Peptide mapping using capillary electrophoresis offline coupled to matrix-assisted laser desorption ionization time of flight mass spectrometry

This article reports the results of a study carried out to evaluate the offline hyphenation of capillary zone electrophoresis with matrix-assisted lased desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the analysis of low-abundant complex samples, represented by the tryptic phosphorylated peptides of phosphoproteins, such as α-casein, β-casein, and fetuin. The proposed method employs a latex-coated capillary and consists in the online preconcentration of the tryptic peptides by a pH-mediated stacking method, their separation by capillary zone electrophoresis, and subsequent deposition of the separated analytes onto a MALDI target for their MS analysis. The online preconcentration method allows loading a large sample volume (～150?nL), which is introduced into the capillary after the hydrodynamic injection of a short plug of 1.0?M ammonium hydroxide solution and is sandwiched between two plugs of the acidic background electrolyte solution (BGE) filling the capillary. The sample spotting of the separated analytes onto the MALDI target is performed either during or postseparation using an automatic spotting device connected to the exit of the separation capillary. The proposed method allows the separation and identification of multiphosphorylated peptides from other peptides and enables their identification at femtomole level with improved efficiency compared with LC approaches hyphenated to MS.     

Separation of 2-aminobenzoic acid-derivatized glycosaminoglycans and asparagine-linked glycans by capillary electrophoresis.

A capillary electrophoresis method was developed for the analysis of oligosaccharides combined with derivatization with 2-aminobenzoic acid. Glycosaminoglycan delta-disaccharides were effectively resolved on a fused-silica capillary tube using 150 mM borate, pH 8.5, as a running electrolyte solution. This analytical method was applied to the identification of glycosaminoglycan in combination with enzymatic digestion. The separation of N-glycans or glucose-oligomers was performed with a phosphate buffer containing polyethylene glycol or borate as an electrolyte solution. This method is expected to be useful in the determination of oligosaccharide structures in a glycoprotein.     

Rapid Determination of Amino Acids by Capillary Electrophoresis without Preliminary Derivatization

A rapid method was developed for the identification and determination of a mixture of eleven amino acids present together without their preliminary derivatization and without adding modifying agents to the buffer solution. A Kapel'-105 instrument for capillary electrophoresis (Lyumex Company, Russia) was used.     

Identification of a metallothionein in Synechococcus by capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry.

A home-made system hyphenating capillary electrophoresis with an inductively coupled plasma mass spectrometer (CE-ICP-MS) for cadmium speciation of protein-binding and free cadmium ions in solution is presented. The CE-ICP-MS interface consisted of an acrylic block with an internal volume ca. 20 microL in which a platinum electrode, a capillary column, and a connection to an ICP nebulizer were inserted. A make-up electrolyte solution containing 50 mmol L(-1) Tris-HCl buffer solution (pH 9.0) was continuously flowed through the interface to the ICP nebulizer. The separation of free Cd ions, Cd-cysteine, and Cd bounded to metallothionein (MT) isoforms from rabbit liver was carried out by capillary electrophoresis, and the analytes were detected by ICP-MS. The feasibility to isolate metallothionein compounds extracted from the cyanobacterium Synechococcus PCC7942 was demonstrated. The Cd binding proteins were induced in Synechococcus PCC7942 and further analyzed by CE ICP-MS.     

寡糖的毛细管电泳分析

多种寡糖经α－萘胺衍生化后，用硼砂作为电泳介质，实现了高效毛细管电泳分离。比较了毛细管区带电泳和胶束毛细管电动色谱分离寡糖α－萘胺衍生物的电泳行为，对影响分离度的诸因素进行了考察，选择了最佳分离条件。     

pH Change of buffer solution in a microcapillary chip and its suppression

During the electrophoresis separation of B- and T-cells from lymphocytes employing a microcapillary chip, they were found to become inactive in the reservoir after a short time. This was caused by the buffer solution becoming alkaline due to electrolysis. This was considered to take place in chips with small reservoir volumes. The pH change was confirmed by the ISFET (ion-sensitive field effect transistor) embedded in the chip. To suppress the pH change, two methods were studied. One is the insertion of a salt bridge just in front of and behind the capillary, thus introducing an electric potential but stopping flow of the acid and alkaline solutions into the capillary. The other is neutralization of the alkaline solution in the reservoir by injecting the acid solution produced in another capillary with the same structure by employing an electroosmotic flow (EOF) pump. Both methods achieved no pH change during electrophoresis measurements in the microcapillary.     

Identification of bioactive peptides in hypoallergenic infant milk formulas by capillary electrophoresis-mass spectrometry

In this study, we use capillary electrophoresis-mass spectrometry (CE-MS) for the identification of bioactive peptides in hypoallergenic infant milk formulas (IF), which are complex bovine milk protein hydrolysates. A sample clean-up pretreatment with a citrate buffer containing dithiothreitol and urea followed by solid-phase extraction (SPE) with different reversed-phase commercial cartridges was investigated to achieve optimum detection sensitivity in CE-MS. SPE with C18, StrataX and Oasis HLB cartridges allowed detection of the largest number of low molecular mass components, but combination of C18 and StrataX results was enough to achieve an excellent coverage of the studied IF. The monoisotopic molecular mass values of the low molecular mass components obtained by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) allowed the tentative identification of nine bioactive sequences. Only the identification of five of them could be confirmed when accurate mass measurements were performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS), namely LKP, IPY, ALPM, PGPIHN and VAGTWY, which were reported to present angiotensin-converting enzyme (ACE) inhibitory and antimicrobial activity (only VAGTWY).     

