Whole-column Imaging Detection Research Articles

Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection

Because the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is the immunodominant antigen, the S protein and its receptor-binding domain (RBD) are both targets currently to be genetically engineered for designing the broad-spectrum vaccine. In theory, the expressed protein exists as a set of variants that are roughly the same but slightly different, which depends on the protein expression system. The variants can be phenotypically manifested as charge heterogeneity. Here, we attempted to depict the charge heterogeneity of the trimeric SARS-CoV-2 RBD by using capillary isoelectric focusing with whole-column imaging detection (cIEF-WCID). In its nature form, the electropherogram fingerprints of the trimeric RBD were presented under optimized experimental conditions. The peaks of matrix buffers can be fully distinguishable from peaks of trimeric RBD. The isoelectric point (pI) was determined to be within a range of 6.67–9.54 covering the theoretical pI of 9.02. The fingerprints of three batches of trimeric RBDs are completely the same, with the intra-batch and batch-to-batch relative standard deviations (RSDs) of both pI values and area percentage of each peak no more than 1.0%, indicating that the production process is stable and this method can be used to surveillance the batch-to-batch consistency. The fingerprint remained unchanged after incubating at 37 °C for 7 d and oxidizing by 0.015% H2O2. In addition, the fingerprint was destroyed when adjusting the pH value to higher than 10.0 but still stable when the pH was lower than 4.0. In summary, the cIEF-WCID fingerprint can be used for the identification, batch-to-batch consistency evaluation, and stability study of the trimeric SARS-CoV-2 RBD, as part of a quality control strategy during the potential vaccine production.

Selective extraction and enrichment of glycoproteins based on boronate affinity SPME and determination by CIEF-WCID

In this study, a new thin-film boronic acid coating was developed for solid-phase microextraction (SPME) followed by capillary isoelectric focusing with whole-column imaging detection (CIEF-WCID). Boronate functionalized particles of phenylboronic acid (PBA) and 3-aminophenylboronic acid (3-aPBA) were utilized as boronate affinity solid phase coating on thin-film stainless steel blades for selective extraction and enrichment of glycoproteins. The process of extraction and elution could be easily controlled by adjusting pH. To test specificity, asialofetuin and lactoferrin were selected as glycoproteins test molecules, while BSA and myoglobin were used as control non-glycoproteins in this study. The boronate affinity coating was characterized. The effect of buffer, pH, extraction profiles and elution profiles were investigated. The developed method was successfully applied to extract glycoproteins from standard buffer, PBS, human plasma and 10-fold diluted human blood using two kinds of boronate affinity blades. Boronate affinity SPME could be a promising tool for selective extraction and enrichment of low-abundance glycoproteins in real biological samples.

Characterization of human papillomavirus by capillary isoelectric focusing with whole‐column imaging detection

CIEF with whole-column imaging detection (WCID) can be a useful tool for the characterization and identification of human papillomavirus (HPV). This article is the initial report of the determination of the pI of HPV by CIEF-WCID method. In this study, components of the assay selected for optimization were ampholytes, additives, methylcellulose concentration, HPV concentration, salt concentration, and focusing time. Then the optimization CIEF-WCID method was validated for HPV 16L1 and HPV 18L1. As a result, a precise method to analyze the pI values of HPVs was achieved with RSD < 1.0%. The HPV peak pattern was reproducible. CIEF-WCID had great potential for HPV quality control, as WCID eliminated the mobilization step required by the conventional single-point detection. In the example, the five HPVs displayed pI values of 8.43 ± 0.06 (n = 10; HPV 6L1), 8.70 ± 0.04 (n = 10; HPV 11L1), 7.94 ± 0.05 (n = 18; HPV 16L1), 7.57 ± 0.04 (n = 18; HPV 18L1), and 8.45 ± 0.05 (n = 10; HPV 58L1). This CIEF-WCID platform could be a powerful analytical tool for characterization, process development support, release testing, and stability study in pharmaceutical industry.

