Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies.

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

Capillary electrophoresis-mass spectrometry for protein analyses under native conditions: Current progress and perspectives.

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigration separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out.

Capillary zone electrophoresis-high field asymmetric ion mobility spectrometry-tandem mass spectrometry for top-down characterization of histone proteoforms.

Characterization of histone proteoforms with various post-translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)-based top-down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's-eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi-dimensional separations of histone proteoforms. Our CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform identified 366 (ProSight PD) and 602 (TopPIC) histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE-FAIMS-MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE-MS/MS alone (without FAIMS). The results indicate that CZE-FAIMS-MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.

Pilot Study for Deciphering Post-Translational Modifications and Proteoforms of Tau Protein by Capillary Electrophoresis-Mass Spectrometry.

Abnormal accumulation of tau protein in the brain is one pathological hallmark of Alzheimer's disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of the PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant human tau-0N3R. We identified 50 phosphorylation sites of tau-0N3R in total, which is about 25% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird's-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to nine phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under native-like cIEF-MS conditions, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.

Predictive analysis of breast cancer response to neoadjuvant chemotherapy through plasma metabolomics.

PURPOSE: Preoperative chemotherapy is a critical component of breast cancer management, yet its effectiveness is not uniform. Moreover, the adverse effects associated with chemotherapy necessitate the identification of a patient subgroup that would derive the maximum benefit from this treatment. This study aimed to establish a method for predicting the response to neoadjuvant chemotherapy in breast cancer patients utilizing a metabolomic approach. METHODS: Plasma samples were obtained from 87 breast cancer patients undergoing neoadjuvant chemotherapy at our facility, collected both before the commencement of the treatment and before the second treatment cycle. Metabolite analysis was conducted using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS). We performed comparative profiling of metabolite concentrations by assessing the metabolite profiles of patients who achieved a pathological complete response (pCR) against those who did not, both in initial and subsequent treatment cycles. RESULTS: Significant variances were observed in the metabolite profiles between pCR and non-pCR cases, both at the onset of preoperative chemotherapy and before the second cycle. Noteworthy distinctions were also evident between the metabolite profiles from the initial and the second neoadjuvant chemotherapy courses. Furthermore, metabolite profiles exhibited variations associated with intrinsic subtypes at all assessed time points. CONCLUSION: The application of plasma metabolomics, utilizing CE-MS and LC-MS, may serve as a tool for predicting the efficacy of neoadjuvant chemotherapy in breast cancer in the future after all necessary validations have been completed.

Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

Analytical Techniques for Single-Cell Biochemical Assays of Lipids.

Lipids are essential cellular components forming membranes, serving as energy reserves, and acting as chemical messengers. Dysfunction in lipid metabolism and signaling is associated with a wide range of diseases including cancer and autoimmunity. Heterogeneity in cell behavior including lipid signaling is increasingly recognized as a driver of disease and drug resistance. This diversity in cellular responses as well as the roles of lipids in health and disease drive the need to quantify lipids within single cells. Single-cell lipid assays are challenging due to the small size of cells (∼1 pL) and the large numbers of lipid species present at concentrations spanning orders of magnitude. A growing number of methodologies enable assay of large numbers of lipid analytes, perform high-resolution spatial measurements, or permit highly sensitive lipid assays in single cells. Covered in this review are mass spectrometry, Raman imaging, and fluorescence-based assays including microscopy and microseparations.

Recent advances in top-down proteome sample processing ahead of MS analysis.

Top-down proteomics is emerging as a preferred approach to investigate biological systems, with objectives ranging from the detailed assessment of a single protein therapeutic, to the complete characterization of every possible protein including their modifications, which define the human proteoform. Given the controlling influence of protein modifications on their biological function, understanding how gene products manifest or respond to disease is most precisely achieved by characterization at the intact protein level. Top-down mass spectrometry (MS) analysis of proteins entails unique challenges associated with processing whole proteins while maintaining their integrity throughout the processes of extraction, enrichment, purification, and fractionation. Recent advances in each of these critical front-end preparation processes, including minimalistic workflows, have greatly expanded the capacity of MS for top-down proteome analysis. Acknowledging the many contributions in MS technology and sample processing, the present review aims to highlight the diverse strategies that have forged a pathway for top-down proteomics. We comprehensively discuss the evolution of front-end workflows that today facilitate optimal characterization of proteoform-driven biology, including a brief description of the clinical applications that have motivated these impactful contributions.