Assay of protein drug substances present in solution mixtures by fluorescamine derivatization and capillary electrophoresis.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.     

Prevention of evaporation of small-volume sample solutions for capillary electrophoresis using a mineral-oil overlay.

The suppression of evaporation of water from small volumes of sample solutions or reagents for capillary electrophoresis by the use of a mineral-oil overlay was investigated in affinophoresis applications, in which the affinity constant of a mutant protein of recombinant human galectin-1 to a lactose affinophore, a triply negative charged ion having a lactoside as an affinity ligand, was determined. When an injection was carried out from a minimum of 20 microL of an aqueous solution beneath the oil overlay, no oil contamination inside the capillary was observed, provided the capillary was cleanly cut so that the end was flat, and the polyimide coating had been removed for a distance of about 2 mm from the end. Affinophoresis was carried out using 20 microL of an affinophore solution covered with an oil overlay. The abnormalities in the electropherograms as the result of the evaporation of the water from the solution during storage prior to use in an automatic operation of a capillary electrophoresis instrument were suppressed, with respect to the formation of a base line gap, an increase in the detection time of a marker ion and an increase in the initial current. A solution in a vial could be used repeatedly for a longer period of time when overlaid with mineral oil than in the absence of an overlay. The use of a mineral-oil overlay is a simple but very efficient technique for solving the problem of the evaporation of water from small volumes of aqueous solutions for use in capillary electrophoresis.     

Off-line and in-line determination of catechol- O-methyltransferase activity in a capillary electrophoretic system

The use of capillary electrophoresis for the determination of catechol-O-methyltransferase (COMT) activity with dihydroxybenzoic acid as a substrate was investigated. Both an off-line and in-line capillary electrophoresis determination of COMT activity was developed and the two approaches are discussed. In the presented methods, substrate and reaction products are monitored at the same time. The initial velocity of the reaction is quantified spectrophotometrically by the corrected peak area of the products at 200 nm. In the off-line setup, capillary zone electrophoresis is used to separate and quantify the different reaction compounds. Each electrophoretic run required only 37 nL of the enzymatic reaction solution. Based on the off-line assay, an in-line determination of COMT activity was developed by a methodology known as electrophoretically mediated microanalysis (EMMA). All the different steps (i.e. mixing, incubation, separation and in-line quantitation) are combined in the capillary, which is used as a microreactor for the enzymatic reaction. Full automation of the assay is achieved with this microscale approach.     

Elimination of adsorption effects in the analysis of water-soluble vitamins in pharmaceutical formulations by capillary electrophoresis.

A tendency to an increase in migration times was observed when different water-soluble vitamins were analysed repeatedly in pharmaceutical preparations by capillary electrophoresis. In order to better understand the origin of this effect, the influence of the vitamins and the excipients, such as cellulose derivatives, was investigated. These studies indicated that the increase in analyte migration times was most probably due to the adsorption of different kinds of constituents to the capillary wall. Different rinsing procedures were tested in order to eliminate these unfavourable effects. A rinse of the capillary with a 25 mM sodium dodecyl sulfate (SDS) solution in the running buffer between successive runs was found to be particularly effective when the analysis was performed by free solution capillary zone electrophoresis (CZE). When the vitamins were determined by micellar electrokinetic chromatography (MEKC) using SDS as surfactant, a short capillary rinse with the running buffer was sufficient to obtain reproducible migration times. The CZE and MEKC methods developed were validated and compared. Both methods could be applied to the determination of water-soluble vitamins in different multivitamin formulations.     

Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis.

A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross-linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross-linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte-wall interactions. The long-term stability of cross-linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross-linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.     

Analysis of synthetic chemical drugs in adulterated Chinese medicines by capillary electrophoresis/electrospray ionization mass spectrometry

Sixteen synthetic chemical drugs, often found in adulterated Chinese medicines, were studied by capillary electrophoresis/UV absorbance (CE/UV) and capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS). Only nine peaks were detected with CZE/UV, but on-line CZE/MS provided clear identification for most compounds. For a real sample of a Chinese medicinal preparation, a few adulterants were identified by their migration times and protonated molecular ions. For coeluting compounds, more reliable identification was achieved by MS/MS in selected reaction monitoring mode. Micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) provided better separation than capillary zone electrophoresis (CZE), and, under optimal conditions, fourteen peaks were detected using UV detection. In ESI, the interference of SDS was less severe in positive ion mode than in negative ion mode. Up to 20 mM SDS could be used in direct coupling of MEKC with ESI-MS if the mass spectrometer was operated in positive ion mode. Because of better resolution in MEKC, adulterants can be identified without the use of MS/MS.     

