CE-MS Method Research Articles

Microseparation techniques coupled to mass spectrometry for a sensitive and unequivocal identification of glycopeptides

Capillary electrophoresis (CE) and low-flow liquid chromatography (i.e., micro, capillary, and nanoLC) both coupled to mass spectrometry (MS) are nowadays widely accepted for glycopeptide analysis. However, there are still analytical challenges related to the separation of glycopeptide glycoforms and their unequivocal identification. In the present study, CE-MS and capillary LC-MS (capLC-MS) were evaluated for a robust and high-throughput analysis of glycopeptides. Recombinant human erythropoietin (rhEPO) was selected as model glycoprotein due to its relevance as a biopharmaceutical and its high glycopeptide microheterogeneity. The CE-MS method provided adequate glycopeptide separation for those glycoforms differing in the number of sialic acids. However, the separation of glycoforms with the same sialic acid content but different glycan branching (number of antennas) was not achieved. In contrast, capLC-MS with an appropriate reversed phase C18 column enabled to improve their separation. Furthermore, the analysis of rhEPO glycopeptides with the established microseparation methods coupled to tandem mass spectrometry (CE-MS/MS and capLC-MS/MS) provided specific fragmentation patterns and a more reliable glycoform identification. The capLC-MS/MS method was the most promising as, it was able to detect and unequivocally identify a greater number of glycopeptide glycoforms compared to the CE-MS/MS method, including those with glycolylneuraminic acid, which are necessary to differentiate between endogenous and recombinant hEPO.Data are available via ProteomeXchange with identifier PXD047106.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Coupling capillary electrophoresis with mass spectrometry for the analysis of oxidized phospholipids in human high-density lipoproteins.

The oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human high-density lipoproteins (HDLs) were investigated by low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS). To accelerate the optimization, native PAPC (n-PAPC) standard was first analyzed by a commercial CE instrument with a photodiode array detector. The optimal separation buffer contained 60% (v/v) acetonitrile, 40% (v/v) methanol, 20mM ammonium acetate, 0.5% (v/v) formic acid, and 0.1% (v/v) water. The selected separation voltage and capillary temperature were 20kV and 23°C. The optimal CE separation buffer was then used for the low-flow CE-MS analysis. The selected MS conditions contained heated capillary temperature (250°C), capillary voltage (10V), and injection time (1 s). No sheath gas was used for MS. The linear range for n-PAPC was 2.5-100.0µg/mL. The coefficient of determination (R2 ) was 0.9918. The concentration limit of detection was 1.52µg/mL, and the concentration limit of quantitation was 4.60µg/mL. The optimal low-flow CE-MS method showed good repeatability and sensitivity. The ox-PAPC products in human HDLs were determined based on the in vitro ox-PAPC products of n-PAPC standard. Twenty-one ox-PAPC products have been analyzed in human HDLs. Uremic patients showed significantly higher levels of 15 ox-PAPC products than healthy subjects.

Understanding How CNNs Recognize Facial Expressions: A Case Study with LIME and CEM.

Recognizing facial expressions has been a persistent goal in the scientific community. Since the rise of artificial intelligence, convolutional neural networks (CNN) have become popular to recognize facial expressions, as images can be directly used as input. Current CNN models can achieve high recognition rates, but they give no clue about their reasoning process. Explainable artificial intelligence (XAI) has been developed as a means to help to interpret the results obtained by machine learning models. When dealing with images, one of the most-used XAI techniques is LIME. LIME highlights the areas of the image that contribute to a classification. As an alternative to LIME, the CEM method appeared, providing explanations in a way that is natural for human classification: besides highlighting what is sufficient to justify a classification, it also identifies what should be absent to maintain it and to distinguish it from another classification. This study presents the results of comparing LIME and CEM applied over complex images such as facial expression images. While CEM could be used to explain the results on images described with a reduced number of features, LIME would be the method of choice when dealing with images described with a huge number of features.