Charge Heterogeneity of Monoclonal Antibodies by Multiplexed Imaged Capillary Isoelectric Focusing Immunoassay with Chemiluminescence Detection

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.

Human serum albumin interaction with oxaliplatin studied by capillary isoelectric focusing with the whole column imaging detection and spectroscopic method

Capillary isoelectric focusing (CIEF) with whole column imaging detection (WCID) was used to investigate drug-protein interactions. This study was designed to examine the interaction between the platinum-based anticancer drug, oxaliplatin, with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 10 to 100 μM and a constant concentration of HSA (5.0 × 10 −5 M). The reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48 and 72 h at 37 °C in a water bath. The CIEF results indicate that with increasing the drug concentration, the complex formation of protein adducts increased compared to low-drug concentrations and major structural changes were observed as the incubation time progressed. The altered CIEF profile demonstrated the possible conformation change due to the binding of the drug. Results also showed a significant protein's p I shift for higher HSA–oxaliplatin incubation ratios. Furthermore, spectroscopic evidence shows that oxaliplatin caused the fluorescence quenching of HSA by formation of HSA–oxaliplatin complex. Using the Stern–Volmer equation, the quenching constants were calculated in the linear range. The quenching rate constants K q at three different temperatures indicating the presence of static quenching mechanism in the interactions of oxaliplatin with HSA. This paper describes the validity of the CIEF-WCID technique for the study of protein–drug interactions and provides useful information and insight into the interaction of anticancer drugs with HSA.

Investigation of interaction between human hemoglobin A0 and platinum anticancer drugs by capillary isoelectric focusing with whole column imaging detection

CIEF with whole column imaging detection (WCID) was used to investigate the interaction of platinum-based anticancer drugs, cis-platinum(II) diamine dichloride (cisplatin) and [SP-4-2-{1R-trans)]-(1,2-cyclohexanediamine-N,N')[ethanedioata(2-)-O,O']platinum (oxaliplatin), with human hemoglobin A(0) (Hb). This technique facilitates the investigation and characterization of the formation of adducts between drugs and proteins. Cisplatin and oxaliplatin were mixed with the target protein at different concentrations (0:1, 1:1, 1:10, 1:50, and 1:100), and the reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48, and 72 h at 37 degrees C in a water-bath. The focused Hb-drug adduct profiles were imaged by WCID. At higher drug to protein molar ratios (for both oxaliplatin and cisplatin), the results exhibit significant changes in the peak shapes and heights, which may indicate the destabilization of the protein. However, the conformational change was less evident at lower molar ratios. In addition, a major pI shift was observed for the oxaliplatin reaction mixtures (for 1:10, 1:50, and 1:100 ratios). In comparison with previously reported findings obtained by other analytical methods, conclusions were drawn about the validity of CIEF as a simple and convenient method for the investigation of protein-drug interactions. These results may provide useful information for further understanding the activity and toxicity of these chemotherapeutic drugs and improving their clinical performance.

Rapid and high-resolution glycoform profiling of recombinant human erythropoietin by capillary isoelectric focusing with whole column imaging detection

Human erythropoietin (hEPO) is a glycoprotein hormone produced primarily by the kidney, which stimulates red blood cell production. Recombinant human erythropoietin (rhEPO), generally produced in Chinese hamster ovary (CHO) cells, can be used as not only a therapeutic protein but also a doping agent in sports. Profiling of EPO glycoforms is a critical means for quality control in pharmaceutical industrial and anti-doping analysis of misuse in sports. However, the existing methods for the analysis of EPO are associated with either time consuming or poor resolution. In this work, a rapid and high-resolution glycoform profiling method was presented based on capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID). Experimental conditions that influence the separation were investigated. Under optimized conditions, rhEPO from three different sources were resolved into distinct populations within 5 min with excellent reproducibility. As compared with existing methods, the presented method exhibited the advantages of speed and high resolution. If combined with an effective sample enrichment step and a much more sensitive WCID version, the method can be a potential alternative for the detection of rhEPO misuse in sports.

