Capillary electrophoresis-mass spectrometry for the analysis of intact proteins 2007-2010

CE coupled to MS has proven to be a powerful analytical tool for the characterization of intact proteins, as it combines the high separation efficiency of CE with the selectivity of MS. This review provides an overview of the development and application of CE-MS methods within the field of intact protein analysis as published between January 2007 and June 2010. Ongoing technological developments with respect to CE-MS interfacing, capillary coatings for CE-MS, coupling of CIEF with MS and chip-based CE-MS are treated. Furthermore, CE-MS of intact proteins involving ESI, MALDI and ICP ionization is outlined and overviews of the use of the various CE-MS methods are provided by tables. Representative examples illustrate the applicability of CE-MS for the characterization of proteins, including glycoproteins, biopharmaceuticals, protein-ligand complexes, biomarkers and dietary proteins. It is concluded that CE-MS is a valuable technique with high potential for intact protein analysis, providing useful information on protein identity and purity, including modifications and degradation products.     

Capillary electrophoresis-mass spectrometry for the analysis of intact proteins

Developments in the fields of protein chemistry, proteomics and biotechnology have increased the demand for suitable analytical techniques for the analysis of intact proteins. In 1989, capillary electrophoresis (CE) was combined with mass spectrometry (MS) for the first time and its potential usefulness for the analysis of intact (i.e. non-digested) proteins was shown. This article provides an overview of the applications of CE-MS within the field of intact protein analysis. The principles of the applied CE modes and ionization techniques used for CE-MS of intact proteins are shortly described. It is shown that separations are predominantly carried out by capillary zone electrophoresis and capillary isoelectric focusing, whereas electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the most popular ionization techniques used for interfacing. The combination of CE with inductively coupled plasma (ICP) MS for the analysis of metalloproteins is also discussed. The various CE-MS combinations are systematically outlined and tables provide extensive overviews of the applications of each technique for intact protein analysis. Selected examples are given to illustrate the usefulness of the CE-MS techniques. Examples include protein isoform assignment, single cell analysis, metalloprotein characterization, proteomics and biomarker screening. Finally, chip-based electrophoresis combined with MS is shortly treated and some of its applications are described. It is concluded that CE-MS represents a powerful tool for the analysis of intact proteins yielding unique separations and information.     

Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.     

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.     

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.     

Biomarker discovery by CE-MS enables sequence analysis via MS/MS with platform-independent separation

CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.     

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。     

Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion trap-mass spectrometry and capillary electrophoresis-time of flight-mass spectrometry

In this work, a new capillary electrophoresis-mass spectrometry (CE-MS) procedure is developed to analyze proteins in Spirulina platensis microalgae. It is demonstrated that a fine optimization of several separation parameters is essential in order to achieve suitable CE-MS analysis of these proteins in natural extracts from microalgae. Namely, optimization of the composition of the separation buffer, electrospray conditions, and washing routine between runs are required in order to obtain reliable and reproducible CE-MS analyses of the main proteins found in this microalga (namely, allophycocyanin-alpha chain, allophycocyanin-beta, c-phycocyanin-alpha, and c-phycocyanin-beta). The relative molecular mass of these biopolymers is determined using two different MS instruments coupled to CE, i.e., CE-ion trap-MS and CE-time of flight-MS (CE-TOF-MS). A comparison between the results obtained with both instruments is carried out. The high resolution of the TOF-MS enables the distinction of small modifications in proteins and, thus, a more accurate mass determination. Interestingly, molecular mass values obtained by both CE-MS procedures agree very well while these experimental values are only in partial agreement with those theoretically expected (i.e., genetically derived masses). Some protein modifications due to amino acids exchange induced by nucleotide codon mutations are proposed to explain this difference.     

