Characterization of transferrin glycoforms in human serum by CE-UV and CE-ESI-MS

Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

Chiral capillary electrophoresis-mass spectrometry of amino acids in foods

In this work, the development of a new chiral capillary electrophoresis-mass spectrometry (CE-MS) method to separate D- and L-amino acids is shown. On-line coupling between CE and MS is established through an electrospray-coaxial sheath flow interface. Enantiomer separation is achieved by using a cheap, nonvolatile, chiral selector as beta-cyclodextrin in the background electrolyte (BGE) together with a physically coated capillary that is aimed to prevent contamination of the electrospray. The capillary coating is simple and easy to obtain as it only requires flushing of the capillary with a polymer aqueous solution for 3 min. Optimization of CE parameters (pH of BGE, type and concentration of chiral selector, and capillary inner diameter) and electrospray-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure) is carried out. Two different derivatization protocols of amino acids using dansyl chloride (DNS) and fluorescein isothiocyanate (FITC) are compared in terms of MS sensitivity and chiral resolution. Under optimum CE-MS conditions it is observed that the MS sensitivity obtained for FITC- and DNS-amino acids is similar (with limit of detection (LOD) in the microM range, corresponding to amounts injected in the fmol range) while chiral resolution is better for FITC-amino acids. The optimized method is demonstrated to provide the simultaneous analysis of 15 selected amino acids (i.e., FITC-D/L-Asp, -Glu, -Ser, -Asn, -Ala, -Pro, -Arg, and FITC-gamma-aminobutyric acid (GABA) in a single chiral CE-MS run, corresponding to the main amino acids that can be found in orange. Moreover, as a result of the high resolution achieved, it is possible to detect down to 2% of D-Asp in the presence of 98% of L-Asp. The good possibilities of chiral CE-MS in food analysis are corroborated through the detection of the main amino acids in a commercial orange juice (i.e., FITC-L-Asp, -Glu, -Ser, -Asn, -Pro, -Arg, and the nonchiral FITC-GABA) as well as the determination of the fraudulent addition of synthetic amino acids (containing D- and L-forms) to a fresh orange juice.     

Sensitivity enhancement in capillary electrophoresis-mass spectrometry of anionic metabolites using a triethylamine-containing background electrolyte and sheath liquid

Analyte responses in CE‐ESI‐MS using negative ionization are frequently relatively low, thereby limiting sensitivity in metabolomics applications. In order to enhance the ionization efficiency of anionic metabolites, BGEs and sheath liquids (SLs) of various compositions were evaluated. Pressure‐induced infusion and CE‐MS experiments showed that addition of triethylamine (TEA) to the BGE and SL enhanced analyte intensities. A BGE consisting of 25 mM TEA (pH 11.7) and an SL of water–methanol (1:1, v/v) containing 5 mM TEA was selected, providing separation and detection of ten representative test metabolites with good reproducibility (migration time RSDs<1%) and linearity (R²>0.99). This BGE yielded lower limits of detection (0.7–9.1 μM) for most test compounds when compared with common CE‐MS methods using a BGE and SL containing ammonium acetate (NH₄Ac) (25 and 5 mM, respectively). CE‐MS of human urine revealed an average amount of 231 molecular features in negative ionization mode when TEA was used in the BGE and SL, whereas 115 and 102 molecular features were found with an NH₄Ac‐containing BGE and SL, employing a bare fused‐silica (BFS) and Polybrene‐dextran sulfate‐Polybrene (PB‐DS‐PB)‐coated capillary, respectively. With the CE‐MS method using TEA, about 170 molecular features were observed that were not detected with the NH₄Ac‐based CE‐MS methods. For more than 82% of the molecular features that were detected with the TEA as well as the NH₄Ac‐containg BGEs (i.e. common features), the peak intensities were higher using TEA with gain factors up to 7. Overall, the results demonstrate that BGEs and SLs containing TEA are quite favorable for the analysis of anionic metabolites in CE‐MS.     