Fatty acids analysis by capillary electrophoresis: Fundamentals, advantages and applications.

This review covers the know-how of the Grupo de Química Analítica e Quimiometria regarding the analysis of fatty acids by capillary electrophoresis acquired over its 20 years of existence. Therefore, the fundamentals, advantages, and applications of this technique for analyzing different fatty acids in samples such as food, oils, cosmetics, and biological matrices are presented and discussed. Capillary electrophoresis is, thus, shown as an interesting and valuable separation technique for the target analysis of these analytes as an alternative to the gas chromatography coupled to flame ionization detection, as it offers advantages over the latter such as low analysis times, low sample and reagent consumption, the use of a nondedicated column, and simpler sample preparation. In addition, the methods shown in this literature review can be useful for quality control, adulteration, and health-related research by regulatory agencies.

Sorting and simultaneous quantitation of intact mixed-cell samples via capillary isotachophoresis.

A great need currently exists for rapid, inexpensive, and accurate methods for microbial analysis in the medical, food, industrial, and water quality fields. Here, a novel capillary isotachophoresis (CITP) method is presented for the focusing, sorting, and quantitation of intact cells in mixed samples based on their electrophoretic mobility ranges. Using a series of ion spacers dissolved in the sample, this technique results in several efficient cell peaks in the electropherogram corresponding to specific cell electrophoretic mobility ranges. The concentrations of different species in mixed-cell samples are determined from the cell peak areas and the known peak response factors for the cell species using a series of linear equations. Method design and optimization are discussed, including the choice of running buffer, pH, and ion spacers. Mixed-cell samples of up to four different species were focused and quantified as a proof-of-principle of the method. When sample cell concentrations were toward the middle of the linear response range, accuracies between 1% and 11% and relative standard deviations of 1%-14% were obtained, depending on the number of cell species in the mixture. This work provides a useful basis for future studies of cell quantitation using CITP, which could be potentially applied to a variety of fields including cell growth studies, microbial contamination testing, and sterility testing.

Recent developments in capillary and microchip electroseparations of peptides (2021-mid-2023).

This review article brings a comprehensive survey of developments and applications of high-performance capillary and microchip electromigration methods (zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical characterization of peptides in the period from 2021 up to ca. the middle of 2023. Progress in the study of electromigration properties of peptides and various aspects of their analysis, such as sample preparation, adsorption suppression, electroosmotic flow regulation, and detection, are presented. New developments in the particular capillary electromigration methods are demonstrated, and several types of their applications are reported. They cover qualitative and quantitative analysis of synthetic or isolated peptides and determination of peptides in complex biomatrices, peptide profiling of biofluids and tissues, and monitoring of chemical and enzymatic reactions and physicochemical changes of peptides. They include also amino acid and sequence analysis of peptides, peptide mapping of proteins, separation of stereoisomers of peptides, and their chiral analyses. In addition, micropreparative separations and physicochemical characterization of peptides and their interactions with other (bio)molecules by the above CE methods are described.

Nucleic acid strand displacement for indirect determination of foodborne bacteria by capillary electrophoresis and its application in antagonism and bacteriostasis studies.

Foodborne bacteria threaten human's health. Capillary electrophoresis (CE) is a powerful separation means for the determination of bacteria. Direct separation of bacteria suffers from the shortages of low resolution, channel adsorption, and bacterial aggregation. In this work, a method of nucleic acid strand displacement was developed to indirect separate the bacteria by CE. DNA complexes, consisting of probes and aptamers, were mixed with the three bacteria Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The aptamers could specifically bond with bacteria and release the probes. Through the separation of the probes, the bacteria could be indirectly determined by CE. This method avoided the shortages of direct separation of bacteria. Under the optimized conditions, the three probes for the bacteria could be separated and detected within 2.5 min by high-speed CE with laser-induced fluorescence detection. The limits of detection for the bacteria were in the range 4.20 × 106 to 1.75 × 107  CFU/mL. Finally, the developed method was applied on the study of antagonism of the coexistent bacteria to reveal the relationship between them. Furthermore, the efficiency of bacteriostasis of three traditional Chinese medicines, Coptis chinensis, Schisandra chinensis, and honeysuckle, was also studied by this method.

