Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.     

Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of delta and omega-conotoxins

The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.     

Capillary electrophoresis—matrix-assisted laser-desorption ionization mass spectrometry of proteins

An “off-line” combination of capillary electrophoresis (CE) with matrix-assisted laser-desorption mass spectrometry (MALDI-MS) has been developed for the structural characterization of CE-separated peptides and proteins. Using a sheath flow interface, similar to that developed for “on-line” CE—fast atom bombardment MS and CE—electrospray MS, an efficient sample isolation procedure has been developed which is applicable to bioorganic compounds in aqueous buffer solutions. This isolation procedure, with subsequent transfer to the MALDI-MS sample target, has been successfully used for the direct analysis of CE-separated proteins of M _r up to 67 000, and a mixture of apolipoprotein AII monomer and homodimer, using sample amounts of less than 1 pmol.     

Derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The characterization of glycosylation in proteins by mass spectrometry (MS) is often impeded by strong suppression of ionization of glycopeptides in the presence of non-glycosylated peptides. Glycopeptides with a large carbohydrate part and a short peptide backbone are particularly affected by this problem. To meet the goal of generating mass spectra exhibiting glycopeptide coverages as complete as possible, derivatization of glycopeptides offers a practical way to increase their ionization yield. This paper investigated derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) which is a rapid labeling technique commonly used for fluorescence detection in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). As test samples we used peptides and glycopeptides obtained by enzymatic digestion of three different glycoproteins, i.e., human antithrombin, chicken ovalbumin, and bovine alpha1-acid-glycoprotein. It was found that AQC derivatization resulted in strongly increased signal intensities when analyzing small peptides and glycopeptides by matrix-assisted laser desorption/ionization (MALDI)-MS. For these compounds the limit of detection could be reduced to low fmol amounts. Without derivatization only glycopeptides containing large peptide backbones were detected by MALDI-MS. This effect was even significant when glycopeptides were pre-separated and enriched by means of lectin affinity chromatography before MALDI-MS analysis and when using electrospray ionization (ESI). This labeling method, applied in combination with MS detection for the first time, was found to be well suited for the enhancement of detection sensitivity for small glycopeptides in MALDI-MS analysis and thus for reducing the need for pre-separation steps.     

Characterization of protein hydrolysates of cosmetic use by CE-MS

Protein hydrolysates have been used as active principles in cosmetic products conferring different properties to the final formulations, which are mostly controlled by the peptide size and its amino acid sequence. In this work, capillary electrophoresis coupled to mass spectrometry analyses were carried out in order to investigate such characteristics of protein hydrolysates. Samples of different origins (milk, soy and rice) were obtained from a local company, and were analyzed without a previous preparation step. The background electrolyte (BGE) and sheath liquid compositions were optimized for each sample. The best BGE composition (860 mmol/L formic acid--pH 1.8--in 70:30 v/v water/methanol hydro-organic solvent) was chosen based on the overall peak resolution whereas the best sheath liquid was selected based on increased sensitivity and presented different compositions to each sample (10.9-217 mmol/L formic acid in 75:25-25:75 v/v water/methanol hydro-organic solvent). Most of the putative peptides in the hydrolysate samples under investigation presented molecular masses of 1000 Da or less. De novo sequencing was carried out for some of the analytes, revealing the hydrophobicity/polarity of the peptides. Hence, the technique has proved to be an advantageous tool for the quality control of industrial protein hydrolysates.     

Rapid determination of 4‐aminobutyric acid and L‐glutamic acid in biological decarboxylation process by capillary electrophoresis‐mass spectrometry

4‐Aminobutylic acid (GABA) is a monomer of plastic polyamide 4. Bio‐based polyamide 4 can be produced by using GABA obtained from biomass. The production of L ‐glutamic acid (Glu) from biomass has been established. GABA is produced by decarboxylation of Glu in biological process. High‐performance liquid chromatography (HPLC) with derivatization is generally used to determine the concentration of GABA and Glu in reacted solution samples for the efficient production of GABA. In this study, we have investigated the rapid determination of GABA and Glu by capillary electrophoresis‐mass spectrometry (CE‐MS) without derivatization. The determination was achieved with the use of a shortened capillary, a new internal standard for GABA, and optimization of sheath liquid composition. Determined concentrations of GABA and Glu by CE‐MS were compared with those by pre‐column derivatization HPLC with phenylisothiocyanate. The determined values by CE‐MS were close to those by HPLC with pre‐column derivatization. These results suggest that the determination of GABA and Glu in reacted solution is rapid and simplified by the use of CE‐MS.     