Highly sensitive analysis of multiple pesticides in foods combining solid-phase microextraction, capillary electrophoresis-mass spectrometry, and chemometrics

A highly sensitive procedure to detect multiple pesticides at trace levels in foods is presented. Initially a comparative study between capillary electrophoresis (CE)-UV and CE-mass spectrometry (MS) is carried out analyzing five pesticides not studied up to now (pyrimethanil, pyrifenox, cyprodinil, cyromazine, and pirimicarb). The comparison between CE-UV and CE-MS is established in terms of separation efficiency, speed of analysis, reproducibility, and sensitivity. A good separation of these compounds is achieved by both techniques using a volatile aqueous buffer containing 0.3 M ammonium acetate/acetic acid at pH 4. Time analysis reproducibility is studied for the same day (n = 5) and three different days (n = 15), showing no significant differences between CE-UV and CE-MS. The study on peak areas reproducibility shows a slightly worse reproducibility for CE-MS compared with CE-UV. The best limit of detection (LOD) that can be achieved for these pesticides using CE-UV was 0.6 microg/mL. CE-MS provides LODs one order of magnitude better than CE-UV. Chemometrics are used to optimize the multiple parameters that play a role in solid-phase microextraction (SPME) and CE-MS analysis (e.g., extraction and desorption times, nebulizer pressure, dry gas flow, dry gas temperature, percentage of organic solvent and acid in the sheath liquid, etc.). The combined use of chemometrics and SPME-CE-MS clearly improves the LODs that can be achieved allowing the detection of pesticides at concentrations down to 15 ng/mL. The usefulness of this approach is demonstrated detecting multiple pesticides in different food samples as grapes and orange juice in a single run. The concentrations detected are below the maximum residue limits (MRLs) permitted for these pesticides in foods corroborating the value of our approach. This work demonstrates, to our knowledge for the first time, the good possibilities of the combined use of SPME-CE-MS and chemometrics.     

Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis-mass spectrometry (CE-MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE-MS using the same porous tip capillary and an isopropanol-water-acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE-MS, detection limits were improved by a factor 6.5-20 with sheathless CE-MS. The analyte response in sheathless CE-MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary - equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE-MS of intact proteins.     

Optimization of capillary electrophoretic-electrospray ionization-mass spectrometric analysis of catecholamines

The capillary electrophoretic-mass spectrometric analysis (CE-MS) of catecholamines was optimized with coaxial sheath flow interface and electrospray ionization (ESI). The parameters studied included the sheath liquid composition and its flow rate, separation conditions in ammonium acetate buffer together with the ESI and cone voltages as mass spectrometric parameters. In addition, the effect of ESI voltage on injection as well as the siphoning effect were considered. The optimized conditions were a sheath liquid composition of methanol-water (80:20 v/v) with 0.5% acetic acid, with a flow rate of 6 microL/min. The capillary electrophoretic separation parameters were optimized with 50 mM ammonium acetate buffer, pH 4.0, to +25 kV separation voltage together with a pressure of 0.1 psi. The most intensive signals were obtained with an ESI voltage of +4.0 kV and a cone voltage of +20 V. The nonactive ESI voltage during injection as well as avoidance of the siphoning effect increased the sensitivity of the MS detection considerably. The use of ammonium hydroxide as the CE capillary conditioning solution instead of sodium hydroxide did not affect the CE-MS performance, but allowed the conditioning of the capillary between analyses to be performed in the MS without contaminating the ion source.     

Simplifying capillary electrophoresis-mass spectrometry operation: eliminating capillary derivatization by using self-coating background electrolytes

To simplify capillary electrophoresis-mass spectrometry (CE-MS) operation, a background electrolyte (BGE) containing a polymer additive is introduced that allows the analysis of peptides and protein mixtures in underivatized fused-silica capillaries without any pretreatment, thereby increasing throughput. The most important characteristic of these polymer additives is that they do not significantly suppress the signals of the proteins and peptides under electrospray ionization, thereby allowing them to be used as an additive to common BGEs that are used for CE-MS analysis of peptide and protein mixtures. In addition, because the fused-silica capillary inner wall is continuously coated with the polymer additive, migration irreproducibility, due to the degradation of the capillary inner wall coating, under CE-MS is minimized. High sensitivity of detection, migration reproducibility, and ease of fabrication allow CE-MS analyses that require long analysis time, such as (CE-MS/MS)n, to be performed with ease. The utility of this background electrolyte has been demonstrated for the analysis of complex protein digests and intact proteins.     