Whole column fluorescence imaging on a microchip by using a programmed organic light emitting diode array as a spatial-scanning light source and a single photomultiplier tube as detector

A novel miniaturized, integrated whole-column imaging detection (WCID) system on a microchip is presented. In this system, a program controlled organic light emitting diode (OLED) array was used as a spatial-scanning light source, to achieve imaging by the time sequence of the excited fluorescence. By this mechanism, a photomultiplier tube (PMT) instead of a charge coupled detector (CCD) can be applied to the imaging. Unlike conventional systems, no lenses, fibers or any mechanical components are required either. The novel flat light source provides uniform excitation light without size limitations and outputs a stronger power by pulse driving. The scanning mode greatly reduced the power consumption of the light source, which is valuable for a portable system. Meanwhile, this novel simplified system has a broader linear range, higher sensitivity and higher efficiency in data collection. Isoelectric focusing of R-phycoerythrin (PE) and monitoring of the overall process with WCID were performed on this system. The limit of detection (LOD) was 38 ng mL(-1) or 3.2 pg at 85 nL per column injection of PE. The system provides a technique for WCID capillary isoelectric focusing (cIEF) on chip and can be used for throughput analysis.

Characterization of plant growth-promoting rhizobacteria using capillary isoelectric focusing with whole column imaging detection

Capillary isoelectric focusing (cIEF) can be a useful tool for the characterization and identification of microbes. Based on the whole column imaging detection (WCID) technique and using plant growth-promoting rhizobacteria (PGPR) as test microbes, we present a two-level cIEF characterization method for the characterization and identification of bacteria. Intact bacteria were first characterized according to their apparent isoelectric points measured by cIEF-WCID and then lysed bacteria were further characterized by cIEF profiling of the intracellular proteins. Cellular clustering was found to be the main experimental barrier for the characterization of intact bacteria. The addition of sodium chloride (100 mM) to the sample mixture was found to be an effective way to reduce clustering. Due to the high efficiency and high resolution of cIEF-WCID, characterization of bacteria according to their intracellular proteins can be implemented simply and quickly without optimization of the experimental conditions. To improve the detection sensitivity with laser induced fluorescence (LIF)-WCID, the possibility to label bacteria with a non-covalent fluorescent dye, NanoOrange, was explored.

High-Speed, Whole-Column Fluorescence Imaging Detection for Isoelectric Focusing on a Microchip Using an Organic Light Emitting Diode as Light Source

An integrated and simplified microfluidic device using a 250 microm x 1-4 cm of organic light emitting diode (OLED) array as a two-dimensional light source for single-channel and multichannel whole-column imaging detection was developed. This fluorescence detection system was used for isoelectric focusing (IEF) of R-phycoerythrin in a microchip. The IEF conditions were optimized, and the total analysis time was extremely reduced to 30 s for 2-cm-long microchannels at 700 V/cm of electric field strength without the presence of electroosmotic flow. The compression of pH gradient caused by electrolytes drawing into the microchannels was efficiently restrained when 1% hydroxylpropylmethyl cellulose in 2% ampholyte was used as the carrier for IEF. Under optimized IEF conditions, the detection limit of this system was approximately 0.6 microg/mL or 45 pg at 75 nL/column injection of R-phycoerythrin. This OLED-induced fluorescence detection system for WCID provides a high-speed IEF technique with quantitative ability and the potential for high integration and throughput microchip systems.

