On-line Capillary Isoelectric Focusing Research Articles

Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing - Mass spectrometry

Membranous nephropathy (MN) is one of the most common causes of primary glomerular diseases worldwide. The M-type phospholipase A2 receptor (PLA2R), an antigen expressed in more than 70% of cases of idiopathic membranous nephropathy (IMN), is a biomarker which is now used by physicians for clinical diagnosis. Despite the prevalence of PLA2R in the cases of MN, it is not always effective to use PLA2R for differentiating primary or secondary MNs. On the other hand, urinary albumin assay is one of the de facto tests for kidney function testing for several decades. In this work, urinary albumin species between primary and secondary MN patients are compared using a newly developed capillary isoelectric focusing – mass spectrometry (CIEF-MS) technology. The distinct patterns of cationic and acidic urinary albumin species, as revealed by this novel CIEF-MS technology, suggest potential applications of this differential analysis for subtyping of membranous nephropathy. Further investigation of these cationic human albumin species in urine may provide clues to the disease onset and development of MN, thus facilitating treatment. In addition, this novel workflow of using CIEF-MS for urinary protein analysis may be beneficial to the research, pathology, prognosis, and diagnosis of many other types of kidney diseases, such as chronic kidney disease, diabetic nephrology, etc.

Front cover: Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Front cover: Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time-of-flight mass spectrometry.

Capillary isoelectric focusing (cIEF) was online coupled to a Q-TOF MS by a flow-through microvial interface for the analysis of therapeutic mAb. Intact molecular weights obtained from the mass spectrum deconvolution of separated charge variants provided information on the structural heterogeneity of therapeutic mAbs. A sandwich cIEF-MS configuration composed of anolyte, sample, and catholyte segments sequentially injected into a neutrally coated capillary was used for the charge heterogeneity separation of four mAbs. Acetic acid and ammonium hydroxide were used in places of the non-volatile acids and bases commonly used for IEF but are incompatible with online MS detection. Glycerol was added as the anti-convective reagent. A chemical modifier was mixed with the cIEF effluent in the flow-throw microvial to maintain the ESI stability and to mitigate ion suppression from the co-eluted carrier ampholytes and glycerol. Analysis of mAb samples have shown relative populations of two basic variants originating from C-terminal lysine process and acidic variant of deamidation. The lysine clippings, deamidation, and sialic acid modification in oligosaccharide chains were revealed in infliximab. Two lysine clipping variants and a deamidated variant were observed in adalimumab. The duplicate analyses of a reference mAb demonstrated five charge variants separated by cIEF due to some unidentified modifications, as their mass spectra shared close similarities. The mAb analyses demonstrated the feasibility of the cIEF-MS method, and they demonstrated how charge and structural variants and minor differences in therapeutic mAbs are observed with this technology. Online cIEF-MS is an information rich technology with high throughput, demonstrated by the initial data presented here.

Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

In the presence of methanol and n-decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3-10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M-IPG). In addition, an online self-built platform for protein separation was established on account of the introduction of a cross-shaped unit and two short-off valves. In this platform, a cross-shaped unit was not only used to join the M-IPG column and a six-way injection valve (1.5 μL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short-off valve in the tee unit or cross-shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7-proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M-IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A1c have been successfully resolved with the newly developed M-IPG column.

On-line capillary isoelectric focusing hyphenated to native electrospray ionization mass spectrometry for the characterization of interferon-γ and variants.

The on-line hyphenation of Capillary IsoElectric Focusing (CIEF) with ElectroSpray Ionization Mass Spectrometry (ESI/MS) has been carried out in a non-denaturing detection mode at the CIEF-MS interface. This CIEF-MS coupling methodology relied on the use of 40% glycerol-water medium as anti-convective agent in the CE capillary and the addition of 10 mM ammonium acetate buffer, pH 5, as a volatile aqueous sheath liquid. These CIEF-MS coupling conditions allowed the characterization of the highly basic cytokine human interferon-gamma (IFN-γ) and its detection as a non-covalent homodimer (33,814.3 g mol(-1)) corresponding to the active form of this immune-regulatory protein. An experimental pI value of 9.95 was determined for the human IFN-γ homodimer in these conditions. The CIEF-MS analysis of several variants bearing punctual or deletion mutations within the two D1 and D2 basic clusters at the C-terminal end of IFN-γ revealed the different contribution of these domains to the charge properties of this heparan sulfate-binding protein.

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing—Electrospray ionization mass spectrometry in glycerol–water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol–water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close p I values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close p I values (4.5–5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on p I determination. After optimization of sheath liquid composition (80:20 (v/v) methanol–water + 1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57–136 nM and 11–68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF–MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.