Design, optimisation, and evaluation of a sheath flow interface for automated capillary electrophoresis-electrospray-mass spectrometry

A sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) system utilizing a fully integrated large-bore stainless-steel emitter electrode tapered at the end for micro-ionspray operation has been developed and evaluated. A separation capillary with an outer diameter of up to 360 microm was inserted into the electrode thus forming a void volume of less than 15 nL between the capillary end and the electrospray ionisation (ESI) tip. The sheath liquid, usually methanol-water (80:20) with 0.1% formic acid for positive ion mode or methanol for negative ion mode, was delivered at 0.5-1.0 microL/min. Unlike previously reported CE-MS interfaces, the CE-MS probe was incorporated directly onto an Applied Biosystems/MDS SCIEX orthogonal-spray Turbo "V" ion source for ease of use and automatic operation. This integration enables fast and facile coupling and replacement of the separation capillary without interrupting the ion source configuration, and the sheath liquid supply. The reusable electrospray electrode was precisely fabricated and aligned with the length of the nebulizing gas tube for improved reproducibility. Automation was achieved through software control of both CE and tandem MS (MS/MS) for unattended batch sample analysis. The system was evaluated for attomole- to low femtomole-level profiling of model peptides and protein mixtures, bisphosphates, as well as antiviral nucleosidic drugs in cellular extracts.     

Influence of capillary dimensions on the performance of a coaxial capillary electrophoresis-electrospray mass spectrometry interface

The dimensions of the capillaries used to construct a typical coaxial capillary electrophoresis-mass spectrometry (CE-MS) interface, i.e. the inner diameter, the outer diameter and the wall thickness, have been shown to affect the performance of the CE-MS system. The influence of these parameters has been investigated in both MS and MS-MS modes. The initial results indicate that by reducing all the sheath capillaries' dimensions and the CE capillary outer diameter, better operation and increased sensitivity can be achieved. The capillary arrangement which gives optimum sensitivity and stable operation has been suggested.     

Coupling CE with atmospheric pressure photoionization MS for pharmaceutical basic compounds: optimization of operating parameters

The use of CE coupled with MS (CE-MS) has evolved as a useful tool to analyze charged species in small sample volumes. Because of its sensitivity, versatility and ease of implementation, the ESI interface is currently the method of choice to hyphenate CE to MS. An alternative can be the atmospheric pressure photoionization (APPI) source, however, numerous parameters must be optimized for its coupling to CE. After evaluation of the sheath liquid composition and the CE capillary outlet position, an experimental design methodology was assessed for optimizing other ionization source parameters, such as sheath liquid flow rate, drying gas flow rate and temperature, nebulizing gas pressure, vaporizer temperature, and capillary voltage. For this purpose, a fractional factorial design (FFD) was selected as a screening procedure to identify factors which significantly influence sensitivity and efficiency. A face-centered central composite design (CCD) was then used to predict and optimize sensitivity, taking into account the most relevant variables. Sensitivity was finally evaluated with the optimized conditions and height-to-noise ratios (H/N) around 10 were achieved for an injection of 200 ng/mL of each analyte.     

A new insight into suction and dilution effects in capillary electrophoresis coupled to mass spectrometry via an electrospray ionization interface. Part I-Suction effect

The hyphenation of CE with MS is nowadays accepted as a powerful analytical approach. Employing ESI, the most common interface, one challenge is to provide quantitative information, which is quite a difficult task, as it is linked, among other factors, to suction and dilution effects. In the coaxial ESI configuration, the suction effect has been presented in literature as stemming from nebulizing gas (NG) flow rate and drying gas temperature. But as this interface consists in three concentric capillaries, allowing for BGE, sheath liquid (SL) and NG mixing, it is demonstrated herein that other parameters are also involved in this suction effect: the CE capillary protrusion from the interface needle, SL flow rate, and overall BGE flow rate and velocity profile. Whereas NG flow rate is the parameter affecting suction to a greater extent, separation capillary protruding length, SL, and overall BGE flow rate have a significant additional impact on this phenomenon. It is shown that SL flow rate can affect suction differently according to the NG velocity, which may be explained by modification of the Taylor cone geometry. Furthermore, it appears that suction effect is noticeably favored by a parabolic velocity profile of the BGE, again probably due to the Taylor cone shape modification. Finally, the temperature gradient created by the contact between the heated NG and the separation capillary enhances this effect.     