Study of posttranslational non-enzymatic modifications of collagen using capillary electrophoresis/mass spectrometry and high performance liquid chromatography/mass spectrometry

The depository effects that occur in slowly metabolized proteins (typically glycation) are very difficult to assess, owing to their extremely low concentration in the protein matrix. Collagen accumulates reactive metabolites through reactions that are not regulated by enzymes. A typical example of these non-enzymatic changes is glycation (the Maillard reaction, the formation of advanced glycation end products), resulting from the reaction of the oxo-group of sugars with the epsilon-amino group of lysine and arginine. Collagen samples (type I) as a test protein were incubated separately with glucose, ribose and malondialdehyde. Collagen was fragmented with cyanogen bromide and then digested with trypsin. This peptide digest was separated by CE, CE-MS/MS, and HPLC-MS/MS. An ion trap MS was used and MS conditions were optimized for both methods. These on-line CE-MS/MS and HPLC-MS/MS couplings made it possible to discover specific modifications such as (N(epsilon)-(carboxymethyl)-lysine) in the precise location in the structure of collagen corresponding to posttranslational non-enzymatic modifications. A new CE-MS/MS technique for peptide analysis was developed, and applied in the identification of posttranslational modifications in slowly metabolized test proteins.     

Toward a screening method for the analysis of small intact proteins by CE-ESI-TOF MS

Capillary electrophoresis-mass spectrometry (CE-MS) more and more gains in importance as an analytical technique for the identification and characterization of intact proteins in the biopharmaceutical area. Thus, a CE-ESI-MS method was optimized and validated systematically with respect to the improved screening and characterization of intact proteins. The optimization was accomplished by variation of different CE-MS parameters, such as capillary coating, background electrolyte, sheath liquid, and nebulizer gas pressure, while monitoring both the resolution and signal intensities. Achievable separation is discussed quantitatively in the context of the coating and the resulting EOF, the protein mobilities, and the suction effect of the sprayer. The observed precisions of the optimized method regarding the migration times (mean RSD = 1.4%) and peak areas (mean RSD = 12.3%) and an extensive principal component analysis revealed that the presented method is reliable and useful for the quantitation of intact proteins and protein isoforms. The applicability of this method to various proteins showing different characteristics (pI value, molecular mass, hydrophobicity, etc.) is discussed. The presented method will contribute to the improved characterization of a large variety of intact proteins in the biomedical and pharmaceutical area.     

Capillary electrophoresis-mass spectrometry for glycoscreening in biomedical research

Application of capillary electrophoresis (CE) in combination with mass spectrometry (MS) and tandem MS to glycoscreening in biomedical projects is highlighted. In the first part recent CE-MS experiments by sheath liquid CE and multiple stage MS are reported. Neutral and negatively charged N-glycan mixtures from ribonuclease B and fetuin, high-mannose type N-glycoforms, oligosaccharides from lipopolysaccharides (LPS) of Haemophilus influenzae, polysaccharides of Pseudomonas aeruginosa and Staphylococcus aureus were analyzed. A particular emphasis is devoted to the applicability of novel off- and on-line CE-MS and tandem MS methods for screening of proteoglycan-derived oligosaccharides, glycosaminoglycans (GAGs), such as hyaluronates from Streptococcus agalactiae, chondroitin/dermatan sulfates (CS/DS) from bovine aorta and human skin fibroblast decorin, and heparin/heparan sulfate (HS) from porcine and bovine mucosa. The performance of CE-MS/MS for identification of glycoforms in glycopeptides and glycoproteins is illustrated by experiments performed on complex mixtures from urine of patients suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease) and urine of patients suffering from cancer cachexia. For determination of glycosylation patterns in glycoproteins like enzymes and antibodies by CE/MS, both CE-matrix assisted laser desorption/ionization (MALDI) and CE-electrospray ionization (ESI)-MS were functional. Finally, the potential of CE-ESI-MS strategy in glycolipid analysis is demonstrated for gangliosides from bovine brain for which particular CE buffer conditions are required.     