Analysis of major milk whey proteins by immunoaffinity capillary electrophoresis coupled with MALDI-MS

Two major milk whey proteins, β-lactoglobulin and α-lactalbumin, are among the main cow milk allergens and can cause allergy even at a very low concentrations. Therefore, these proteins are interesting targets in food analysis, not only for food quality control but also for highlighting the presence of allergens. Herein, a sensitive analysis for β-lactoglobulin and α-lactalbumin was developed using immunoaffinity capillary electrophoresis hyphenated with MALDI-MS. Magnetic beads functionalized with appropriate antibodies were used for β-lactoglobulin and α-lactalbumin immunocapture inside the capillary. After elution from the beads, analyte focusing and separation were performed by transient isotachophoresis followed by MALDI-MS analysis performed through an automated iontophoretic fraction collection interface. A LOD in the low nanomolar range was attained for both whey proteins. The method developed was further applied to the analysis of different milk samples including fortified soy milk.     

Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.     

Capillary electrophoresis using a surfactant-treated capillary coupled with offline matrix-assisted laser desorption ionization mass spectrometry for high efficiency and sensitivity detection of proteins.

A method of combining capillary electrophoresis (CE) using a surfactant-modified capillary with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is described for protein analysis. The CE-MALDI-MS coupling is based on CE fraction collection of nanoliter volume samples in less than 5 microl of dilute acid. This offline coupling does not require any special instrumentation and can be readily performed with commercial instruments. Protein adsorption during CE separation is prevented by coating the capillary with the surfactant didodecyldimethylammonium bromide. This surfactant binds strongly with the capillary wall, hence it does not desorb significantly to interfere with subsequent MALDI-MS analysis. It is shown that the use of a dilute acid for CE fraction collection is advantageous in lowering the detection limit of MALDI-MS compared to using an electrophoretic buffer. The detection limit for proteins such as cytochrome c is 23 fmol injected for CE, or 1.2 fmol spotted for MALDI-MS. This sensitivity is comparable to alternative CE-MALDI-MS coupling techniques using direct CE sample deposition on the MALDI target. In addition, the fraction collection approach has the advantage of allowing multiple reactions to be carried out on the fractioned sample. These reactions are very important in protein identification and structure analysis.     

Comparing MALDI-MS, RP-LC-MALDI-MS and RP-LC-ESI-MS glycomic profiles of permethylated N-glycans derived from model glycoproteins and human blood serum

The glycomic profiling of purified glycoproteins and biological specimen is routinely achieved through different analytical methods, but mainly through MS and LC-MS. The enhanced ionization efficiency and improved tandem MS interpretation of permethylated glycans have prompted the popularity of this approach. This study focuses on comparing the glycomic profiling of permethylated N-glycans derived from model glycoproteins and human blood serum using MALDI-MS as well as RP-LC-MALDI-MS and RP-LC-ESI-MS. In the case of model glycoproteins, the glycomic profiles acquired using the three methods were very comparable. However, this was not completely true in the case of glycans derived from blood serum. RP-LC-ESI-MS analysis of reduced and permethylated N-glycans derived from 250 nl of blood serum allowed the confident detection of 73 glycans (the structures of which were confirmed by mass accuracy and tandem MS), while 53 and 43 structures were identified in the case of RP-LC-MALDI-MS and MALDI-MS analyses of the same sample, respectively. RP-LC-ESI-MS analysis facilitates automated and sensitive tandem MS acquisitions. The glycan structures that were detected only in the RP-LC-ESI-MS analysis were glycans existing at low abundances. This is suggesting the higher detection sensitivity of RP-LC-ESI-MS analysis, originating from both reduced competitive ionization and saturation of detectors, facilitated by the chromatographic separation. The latter also permitted the separation of several structural isomers; however, isomeric separations pertaining to linkages were not detected.     

Synchronization of separation and determination based on multichannel mode-filtered light detection with capillary electrophoresis.

A novel method for the synchronization of separation and determination is described, in which a mode-filtered light detector is used as an online detector in capillary electrophoresis. An instrument is described which has been developed for this purpose. The round capillary used in conventional capillary electrophoresis is replaced by an annular column, which is constructed from a naked optical fibre inserted into a fused-silica capillary. In fact, the annular electrophoresis column itself forms part of the mode-filtered light sensor. Along the side of the annular column are several detection channels for gathering and transmitting the mode-filtered light to a charge-coupled device (CCD). Every channel provides information on the sample from the point at which it is located. Using capillary isotachophoresis incorporating the annular column, the analytes in a sample containing alanine (10.0 mM) and glycine (9.7 mM) were simultaneously separated and determined using multichannel mode-filtered light detection with a detection limit of 1.5 mM.     

An improved design to capture magnetic microparticles for capillary electrophoresis based immobilized microenzyme reactors

In this paper, we demonstrate the effectiveness of a new 3D printed magnet holder that enables capture of magnetic microparticles in commercially available capillary electrophoresis equipment with a liquid or air based coolant system. The design as well as the method to capture magnetic microparticles inside the capillary are discussed. This setup was tested at temperature and pH values suitable for performing enzymatic reactions. To demonstrate its applicability in CE‐ immobilized microenzyme reactors (IMER) development, human flavin‐containing monooxygenase 3 and bovine serum albumin were immobilized on amino functionalized magnetic microparticles using glutaraldehyde. These microparticles were subsequently used to perform in‐line capillary electrophoresis with clozapine as a model substrate. This setup could be used further to establish CE‐IMERs of other drug metabolic enzymes in a commercially available liquid based capillary coolant system. The CE‐IMER setup was successful, although a subsequent decrease in enzyme activity was observed on repeated runs.     