On-line capillary isoelectric focusing–mass spectrometry for quantitative analysis of peptides and proteins

On-line capillary isoelectric focusing–mass spectrometry (cIEF–MS) was applied to determine concentrations of peptides and proteins using angiotensin II and human tetrasialo-transferrin as the model samples. The concentration of the carrier ampholyte was optimized for both resolution and ion intensity. cIEF–MS employing 1% Pharmalyte 3–10 and a sheath liquid containing water/methanol/acetic acid (50/49/1) resolved angiotensin I and II (5 μM each, Δp I = 0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1–5 μM) well correlated ( R = 0.999) with that obtained by the conventional RP–HPLC method. The limit of detection was 0.22 μM, which was about 10 times lower than that by UV detection (2 μM). The repeatability and accuracy were <15 and <11%, respectively. cIEF–MS was also applied to determine human tetrasialo-transferrin concentration. The good linearity ( R 2 = 0.998) was also observed between the transferrin concentration (0.5–1.2 g/L) and peak area ratio (IS; β-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability (∼10% at 1 g/L).

Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry.

Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h.

Capillary isoelectric focusing-electrospray ionization time-of-flight mass spectrometry for protein analysis

On-line capillary isoelectric focusing (CIEF)–electrospray ionization time-of-flight mass spectrometry (ESI–TOFMS) as a two-dimensional separation system is employed for high-resolution analysis of model proteins (myoglobin and β-lactoglobulin) and human hemoglobin variants C, S, F, and A. The focused proteins in a polyacrylamide-coated capillary are mobilized by replacing the sodium hydroxide catholyte with a sheath liquid solution containing methanol–water–acetic acid in a volume ratio of 80:19:1. The use of sheath liquid also establishes the electrical connection at the CIEF capillary terminus, which serves to define the electric field along the CIEF capillary and apply an electric voltage for electrospray ionization. At the end of the CIEF capillary, the mobilized protein zones are analyzed and directly identified by ESI–TOFMS. The TOFMS accumulates spectra at 5000 Hz frequency and one averaged spectrum is acquired per second during the CIEF–ESI–TOFMS measurements. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 193–197, 1999

High resolution analysis of protein phosphorylation using capillary isoelectric focusing - electrospray ionization - mass spectrometry.

On-line capillary isoelectric focusing (CIEF)-electrospray ionization - mass spectrometry (ESI-MS) as a two-dimensional separation system is employed for high resolution analysis of ovalbumin phosphorylation. On the basis of their differences in isoelectric point (pI), the mono- and diphosphoovalbumins are separated and resolved in CIEF. The focused protein zones of mono- and diphosphoovalbumins are eluted by combining gravity with cathodic mobilization. At the end of the CIEF capillary, the mobilized ovalbumin zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. Additional ovalbumin variants within each of the mono- and diphosphoovalbumins, differing in their molecular masses due to glycosylation microheterogeneity, are easily distinguished by ESI-MS.

Capillary Isoelectric Focusing–Electrospray Ionization Mass Spectrometry for Transferrin Glycoforms Analysis

On-line capillary isoelectric focusing (CIEF)–electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of bovine serum apotransferrin glycoforms. On the basis of their differences in isoelectric point (pI), the di-, tri-, and tetrasialotransferrins are separated and resolved in CIEF. The focused protein zones of di-, tri-, and tetrasialotransferrins are eluted by combining gravity with cathodic mobilization. At the end of CIEF capillary, the mobilized transferrin zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. Additional transferrin variants within each of di-, tri-, and tetrasialotransferrins, differing in their molecular weights, are easily distinguished by ESIMS. In combination with sialidase digestion, the distribution of pIand molecular weight of asialo-, mono-, di-, tri-, and tetrasialotransferrin variants was obtained from the CIEF–ESIMS measurements. In addition to the differences in the number of sialic acid, the microheterogeneity of bovine serum apotransferrin glycans might be complicated by the partial fucosylated structure and the α-Gal (1–3)-β-Gal on the α-Man-(1–6) antenna.

High-Resolution Capillary Isoelectric Focusing−Electrospray Ionization Mass Spectrometry for Hemoglobin Variants Analysis

On-line capillary isoelectric focusing (CIEF)-electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of hemoglobin variants A, C, S, and F. The effects of moving ionic boundary inside the CIEF capillary and MS scan rate on the separation resolution and mass detection of hemoglobin variants are investigated. The formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. Hemoglobin variants F and A, with a pI difference of 0.05 pH unit, are almost baseline resolved and identified in CIEF-ESIMS. The concentration detection limit for each hemoglobin variant is in the range of 10(-)(8) M, comparable to that obtained in two-dimensional gel electrophoresis using silver staining. Initial preconcentration during the focusing step and the use of single-ion monitoring scan mode are responsible for improving detection limits.

A CE/ESI-MS interface for stable, low-flow operation.

A rugged, stable liquid sheath interfacing procedure for on-line CE/MS is described. This procedure combines optimized component sizes, tapered capillary tips, and adjustment of the capillary with respect to the liquid sheath tube during electrospray operation (active capillary positioning) to establish a stable electrospray quickly and reliably. The interface is especially effective at low flow rates, and CE/MS with a liquid sheath flow rate of 250 nL/min has been achieved. Active capillary positioning also allows on-line capillary isoelectric focusing mass spectrometry with in-probe focusing. In this application, the capillary is retreated into the liquid sheath tube, which becomes a microreservoir. After focusing, the capillary is returned to its proper position for electrospray, and focused zones are mobilized into the mass spectrometer for on-line detection. Both active capillary positioning and in-probe focusing readily lend themselves to automation.