CE-MS of zein proteins from conventional and transgenic maize

In this work, an original CE-MS method has been developed to analyze the complex zein protein fractions from maize. A thorough optimization of: (i) zein protein extraction, (ii) CE separation, and (iii) electrospray-MS (ESI-MS) detection is carried out in order to obtain highly informative CE-MS profiles of this fraction. The developed CE-MS method provides good separation of multiple zein proteins based on their electrophoretic mobilities as well as adequate characterization of these proteins based on their M(r). Zein proteins with small M(r) differences (below 100 Da) were easily separated and successfully analyzed by CE-MS. Thus, apart of the so-called 15-kDa-beta-zein and 16-kDa-gamma-zein, which are demonstrated to be formed by a heterogeneous group of proteins, numerous alpha-zeins belonging to the 19- and 22-kDa fraction were also identified for the first time in this work. The usefulness of this CE-MS method was corroborated by comparing the zein-protein fingerprints of various maize lines including transgenic and their corresponding nontransgenic isogenic lines cultivated under the same conditions.     

Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

 Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection     

Comparison of CE-MS and LC-MS Analyses of Avian Eggshell Matrix Proteins

CE-MS and HPLC-MS methods were developed and compared for the analysis of insoluble proteins in an avian eggshell matrix. The eggshell was gradually decalcified to obtain four distinct layers (cuticle, two palisade and a mammillary layer). The insoluble proteinaceous films from these layers were chemically and/or enzymatically splitted with CNBr/trypsin and proteinase K. The generated peptides were separated by CE and HPLC on-line coupled to MS detection. Capillary electrophoresis (CE) was coupled to an ion-trap electrospray ionization mass spectrometer (Agilent LC-MSD Trap XCT-Ultra) using a grounded needle carrying a flow of sheath liquid (5 mM ammonium acetate/2-propanol, 1:1, at flow-rate 3 μL min^?1). Five main proteins were identified: ovocleidin-116, ovocalyxin-32, ovocalyxin-36, ovocleidin-17 and ovalbumin. The distribution of these proteins in the eggshell was found to be dependent on the location/layer. In the outermost layer (the cuticle layer) the dominant protein is ovocalyxin-32; ovocleidin-116 is distributed throughout all layers while ovalbumin is present only in the internal mammillary layer. The CE-MS peptide maps of eggshell proteins were compared to the HPLC-MS ones, and a different mechanism of separation (migration/elution order) was demonstrated for both methods.     

Comparison of CE-MS and LC-MS Analyses of Avian Eggshell Matrix Proteins

CE-MS and HPLC-MS methods were developed and compared for the analysis of insoluble proteins in an avian eggshell matrix. The eggshell was gradually decalcified to obtain four distinct layers (cuticle, two palisade and a mammillary layer). The insoluble proteinaceous films from these layers were chemically and/or enzymatically splitted with CNBr/trypsin and proteinase K. The generated peptides were separated by CE and HPLC on-line coupled to MS detection. Capillary electrophoresis (CE) was coupled to an ion-trap electrospray ionization mass spectrometer (Agilent LC-MSD Trap XCT-Ultra) using a grounded needle carrying a flow of sheath liquid (5 mM ammonium acetate/2-propanol, 1:1, at flow-rate 3 μL min⁻¹). Five main proteins were identified: ovocleidin-116, ovocalyxin-32, ovocalyxin-36, ovocleidin-17 and ovalbumin. The distribution of these proteins in the eggshell was found to be dependent on the location/layer. In the outermost layer (the cuticle layer) the dominant protein is ovocalyxin-32; ovocleidin-116 is distributed throughout all layers while ovalbumin is present only in the internal mammillary layer. The CE-MS peptide maps of eggshell proteins were compared to the HPLC-MS ones, and a different mechanism of separation (migration/elution order) was demonstrated for both methods.

     