Capillary electrophoresis-chemiluminescence detection system equipped with a consecutive sample-injection device.

We have developed a consecutive sample-injection device for capillary electrophoresis, which comprises one four-way cock, two syringe pumps, and an interface part taking advantage of two three-way Teflon joints. Sample introduction into the capillary is made hydrodynamically by pressure, caused by the flow of the sample solution at the tip of the capillary inlet. We combined the injection device with a capillary electrophoresis-chemiluminescence detection system. A mixture solution of N-(4-aminobutyl)-N-ethylisoluminol, isoluminol isothiocyanate, and luminol was analyzed as a model sample by the present system. The sample solution was consecutively injected and detected with about a 230 s interval. The present capillary electrophoresis-chemiluminescence detection system with the consecutive sample injection device features easy and rapid operation, an inexpensive apparatus, high sensitivity, as well as consecutive analysis.     

Separation efficiency of free‐solution conjugated electrophoresis with drag‐tags incorporating a synthetic amino acid

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge‐to‐friction ratio in free solution, DNA fragments cannot be separated by size. However, size‐based separation of DNA is possible in free‐solution conjugate electrophoresis (FSCE) if a “drag‐tag” is attached to DNA fragments because the tag breaks the linear charge‐to‐friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free‐solution conjugated electrophoresis, generation of a monodisperse drag‐tag and identification of a strong, site‐specific conjugation method between a DNA fragment and a drag‐tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin‐like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site‐specific conjugation, a methionine residue in drag‐tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site.     

High performance capillary electrophoresis of calmodulin

The electrophoretic properties of purified calmodulin were investigated. High performance capillary electrophoresis of this Ca2+(-)binding protein in free solution at pH 2.5 resulted in an elution of a single peak with a retention time of approximately 4.7 min. Addition of [ethylene-bis(oxyethylenenitrilo]) N,N,N',N'-tetraacetic acid (EGTA) to the protein prior to capillary electrophoresis completely abolished this electrophoretic profile. Polyacrylamide gel electrophoresis of calmodulin under denaturing and nondenaturing conditions also revealed a single polypeptide band. However, the relative electrophoretic mobilities of this protein could vary, depending on the presence or absence of Ca2+. The pI of calmodulin was estimated to be 3.7 by using isoelectric focusing techniques. Analysis of this acidic protein by high performance capillary electrophoresis at pH 8.0 revealed that it could be resolved into two major and one minor polypeptide peaks, regardless of the presence or absence of Ca2+. These findings suggest that capillary electrophoresis at near physiological pH may differentiate the microheterogeneity of calmodulin.     

Analysis of choline and atropine in hairy root cultures of Cannabis sativa L. by capillary electrophoresis-electrospray mass spectrometry

We describe a capillary zone electrophoresis method coupled to electrospray ionization (ion trap) mass spectrometry (CZE-ESI-MS) for the identification and determination of choline and atropine compounds in hairy root extracts from Cannabis sativa L. Fused-silica capillary and an alkaline solution of 20 mM ammonium acetate at pH 8.5 are used being the most suitable for the analysis of choline and atropine in less than 10 min. Under the optimized conditions, including CE and ESI-MS parameters, the method resolved both compounds with very high sensitivity. The system exhibited good linear response in the range of 25-500 mg/L and 500-1000 microg/L for choline and atropine, respectively. The detection limit of choline was 18 mg/L and 320 microg/L for atropine. Finally, the developed method was applied to the analysis of these compounds in transgenic root cultures of Cannabis sativa L.     

苯胺及其衍生物的毛细管电泳行为研究

采用紫外吸收检测，毛细管电泳分离，研究了九种苯胺及其衍生物在毛细管区带电泳(CZE)体系和胶束电动毛细管色谱(MECC)体系中的行为特征。讨论了缓冲溶液的浓度与pH、胶束浓度及混合胶束等在不同体系中对分离组分的影响，发现在CZE体系中，控制分离的主要因素是pKb值；在MECC体系中，控制分离的主要因素是溶质分子中碳原子数。建立了一种分离测定九种苯胺及其衍生物的高效毛细管电泳方法。     

Pre-concentration of non-uniform field electrophoresis for sample introduction of capillary electrophoresis.

A new sample introduction method of capillary electrophoresis, in which field-amplified sample injection was combined with a pre-concentration of non-uniform field electrophoresis, is presented in this paper. With an additional pre-concentration voltage applied to sample solution, a non-uniform electric field was generated, with which analytical cations or anions were pre-concentrated around an electrode adjacent to the injection end of capillary. After the pre-concentration, analytical ions were injected into the capillary and stacked at the boundary between sample and buffer solution inside capillary by field-amplified injection technique. In contrast to the conventional field-amplified injection, larger concentration factor and higher analytical sensitivity were obtained with the improved pre-concentration method. Its concentration factor was about 10 approximately 15 fold as that of field-amplified sample injection.