmin−1 flow-rates or lower reproducibly, and resolve a wide size range of DNA fragments rapidly in free solution at the single-molecule level.

Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection

Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary - hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments.

Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.

Resolving DNA at efficiencies of more than a million plates per meter using bare narrow open capillaries without sieving matrices

We report a novel approach for effectively separating DNA molecules in free solution. The method uses a bare narrow open capillary without any sieving matrices to resolve a wide size-range of DNA fragments at efficiencies of more than a million plates per meter routinely.

Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins

We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem - limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis.

Simple Means for Fractionating Protein Based on Isoelectric Point without Ampholyte

In this paper, we develop a simple electrokinetic means for fractionating protein samples according to their pI values without using ampholytes. The method uses inexpensive equipment, and its consumables are primarily ammonium acetate buffers. A key component of its apparatus is a dialysis membrane interface that eliminates electrolysis-caused protein oxidation/reduction and constrains proteins in the desired places. We demonstrate its feasibility for fractionating standard proteins and real-world samples. With the elimination of ampholytes, we can analyze the fractionated proteins directly by a matrix assisted laser desorption/ionization time-of-flight mass spectrometer. Important experimental parameters are also discussed in order to obtain good fractionation results.