Determination of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine products in human very low-density lipoproteins by nonaqueous low-flow capillary electrophoresis-mass spectrometry

A simple and fast low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS) method has been developed to analyze oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human very low-density lipoproteins (VLDLs). Native PAPC standard was analyzed to optimize the low-flow CE-MS method. The optimal CE conditions included separation buffer (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 0.5% (v/v) formic acid, 20 mM ammonium acetate), sheath liquid (60% (v/v) acetonitrile, 40% (v/v) methanol, 0.1% (v/v) water, 20 mM ammonium acetate), separation voltage (20 kV), separation capillary internal diameter (i.d.) (75 µm), separation capillary temperature (23˚C) and sample injection time (6 s). The selected MS conditions included heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). Sheath gas was not used in this study. The total ion chromatograms (TICs), extracted ion chromatograms (EICs) and MS spectra of native PAPC standard and its in vitro oxidation products showed good repeatability and sensitivity. To determine the ox-PAPC products in human VLDLs, the EICs and MS spectra of VLDLs were compared with the in vitro oxidation products of native PAPC standard. For native PAPC standard, the measured linear range was 2.5 - 100.0 µg/mL, and the coefficients of determination (R2) was 0.9994. The concentration limit of detection (LOD) was 0.44 µg/mL, and the concentration limit of quantitation (LOQ) was 1.34 µg/mL. A total of 21 ox-PAPC products were analyzed for the VLDLs of healthy and uremic subjects. The levels of 7 short-chain and 5 long-chain ox-PAPC products on uremic VLDLs were significantly higher than healthy VLDLs. This simple low-flow CE-MS method might be a good alternative for LC-MS for the analysis of ox-PAPC products. Furthermore, it might also help scientists to expedite the search for uremic biomarkers.

Chemical Derivatization to Enhance Capillary Electrophoresis Mass Spectrometric Analysis of Acidic Metabolites in Mammalian Cells.

The simultaneous analysis of cationic and anionic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) has been considered challenging, as often two different analytical methods are required. Although CE-MS methods for cationic metabolite profiling have already shown good performance metrics, the profiling of anionic metabolites often results in relatively low sensitivity and poor repeatability caused by problems related to unstable electrospray and corona discharge when using reversed CE polarity and detection by MS in negative ionization mode. In this protocol, we describe a chemical derivatization procedure that provides a permanent positive charge to acidic metabolites, thereby allowing us to profile anionic metabolites by CE-MS using exactly the same separation conditions as employed for the analysis of basic metabolites. The utility of the overall approach is demonstrated for the analysis of energy metabolism-related metabolites in low numbers of HepG2 cells.

A rapid, simple and sensitive microfluidic chip electrophoresis mass spectrometry method for monitoring amino acids in cell culture media

The development and optimization of cell culture media for biotech applications is a fundamental step of process development. The composition of cell culture media requires an ideal blend of amino acids, vitamins, nucleosides, lipids, carbohydrates, trace elements and other components. The ability to monitor these constituents is required to ensure that cells receive sufficient nutrients to facilitate growth, viability and productivity. Analysis of cell culture media is challenging due to the range and diversity of compounds contained in this matrix and normally requires time consuming methods. A rapid, simple and sensitive microfluidic chip CE-MS method is described to monitor amino acids in chemically defined cell culture media from a Chinese hamster ovary cell line cultured over a period of 10 days. The described platform enabled the separation of 16 amino acids in less than 2 minutes and without the requirement for extensive sample preparation. The analytical parameters evaluated were precision, linearity, limit of detection and limit of quantification. The majority of essential amino acids were present in cell culture growth in high concentrations compared to non-essential amino acids. Over the course of the 10 days cell culture the concentration of certain amino acids declined by up to 100%. Microfluidic chip based CE-MS methods can be used effectively to obtain the consumption rates of amino acids in cell culture media during cell growth and to perform at-line monitoring and screening of cell culture status.

Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip-based capillary electrophoresis-mass spectrometry.

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.

Challenges and applications of isotachophoresis coupled to mass spectrometry: A review.