Protein thermal stability and phospholipid–protein interaction investigated by capillary isoelectric focusing with whole column imaging detection

CIEF with whole column imaging detection (WCID) is an attractive technique for studying protein reaction and protein-ligand interaction due to its fast separation, simple operation, and high efficiency. In this study, two interesting applications by the CIEF-WCID were developed, involving the study of protein thermal stability and phospholipid-protein interaction. Four proteins (beta-lactoglobulin B, trypsin inhibitor, phosphorylase b, and trypsinogen) with different pI, and two types of phospholipids, including phosphatidylcholine (PC) and phosphatidylserine (PS), were used for this purpose. First, the altered CIEF profiles of four proteins were exhibited due to conformational changes resulting from protein denaturation induced by a high incubation temperature at 60 degrees C. It was demonstrated that the addition of a zwitterionic phospholipid (PC) played a crucial role in the thermal stability of targeted proteins, especially for trypsin inhibitor whose thermal stability was promoted with the addition of the PC vesicles at 60 degrees C. Second, the zwitterionic (PC) and acidic (PS) phospholipids displayed completely different interactions with the proteins. The addition of PS vesicles modified the zwitterionic phospholipids to carry negative charges, which correspondingly changed the interaction between the phospholipid and the protein. Our study demonstrates that the CIEF-WCID is a powerful approach to study protein reaction and protein-ligand interaction with high efficiency, high selectivity, and fast separation.

Dynamic process of phospholipid–protein interaction studied by capillary isoelectric focusing with whole‐column imaging detection

Liposomes are vesicles formed by the aggregation of amphiphilic phospholipid molecules, which can mimic natural cell membranes. Interaction between liposome and protein is important for the structure and function of natural cell membranes. In this study, a CIEF method with whole-column imaging detection was developed for monitoring the dynamic process of phospholipid-protein interactions. The CIEF profiles at successive interaction times clearly displayed the formation of the different conjugates between phospholipid and protein at different stages. Due to the diversity of the chemical and physical properties of targeted proteins in this study (trypsin inhibitor, beta-lactoglobulin B, phosphorylase b, and trypsinogen), different dynamic processes of phospholipid-protein interactions were exhibited. The type of phospholipids played an important role in the dynamic process of phospholipid-protein interaction, as noted by the use of zwitterionic phospholipid (phosphatidylcholine) and acidic phospholipid (phosphatidylserine). Mechanisms involved in these interactions were discussed by monitoring the dynamic processes in this study.

Array based capillary IEF with a whole column image of laser‐induced fluorescence in coupling to capillary RPLC as a comprehensive 2‐D separation system for proteome analysis

Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), a comprehensive 2-D system with laser-induced fluorescence was developed for protein mapping. By coupling capillary RPLC (CRPLC) as the first dimension and ACIEF as the second dimension, a high-throughput and high-resolution proteomic expression profiling was obtained. An array of up to 60 capillaries was assembled, with electrical connections made through filling small breaks, created on each capillary at positions of buffer reservoirs, with a porous polymer. A whole column image system with laser-induced fluorescence (LIF) was devised. Spot excitation was performed with a laser converted to produce linear light, and a CCD camera was employed to take images of the protein fluorescence during line laser scanning of the capillary array. Quantitative detection of thousands of focusing protein bands in the capillary array was achieved. Details on the capillary array fabrication and scanning LIF detection system devices are discussed. The efficiency of this CRPLC-ACIEF-LIF-WCID system was further demonstrated using samples of soluble proteins extracted from liver cancer tissue. The overall peak capacity was estimated to be around 18 000 in an analysis time of less than 3 h. The reproducibility of consecutive runs and different columns were assessed as having an RSD of 1.5% and 2.2% in focusing positions, respectively.