Characterization of conformers and dimers of antithrombin by capillary electrophoresis-quadrupole-time-of-flight mass spectrometry

Antithrombin (AT) is a plasma glycoprotein which possesses anticoagulant and anti-inflammatory properties. AT exhibits various forms, among which are native, latent and heterodimeric ones. We studied the potential of capillary electrophoresis-mass spectrometry (CE-MS) using a sheath liquid interface, electrospray ionization (ESI), and a quadrupole-time-of-flight (Q-TOF) mass spectrometer to separate and quantify the different AT forms. For CE separation, a neutral polyvinyl alcohol (PVA) coated capillary was employed. The protein conformation was preserved by using a background electrolyte (BGE) at physiological pH. A sheath liquid of isopropanol-water 50:50 (v/v) with 14 mM ammonium acetate delivered at a flow rate of 120 μL h⁻¹ resulted in optimal signal intensities. Each AT form exhibited a specific mass spectrum, allowing unambiguous distinction. Several co-injection experiments proved that latent AT had a higher electrophoretic mobility (μ_ep) than native AT, and that these conformers could associate to form a heterodimer during the CE analysis. The developed CE-MS method enabled the detection and quantitation of latent and heterodimeric forms in a commercial AT preparation stored at room temperature for three weeks.     

Capillary electrophoresis-atmospheric pressure chemical ionization-mass spectrometry using an orthogonal interface: Set-up and system parameters

The feasibility of atmospheric pressure chemical ionization (APCI) as an alternative ionization technique for capillary electrophoresis-mass spectrometry (CE-MS) was investigated using a grounded sheath-flow CE-MS sprayer and an orthogonal APCI source. Infusion experiments indicated that highest analyte signals were achieved when the sprayer tip was in close vicinity of the vaporizer entrance. The APCI-MS set-up enabled detection of basic, neutral, and acidic compounds, whereas apolar and ionic compounds could not be detected. In the positive ion mode, analytes could be detected in the entire transfer voltage range (0–5 kV), whereas highest signal intensities were observed when the corona discharge current was between 1000 and 2000 nA. In the negative ion mode, the transfer voltage typically was 500 V and the optimum corona discharge current was 6000 nA. Analyte signals were raised with increasing nebulizing gas pressure, but the pressure was limited to 25 psi to avoid siphoning and current drops. Signal intensities appeared to be optimal and constant over a wide range of sheath liquid flow rate (5–25 μL/min) and vaporizer temperature (200–350 °C). APCI-MS signals were unaffected by the composition of the background electrolyte (BGE), even when it contained sodium phosphate and sodium dodecyl sulfate (SDS). Consequently, BGE composition, sheath-liquid flow rate, and vaporizer temperature can be optimized with respect to the CE separation without affecting the APCI-MS response. The analysis of a mixture of basic compounds and a steroid using volatile and nonvolatile BGEs further demonstrates the feasibility of CE-APCI-MS. Detection limits (S/N = 3) were 1. 6–10 μM injected concentrations.     

Capillary electrophoresis-mass spectrometry analysis of trehalose-6-phosphate in Arabidopsis thaliana seedlings

Trehalose-6-phosphate (T6P) is an intermediate in the plant metabolic pathway that results in trehalose production. T6P has been shown to inhibit the sucrose nonfermenting-1-related protein kinase 1, which is a major regulator of metabolism. The quantitation of T6P has proven difficult due to the complexity of the plant matrix and the low abundance of T6P in plant tissues. The aim of this work was to develop a quantitation method for T6P present in Arabidopsis tissues, with capillary electrophoresis (CE) coupled to electrospray ionization-mass spectrometry (MS) with a sheath liquid (SL) interface. The CE-MS method was first optimized with respect to T6P signal intensity and separation of isomers by studying the composition of the background electrolyte (BGE) and SL. The use of triethylamine (TEA) in the BGE was favorable, providing separation of T6P from sucrose-6-phosphate and minimizing ionization suppression. Replacing ammonium acetate with TEA enhanced T6P signal intensities more than four times. The optimized method allowed quantification of T6P in plant extracts with good linearity (r ² > 0.99) within a biologically relevant concentration range. The limit of quantification was 80 nM in Arabidopsis extracts, corresponding to 33 pmol/g plant fresh weight. The CE-MS method was applied to the determination of T6P in seedlings from wild type (WT) Arabidopsis and mutants lacking the trehalase AtTRE1, tre1-1, challenged with trehalose or sorbitol. T6P accumulation in tre1-1 plants grown on sorbitol was about twice the level of T6P found in WT. CE-MS is shown to be a fast and reliable technique to analyze phosphodisaccharides for seedling extracts. The low sample volume requirement of CE and its direct MS coupling makes it an attractive alternative for anion-exchange liquid chromatography–MS.     