Electrophoretic separations are of growing interest to tackle complex analytical challenges. Nevertheless, capillary electrophoresis, as the most common mode, still suffers from insufficient detection limits due to low capillary loadability. ITP is of growing interest as preconcentration method for capillary electrophoresis and is also interesting to be applied as an independent analytical method. While mass spectrometric detection is common for capillary electrophoresis, the combination of ITP with MS is still a niche technique. In this work, we want to give an overview on isotachophoretic effects in CE-MS and ITP-MS methods, as well as coupling techniques of ITP with CE-MS. The challenges and possibilities associated with mass spectrometric detection in ITP and its coupling to capillary electrophoresis are critically discussed.

Profiling nucleotides in low numbers of mammalian cells by sheathless CE-MS in positive ion mode: Circumventing corona discharge.

Negative ion mode nano-ESI-MS is often considered for the analysis of acidic compounds, including nucleotides. However, under high aqueous separation conditions, corona discharge is frequently observed at emitter tips, which may result in low ion abundances and reduced nano-ESI needle emitter lifetimes. In this work, we introduce a sheathless CE-MS method for the highly efficient and sensitive analysis of nucleotides employing ESI in positive ion mode, thereby fully circumventing corona discharge. By using a background electrolyte of 16mM ammonium acetate (pH 9.7) a mixture of 12 nucleotides, composed of mono-, di-, and tri-phosphates, could be efficiently analyzed with plate numbers per meter above 220000 and with LODs in the range from 0.06 to 1.3nM, corresponding to 0.4 to 8.6 attomole, when using an injection volume of about 6.5 nL only. The utility of the method was demonstrated for the profiling of nucleotides in low numbers of mammalian cells using HepG2 cells as a model system. Endogenous nucleotides could be efficiently analyzed in extracts from 50000 down to 500 HepG2 cells only. Moreover, apart from nucleotides, also some nicotinamide-adenine dinucleotides and amino acids could be analyzed under these conditions, thereby clearly illustrating the utility of this approach for metabolic profiling of low amounts of biological material.

Development of a CE-MS method for the study of riociguat and metabolite M1 in pharmaceutical analysis.

Riociguat is a novel antihypertensive drug for the treatment of pulmonary hypertension. We present electrophoretic characterization, i.e. migration behavior of riociguat and metabolite M1 as support for optimized CZE/MS assay. Fundamental separation parameters, such as peak width, symmetry, and resolution are studied in a series of ammonium formate buffers within pH range 2.60-5.61. The narrow region of peak symmetry lies close to pH 4.0 for both analytes. Accordingly, the value of resolution maximizes in a background electrolyte adjusted to pH 4.10. Basic calibration parameters estimated from CZE experiments with absorption photometric and mass spectrometric detection of riociguat and metabolite M1 were evaluated. More than three orders lower LOD was achieved with high resolution mass spectrometric detection. The observed difference in the sensitivity of both detection techniques gives priority to the utilization of CZE/MS in practice. The values of dissociation constants of riociguat and metabolite M1, pKBH , were determined from CZE measurements in lithium formate and lithium acetate background electrolytes with constant ionic strength. The value of pKBH = 4.30±0.02 for riociguat corresponds well to the value already presented in the literature. According to our observation, metabolite M1 behaves like a slightly stronger base with estimated pKBH = 4.40±0.02.

Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry.

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just “dilute and shoot”), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91–15.12% and with accuracy yielding relative errors in the range of 85.47–112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines.

Development of complementary CE-MS methods for speciation analysis of pyrithione-based antifouling agents.