Microdialysis hollow fiber as a macromolecule trap for on-line coupling of solid phase microextraction and capillary electrophoresis

On-line coupling of solid phase microextraction (SPME) and capillary electrophoresis (CE) is highly desirable due to the apparent advantages of the two techniques particularly in the context of microanalysis. However, the hyphenation is a significant challenge, because of band broadening and analyte carryover caused by the slow kinetics of analyte desorption in liquid phase. A novel strategy was presented in this study to overcome these problems. Analytes desorbed from an SPME fiber, which was held by an adapter, were first transferred by electrophoretic migration into a short piece of microdialysis hollow fiber, which was located at the inlet of a CE system. Analytes with molecular weights greater than the molecular weight cut-off of the microdialysis material were trapped in the microdialysis hollow fiber due to the dialysis effect. Then, under another electric field with different electrode polarity, the analytes trapped in the microdialysis hollow fiber migrated into the separation capillary and were separated. In the coupling approach, the microdialysis hollow fiber functioned as a macromolecule trap and a sample pre-concentrator as well. Band broadening was eliminated because the initial sample volume was very small (at nL level). Meanwhile, analyte carryover was eliminated because the desorption time could be as long as needed. Coupling of SPME with CE including two modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), was successfully demonstrated with proteins as test analytes. High efficiency and high resolution were obtained. The detection limits with UV absorbance whole-column imaging detection were 3.0 x 10(-7) and 3.0 x 10(-8) M (S/N = 3) for beta-lactoglobulin A and ovalbumin, respectively.

Characterization of phospholipid–protein interactions by capillary isoelectric focusing with whole-column imaging detection

The integration of functional proteins in the phospholipid bilayer is one of the most crucial features of biological membrane architecture. Phospholipid–protein interactions play an important role in the functions of bounded proteins in the phospholipid membrane. When the phospholipid–protein interactions occur, the protein structure tends to alter, which can result in a change in the isoelectric points (p I) of protein. Capillary isoelectric focusing (cIEF) with whole-column imaging detection (WCID) is an attractive technique that has the features of simple operation, high resolution, and fast separation without focused band mobility for detection of amphoteric biomolecules. In this study, a cIEF-WCID method was developed to characterize the phospholipids–protein interactions by monitoring the protein cIEF profiles. Seven proteins with different p I and molecular mass , and a zwitterionic phosphatidylcholine (PC) with zwitterionic properties, were used to evaluate the feasibility of the cIEF-WCID approach in the study of phospholipid–protein interactions. The cIEF profiles changed in response to the changes in protein conformation, clearly exhibiting interactions between the PC vesicles and the targeted proteins. The formation of PC–protein complex was observed in the cIEF electropherograms. It was demonstrated that seven proteins displayed distinct interactions with the PC vesicles due to their different chemical and physical properties. The influences of the PC concentration, incubation time, and incubation temperature on the phospholipids–protein interactions were investigated. This study validated a novel analytical approach for the characterization of phospholipid–protein interactions.

Role of calcium binding in protein structural changes and phospholipid–protein interactions studied by capillary isoelectric focusing with whole column imaging detection

A variety of biochemical and physical properties of proteins are regulated by calcium ion (Ca 2+) binding with varying specificity and affinity. Calcium ion binding can adjust the phospholipid–protein interactions through changing the properties of phospholipid membrane. As an attractive detection technique, whole column imaging detection (WCID) coupled to capillary isoelectric focusing (cIEF) displays several advantages in the study of protein–ligand and protein–protein interactions, including fast and high-efficient separation, high resolution, and simple operation. In this study, a cIEF–WCID method was evaluated for studying the effect of Ca 2+ binding on protein structural changes and phospholipid–protein interactions. Four proteins with different isoelectirc point (p I), trypsin inhibitor (p I = 4.5), β-lactoglobulin B (p I = 5.2), phosphorylase b (p I = 6.3), and trypsinogen (p I = 9.3), were used for this purpose. The targeted proteins exhibited altered cIEF profiles due to protein conformation changes resulting from the Ca 2+ binding. The study showed that Ca 2+ can be buried in the phospholipid membrane, modify the membrane property, and change the phospholipid–protein interactions. The utility of the cIEF–WCID technique demonstrates that the calcium binding plays a crucial role in the protein structural changes and the phospholipid–protein interactions, and elucidates the possible mechanisms involved in calcium–protein binding and calcium bound phospholipid–protein interactions.