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix-assisted laser desorption/ionization and electrospray-mass spectrometry detection

High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.     

Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn‐superoxide dismutase

The native form of Cu,Zn‐superoxide dismutase (SOD‐1) is a homodimer that coordinates one Cu²⁺ and one Zn²⁺ per monomer. Cu²⁺ and Zn²⁺ ions play crucial roles in enzyme activity and structural stability, respectively. In addition, dimer formation is essential for SOD‐1 functionality, and in humans several SOD‐1 mutant isoforms have been associated with certain types of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder. In this paper we used capillary electrophoresis and mass spectrometry to study the different structures of bovine SOD‐1. The metal ions of the native enzyme (Cu₂,Zn₂‐dimer SOD‐1) were released in acidic medium in order to obtain apo‐SOD‐1, which is a monomer. Both substances were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and capillary electrophoresis with ultraviolet and electrospray ionization mass spectrometry detection (CE/UV and CE/ESI‐MS, respectively). With MALDI‐TOF‐MS, using matrices of sinapinic acid (SA) or 2,5‐dihydroxybenzoic acid (DHB) with or without trifluoroacetic acid (TFA), similar mass spectra were obtained for the metalated and non‐metalated samples. In both cases, an average molecular mass corresponding to the apo‐monomer SOD‐1 was calculated. This finding indicated that the metals were released from the Cu₂,Zn₂‐dimer SOD‐1 during sample preparation or ionization. For CE/UV and CE/ESI‐MS, two background electrolytes (BGEs) potentially compatible with ESI‐MS detection were used, namely 1 M of acetic acid (pH 2.3) and 10 mM of ammonium acetate (pH 7.3). Using a sheath liquid of 2‐propanol/water (60:40 v/v), with or without 0.1% v/v of formic acid, CE/ESI‐MS sensitivity was enhanced when the acidic BGE and the acidic sheath liquid were used. However, the electrophoretic profiles and the mass spectra obtained suggested that the metals of Cu₂,Zn₂‐dimer SOD‐1 were released, which generated the apo‐monomer during the electrophoretic separation. The neutral BGE provided enhanced conditions for the detection of the native enzyme. The differences between the mass spectra obtained for the Cu₂,Zn₂‐dimer and the apo‐monomer forms were significant and the presence of formic acid in the sheath liquid affected only sensitivity. Our results highlight the importance of selecting appropriate non‐denaturing separation and detection conditions to obtain reliable structural information about non‐covalent protein complexes by CE/ESI‐MS. Copyright © 2010 John Wiley & Sons, Ltd.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.     

CE‐MS method development for peptides analysis,especially hepcidin,an iron metabolism marker

A method for the resolution of a peptides mixture including hepcidin‐25, an iron metabolism marker, was developed by CE‐ESI‐MS. Several strategies were tested to optimize peptide separation, such as the addition of cyclodextrins or organic solvents in the BGE or the use of coated capillaries. Best results in terms of resolution, symmetry and efficiency were obtained with a BGE made of 500 mM ammonium acetate pH 4.5/ACN 70:30 v/v. Using the methodology of experimental design, BGE concentration, sheath liquid composition and MS‐coupling parameters were then optimized in order to obtain the best signal intensity for hepcidin. Finally, a 225 mM BGE and a sheath liquid composed of isopropanol/water 80:20 v/v containing 0.5% v/v formic acid were selected as it constitutes the best compromise for selectivity, peak shape and sensitivity.     

Development of a capillary zone electrophoresis-electrospray ionisation tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics

The applicability of a capillary zone electrophoresis–electrospray ionisation tandem mass spectrometric (CZE–ESI-MS–MS) method for the separation of nine fluoroquinolones was investigated. Method optimisation involved systematic trouble-shooting starting with the type and duration of capillary pre-washing and conditioning, the choice of both the CE run buffer, MS sheath liquid, CE run potential, ESI spray voltage, sheath gas flow-rate, MS capillary voltage and CE capillary and MS capillary temperatures. Another extremely important factor was found to be the degree to which the CE capillary protrudes into the ESI chamber as well as whether or not sheath gas and spray voltage are employed during the CE injection or not. The importance of the latter has, to our knowledge, not been addressed elsewhere. Nine fluoroquinolones have been separated and detected in a single run by this technique.