In the recent decade, metal pyrithione complexes have become important biocides for antifouling purposes in shipping. The analysis of metal pyrithione complexes and their degradation products/species in environmental samples is challenging because they exhibit fast UV degradation, transmetalation, and ligand substitution and are known to be prone to spontaneous species transformation within a chromatographic system. The environmental properties of the pyrithione species, e.g., toxicity to target and non-target organisms, are differing strongly, and it is therefore inevitable to identify as well as quantify all species separately. To cope with the separation of metal pyrithione species with minimum species transformation during analysis, a capillary electrophoresis (CE)-based method was developed. The hyphenation of CE with selective electrospray ionization- and inductively coupled plasma-mass spectrometry (ESI-, ICP-MS) provided complementary molecular and elemental information for the identification and quantification of pyrithione species. To study speciation of pyrithiones, a leaching experiment of several commercial antifouling paints containing zinc pyrithione in ultrapure and river water was conducted. Only the two species pyrithione (HPT) and dipyrithione ((PT)2) were found in the leaching media, in concentrations between 0.086 and 2.4μM (HPT) and between 0.062 and 0.59μM ((PT)2), depending on the paint and leaching medium. The limits of detection were 20nM (HPT) and 10nM ((PT)2). The results show that complementary CE-MS is a suitable tool for mechanistical studies concerning species transformation (e.g., degradation) and the identification of target species of metal pyrithione complexes in real surface water matrices, laying the ground for future environmental studies. Graphical abstract Hyphenation of CE with ESI- and ICP-MS provided complementary molecular and elemental information. Metal pyrithione species released from commercial antifouling paints could be identified and quantified in ultrapure and river water matrices.

CE-MS for anionic metabolic profiling: An overview of methodological developments.

The efficient profiling of highly polar and charged metabolites in biological samples remains a huge analytical challenge in metabolomics. Over the last decade, new analytical techniques have been developed for the selective and sensitive analysis of polar ionogenic compounds in various matrices. Still, the analysis of such compounds, notably for acidic ionogenic metabolites, remains a challenging endeavor, even more when the available sample size becomes an issue for the total analytical workflow. In this paper, we give an overview of the possibilities of capillary electrophoresis‐mass spectrometry (CE–MS) for anionic metabolic profiling by focusing on main methodological developments. Attention is paid to the development of improved separation conditions and new interfacing designs in CE–MS for anionic metabolic profiling. A complete overview of all CE–MS‐based methods developed for this purpose is provided in table format (Table 1) which includes information on sample type, separation conditions, mass analyzer and limits of detection (LODs). Selected applications are discussed to show the utility of CE–MS for anionic metabolic profiling, especially for small‐volume biological samples. On the basis of the examination of the reported literature in this specific field, we conclude that there is still room for the design of a highly sensitive and reliable CE–MS method for anionic metabolic profiling. A rigorous validation and the availability of standard operating procedures would be highly favorable in order to make CE–MS an alternative, viable analytical technique for metabolomics.

Magnetic phase composition of ZnO film heavily implanted with Fe ions

Abstract We have implanted ZnO thin film with 40 keV Fe+ ions to a high fluence of 1.25 × 1017 ions/cm2. Elemental and magnetic phase compositions were investigated by XRD, XPS, CEMS, and VSM methods. The Fe-implanted ZnO film reveals strong ferromagnetism at room temperature. The implanted iron ions are found in different valence states of Fe2+ and Fe3+. It was attributed to the formation of paramagnetic and ferromagnetic iron-based phases. The ferromagnetism is explained with the formation of a magnetic ordered solution of iron ions in ZnO matrix. Post-implantation annealing in a high vacuum results in the decrease of magnetism due to the formation of magnetite nanoparticles.

Enhancing Proteomic Throughput in Capillary Electrophoresis-Mass Spectrometry by Sequential Sample Injection.

In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis-mass spectrometry for rapid and sensitive proteome characterization of human lymphoblastic T-cells (line CCRF-CEM). Proteins were extracted, enzymatically digested, and the resulting peptides fractionated by RP-HPLC. Twenty fractions were thereafter analyzed by CE-MS within a single MS analysis. The CE-MS method was designed so that every 10min a new fraction was injected into the CE system. Without any rinsing or equilibration steps we were able to generate a continuous stream of peptides feeding the mass analyzer. In 250min, the total analysis time of a single sequential injection experiment, we were able to identify roughly 28000 peptide sequences counting for 4800 proteins. These numbers could be increased to 62000 peptides and more than 6100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5h. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from.