Characterization of bovine serum albumin–tryptophan interaction by capillary isoelectric focusing with whole column imaging detection

Capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID) was explored for the characterization of bovine serum albumin (BSA)–tryptophan interaction, to further understand protein–drug interactions. The BSA–tryptophan interaction was dynamically monitored by cIEF–WCID, to provide the cIEF profiles of the BSA–tryptophan interaction system at different focusing times. Our study demonstrated that the cIEF behavior of BSA can serve as a probe into the study of BSA–tryptophan interaction, through monitoring the change in its cIEF profile when the interaction occurred. The study illustrated that the BSA peak split due to the BSA–tryptophan interaction, and the peak of BSA–tryptophan complex was clearly identified in the cIEF electropherograms. By comparing the cIEF profiles of BSA/ l-tryptophan and BSA/ d-tryptophan, respectively, our study demonstrated that BSA interacted with the enantiomers of tryptophan with a chiral recognition. l-Tryptophan demonstrated a very strong interaction with BSA, while d-tryptophan exhibited a much weaker interaction with BSA. The effects of the BSA concentration, the tryptophan concentration, the focusing time and the incubation time on the BSA–tryptophan interaction were investigated. This study offers a novel approach for the study of protein–drug interactions.

Whole-column imaging-detection techniques and their analytical applications

We review developments in whole-column detection, including partial column imaging detection and whole-column imaging detection for high-performance liquid chromatography and capillary electrophoresis. We especially discuss recent topics in whole-column imaging detection.

Behaviors of the MS2 virus and related antibodies in capillary isoelectric focusing with whole-column imaging detection

Capillary isoelectric focusing (CIEF) has potential importance for the study of viruses. CIEF with whole-column imaging detection (WCID) is a novel CIEF mode, providing the advantages of high resolution, high speed, and easy method development. To facilitate the application of CIEF-WCID to the immunoassay of viruses, a basic knowledge of related aspects is necessary. In this study, the MS2 bacteriophage was used as a virus model, and the behaviors of MS2 and related antibodies in CIEF were investigated with UV absorbance-WCID and laser-induced fluorescence (LIF)-WCID. The adsorption of the virus and antibodies on the capillary wall was found to be the critical issue in method development. Addition of salt was found to be an effective way to reduce the adsorption and to improve peak shape. The formation of an immunocomplex, which forms the basis of an immunoassay, was monitored with CIEF-WCID. In comparison with UV-WCID, LIF-WCID was advantageous due to its higher detection sensitivity and the elimination of precipitation. Utilization of the noncovalent fluorescent dye, NanoOrange, was demonstrated to be a potential approach for the fluorescent labeling of the virus model and antibody and the associated immunocomplex. The change in microheterogeneity during the immune interactions at different ratios was also observed.

Coupling of Solid-Phase Microextraction and Capillary Isoelectric Focusing with Laser-Induced Fluorescence Whole Column Imaging Detection for Protein Analysis

A coupling method of solid-phase microextraction (SPME) and capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) was developed for the analysis of proteins. Unlike other liquid-phase separation methods and conventional CIEF, proteins are focused into stationary bands within a pH gradient in CIEF-WCID. Thus, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME, which can effectively resolve the problems associated with the slow desorption kinetics of SPME in a liquid phase. By combining SPME and CIEF-WCID, the desorption time can be as long as necessary, allowing complete desorption without any band broadening and analyte carryover. By using this method, R-phycoerythrin in water can be extracted by SPME in 10 min, and subsequently analyzed by CIEF-LIF-WCID within 20 min, providing a limit of detection of 3.5 x 10(-12) M (S/N = 3). The feasibility of the SPME-CIEF-LIF-WCID method was demonstrated by extracting and analyzing extracellular phycoerythrins in cultured cyanobacteria samples. Extracellular phycoerythrins at the nanomolar level were extracted and analyzed in 30 min, while avoiding the interference of the cyanobacteria cells.