High-sensitive protein analysis by FESI-CE-MALDI-MS

Capillary zone electrophoresis (CZE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) are two techniques highly suitable for the separation and detection of intact proteins. Herein, based on the use of a recently introduced iontophoretic fraction collection interface for the coupling of CE and MALDI-MS, the potential of the combination of both techniques for the analysis of intact proteins is assessed. To further provide a bioanalytical platform with high-sensitivity capabilities, field-enhanced sample injection is integrated as on online preconcentration strategy upstream from the electrokinetic separation. Under optimized conditions, more than 3200- and 4800-fold improvement, respectively in terms of peak height and peak area, as well as LODs ranging from 5 to 10 nM, has been achieved.     

Correlation free-solution capillary electrophoresis migration times of small peptides with physicochemical properties

Summary A series of small peptides containing varying degree of charge and size was used to study the effects of physicochemical properties on migration in free-solution capillary electrophoresis (FSCE). A semiempirical relationship between migration time under acidic conditions and the square root of molecular weight divided by the quantity of the number of the positively ionizable groups has been established. The ionization of the carboxyl terminal group in the polypeptides is negligible under acidic conditions. The relationship developed from this work has been used for the prediction of migration parameters in free solution capillary electrophoresis.     

Analytical potential of enzyme-coated capillary reactors in capillary zone electrophoresis

Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.     

Analysis of serotonin in brain microdialysates using capillary electrophoresis and native laser-induced fluorescence detection

Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.     

21世纪毛细管电泳技术及应用发展趋势

在21世纪，毛细管电泳技术面临着新的挑战和机遇，在其检测手段，仪器的小型化和集成化，以及分离模式上都存在着极大的发展空间，文中针对这三方面的发展趋势和毛细管电泳的应用进行了讨论。     

Simultaneous detection and quantitation of sodium, potassium calcium and magnesium in ocular lenses by high- performance capillary electrophoresis with indirect photometric detection

A high-performance capillary electrophoresis (HPCE) method which can be used to quantitatively determine Na⁺, K⁺, Ca²⁺ and Mg²⁺ simultaneously in ocular lenses has been developed. The proteins in the lens aqueous homogenates were precipitated by 10% trichloroacetic acid. The precipitated proteins were removed after a brief centrifugation, and the supernatant containing the cations was washed with ether and directly used for HPCE analysis. A 50 μm × 75 cm fused-silica capillary was used for separation and the detection wavelength was set at 214 nm. A 20-mM imidazole at pH 6.0 containing 0.1% hydroxypropyl methyl cellulose was used as background electrolyte. Sample solution was injected at 15 kV for 10 s, and the electrophoresis was carried out at 15 kV. All the cations can be separated and quantified from the peak areas within 9 min. The values obtained by this method were comparable with commonly used flame atomic absorption and flame atomic emission spectroscopy. It is demonstrated that this HPCE method can be used to quantify all the cation levels simultaneously within a short time even in a small single rat or mice lens.     

Solid-phase extraction for metabolomic analysis of high-salinity samples by capillary electrophoresis-mass spectrometry

Environmental samples such as soil solutions contain inorganic ions such as NH4(+), K(+), Na(+), NO3(-), and PO4(3-) in high concentrations, which must be removed prior to capillary electrophoresis-mass spectrometry analysis to obtain accurate results. However, the separation of these inorganic ions from ionic metabolites, which are the target compounds in capillary electrophoresis-mass spectrometry analysis, is difficult because the physicochemical properties of the inorganic ions are similar to those of the ionic metabolites. In this study, we used various solid-phase extraction (SPE) columns for the purification of the samples containing inorganic ions in high concentrations. We found that cation-exchange SPE columns successfully filtered out the inorganic ions while retaining most of the organic compounds, which were easily collected with high recovery rates. In addition, 17 cationic metabolites in the soil solution were quantified by CE-MS analysis following the SPE purification process. The results suggest that our method can be used to analyze other environmental samples containing inorganic ions in high concentrations.     

Large-volume sample on-line concentration of angiotensins in non-aqueous capillary electrophoresis

A single step on-line concentration and separation method for peptides in non-aqueous capillary electrophoresis was developed. ACN containing 50 mM tetraethylammonium perchlorate was used as the electrophoretic medium; angiotensins I-IV were separated as a result of the differences in the magnitudes of their interactions with perchlorate anions. When the sample solution (ACN containing 0.5% trifluoroacetic acid and angiotensins) was injected as a large-volume plug, the analytes were concentrated at the inlet end of the capillary by both sweeping and stacking mechanisms; the separation procedure then started automatically without any operations such as polarity change. It was found that the concentration of analytes, injection period, and concentration of tetraethylammonium perchlorate in the electrophoretic medium were important factors for both separation and concentration efficiencies. The angiotensins were concentrated and separated with the large-volume injection of up to 80% of the effective capillary length.