GC-MS based metabolomics of colon cancer cells using different extraction solvents

The increasing incidence of colorectal cancer enforces the development of novel methodologies and protocols to deepen in the molecular mechanisms that govern disease pathophysiological events. The aim of this work is to deepen in the optimum metabolite extraction protocol from adherent mammalian cells of colon cancer for high throughput metabolomics using gas chromatography coupled to mass spectrometry (GC-MS). GC-MS results showed that metabolic information obtained from colon cancer cells was highly dependent on metabolite extraction selection, which at the same time is extremely influenced by the analytical platform. A further purpose of this investigation is to uncover an unexplored portion of HT-29 colon cancer cells metabolome, complementary to other already explored by CE-MS and LC-MS methods. At this respect, a total of 150 metabolites were identified in HT-29 colon cancer cells by GC-MS. The extraction protocol with acetonitrile-isopropanol-water was the most appropriate for fatty acids and related pathways analysis. Most of the metabolites involved in pathways of amino acids, glutathione, amino sugars and other polar metabolites were better extracted with acidified water, although water extraction showed the best overall reproducibility. Although pathways involving nitrogenous bases could be investigated using organic or aqueous extracts, a higher number of metabolites involved in these pathways were identified in the aqueous extracts. In addition, metabolite extraction protocol was observed to be crucial for the determination of potentially interesting clusters of metabolites.

In-capillary approach to eliminate SDS interferences in antibody analysis by capillary electrophoresis coupled to mass spectrometry.

Capillary electrophoresis is an important technique for the characterization of monoclonal antibodies (mAbs), especially in the pharmaceutical context. However, identification is difficult as upscaling and hyphenation of used methods directly to mass spectrometry is often not possible due to separation medium components that are incompatible with MS detection. Here a CE-MS method for the analysis of mAbs is presented analyzing SDS-complexed samples. To obtain narrow and intensive peaks of SDS-treated antibodies, an in-capillary strategy was developed based on the co-injection of positively charged surfactants and methanol as organic solvent. For samples containing 0.2% (v/v) of SDS, recovered MS peak intensities up to 97 and 95% were achieved using cetyltrimethylammonium bromide or benzalkonium chloride, respectively. Successful removal of SDS was shown in neutral coated capillaries but also in a capillary with a positively charged coating applying reversed polarity. The usefulness of this in-capillary strategy was demonstrated also for other proteins and for antibodies dissolved in up to 10% v/v SDS solution, and in other SDS-containing matrices, including the sieving matrix used in a standard CE-SDS method and gel-buffers applied in SDS-PAGE methods. The developed CE-MS approaches enable fast and reproducible characterization of SDS-complexed antibodies.

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS

Rapid and sensitive product quality analysis is important for real-time monitoring during biopharmaceutical development and manufacturing. However, low level of protein concentration and complex cell culture matrix pose challenges for product quality characterization at early stages of cell line selection and process development. Here, we describe a fast and simple microfluidic ZipChip CE-MS method to measure quality attributes of monoclonal antibody protein directly from cell culture supernatant. Cell culture supernatant samples were characterized with charge-based separation using microfluidic capillary electrophoresis coupled to a high-resolution mass spectrometer. Under sample reducing conditions, multiple protein glycosylation attributes were determined on the heavy chain, whereas titer information was obtained from comparison of light chain signal intensity following sample spiking-in with heavy labeled mAb. Therefore, the protein expression and product quality can be monitored using the same method with a single microfluidic device. A total volume of ten to fifty microliter of cell culture supernatant is needed, whereas analysis time is within three minutes per sample. In addition, comparison of new method with traditional RP-LC-MS method using a set of time-course bioreactor cell culture samples has been performed. A good correlation of the levels of N-glycosylation attributes between ZipChip CE-MS of crude samples and RPLCMS analysis following Protein A (ProA) purification step has been demonstrated.

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.