2020年毛细管电泳技术年度回顾

该文为2020年毛细管电泳(capillary electrophoresis, CE)技术年度回顾。归纳总结了以“capillary electrophoresis-mass spectrometry”或“capillary isoelectric focusing”或“micellar electrokinetic chromatography”或“capillary electrophoresis”为关键词在ISI Web of Science数据库中进行主题检索得到的2020年CE技术相关研究论文222篇,以及中文期刊《分析化学》和《色谱》中CE技术相关的研究论文37篇。对2020年影响因子(IF)≥5.0的Analytical Chemistry, Food Chemistry, Analytica Chimica Acta和Talanta等13本期刊的38篇文章报道的科研工作作了逐一介绍;对IF<5.0的期刊中CE技术报道较为集中的Journal of Chromatography A和Electrophoresis两本分析化学类期刊发表40篇文章中的代表性内容作了综合介绍;对重要的中文期刊《分析化学》出版的“核酸适配体专刊”和《色谱》出版的2期CE技术专刊所收录的37篇文章中的工作作了总体介绍。总体来说,2020年CE技术发展趋势仍以毛细管电泳-质谱(CE-MS)的新方法和新应用最为突出,主要集中在CE-MS与电化学检测、固相萃取以及多种毛细管电泳模式的联用方面,CE-MS接口相关的报道较前几年有所减少;常规CE技术则以胶束电动毛细管色谱(MEKC)在复杂样本分析、浓缩富集应用为主,尤其在食品和药品等复杂基质样本分析方面的报道较为集中;此外,我国CE相关领域专家学者的科研成果涵盖了CE在生命科学、临床医学、医药研发、环境科学、天然产物、食品分析等领域的应用,代表了国内CE科研应用水平和现状。     

Utility of membrane preconcentration-capillary electrophoresis-mass spectrometry in overcoming limited sample loading for analysis of biologically derived drug metabolites, peptides, and proteins

The limited loading of capillary electrophoresis (CE) leads to relatively poor concentration limits of detection. In this work a unique method for analyte preconcentration with capillary electrophoresis-mass spectrometry (CE-MS) is described. A cartridge containing an impregnated membrane is installed at the inlet of the CE capillary, and we term this approach membrane preconcentration capillary electrophoresis-mass spectrometry (mPC-CE-MS). The analysis of in vivo derived metabolites, peptides, and proteins is described showing the wide applicability of the technology in the analysis of numerous compound classes ranging in molecular weight from 200–60,000 u. In particular, we describe the direct mPC-CE-MS analysis of urine obtained from a patient receiving the neuroleptic drug haloperidol. Three metabolites were found in the urine, and two of them are implicated in the Parkinsonian-like side effects caused by taking this drug. The technique is also applied to the analysis of major histocompatibility complex class I peptides obtained from EG-7 cells. Furthermore, the clinical potential of this approach is described by the direct analysis of urine from a patient suffering from multiple myeloma, as well as aqueous humor derived from a patient undergoing surgery. Finally we show that the use of mPC-ME-MS in conjunction with either analyte stacking (small organic molecules such as metabolites) or moving-boundary transient isotachophoresis (peptides and proteins) after analytes have been eluted from the adsorptive membrane affords optimal performance and no compromise in CE mass spectrometry performance.     

A simple sheathless CE-MS interface with a sub-micrometer electrical contact fracture for sensitive analysis of peptide and protein samples

Online coupling of capillary electrophoresis (CE) to electrospray ionization mass spectrometry (MS) has shown considerable potential, however, technical challenges have limited its use. In this study, we have developed a simple and sensitive sheathless CE-MS interface based on the novel concept of forming a sub-micrometer fracture directly in the capillary. The simple interface design allowed the generation of a stable ESI spray capable of ionization at low nanoliter flow-rates (45–90 nL/min) for high sensitivity MS analysis of challenging samples like those containing proteins and peptides. By analysis of a model peptide (leucine enkephalin), a limit of detection (LOD) of 0.045 pmol/μL (corresponding to 67 attomol in a sample volume of ∼15 nL) was obtained. The merit of the CE-MS approach was demonstrated by analysis of bovine serum albumin (BSA) tryptic peptides. A well-resolved separation profile was achieved and comparable sequence coverage was obtained by the CE-MS method (73%) compared to a representative UPLC-MS method (77%). The CE-MS interface was subsequently used to analyse a more complex sample of pharmaceutically relevant human proteins including insulin, tissue factor and α-synuclein. Efficient separation and protein ESI mass spectra of adequate quality could be achieved using only a small amount of sample (30 fmol). In addition, analysis of ubiquitin samples under both native and denatured conditions, indicate that the CE-MS setup can facilitate native MS applications to probe the conformational properties of proteins. Thus, the described CE-MS setup should be useful for a wide range of high-sensitivity applications in protein research.