A new device for online nanoscale sampling and capillary electrophoresis analysis of plant sap composition.

In this work, an online sampling of plant xylem sap combined with an efficient (CE)-based method was developed and applied to study the kinetics of changes in the sap composition and to assess plant fitness under stress conditions comprehensively. A laboratory-built CE device was developed to provide online sampling and CE analysis of various ionogenic species in the sap during plant stress response. The rapid online sampling and short CE analysis time allow for real-time monitoring of changes in sap constituents in the living plant during the stress response. The developed device was successfully used to analyze chloride, nitrate, and sulfate ions in the plant xylem during the salt stress or stress caused by nitrate deficiency within short time scales.

Simultaneous determination of cystine and other free aminothiols in blood plasma using capillary electrophoresis with pH-mediated stacking.

We developed a method of sensitive capillary electrophoresis using UV detection for the determination of certain free aminothiols (reduced cysteinylglycine (rCysGly), cysteine (rCys), glutathione (rGln), and cystine (CysS) in human blood plasma. The reduced thiols were derivatized with N-ethylmaleimide. The plasma was purified from proteins via ultrafiltration. Electrophoretic separation was performed using 115 mM Na phosphate with 7.5% (v/v) polyethylene glycol 600, pH 2.3. The in-capillary concentration of the analytes was achieved with a pH gradient created via the preinjection of triethanolamine and postinjection of phosphoric acid. The separation was carried out using a silica capillary (50 µm i.d.; total/effective separation length 42/35 cm) at a 25 kV voltage. The total analysis/regeneration time was 18 min. The quantification limits varied from 1.3 µM (rCysGly) to 5.4 µM (CysS). The accuracy was 95%-99%, and the repeatability and reproducibility were approximately 1.8%-3.8% and 1.9%-5.0%, respectively. An analysis of plasma samples from healthy volunteers (N = 41) showed that the mean levels of rCysGly, rCys, rGln, and CysS were 1.64, 10.6, 2.58, and 46.2 µM, respectively.

Investigation of induced electroosmotic flow in small-scale capillary electrophoresis devices: Strategies for control and reversal.

Electroosmotic flow (EOF) is the bulk flow of solution in a capillary or microchannel induced by an applied electric potential. For capillary and microchip electrophoresis, the EOF enables analysis of both cations and anions in one separation and can be varied to modify separation speed and resolution. The EOF arises from an electrical double layer at the capillary wall and is normally controlled through the pH and ionic strength of the background buffer or with the use of additives. Understanding and controlling the electrical double layer is therefore critical for maintaining acceptable repeatability during method development. Surprisingly, in fused silica capillaries at low pH, studies observe an EOF even though the capillary surface should be neutralized. Previous work has suggested the presence of an "induced electroosmotic flow" from radial electric fields generated across the capillary wall due to the separation voltage and grounded components external to the capillary. Using thin-wall (15 µm) fused silica separation capillaries to facilitate the study of radial fields, we show that the EOF mobility depends on both the separation voltage and the location of external grounds. This is consistent with the induced EOF model, in which radial electric fields embed positive charges at the capillary walls to create an electrical double layer. The magnitude of the effect is characterized and shown to have long-range influences that are difficult to completely null by moving grounded components away from the separation capillary. Instead, active EOF control using externally applied potentials or a passive approach using a negative separation voltage are discussed as two possible methods for controlling the induced EOF. Both methods can reverse the EOF and improve the resolution and peak efficiency in amino acid separations.

Comparison between capillary electrophoresis and fluorescence anisotropy competitive immunoassay for glucagon.

Glucagon plays a crucial role in regulating glucose homeostasis; unfortunately, the mechanisms controlling its release are still unclear. Capillary electrophoresis (CE)- and fluorescence anisotropy (FA)-immunoassays (IA) have been used for online measurements of hormone secretion on microfluidic platforms, although their use in glucagon assays is less common. We set out to compare a glucagon-competitive IA using these two techniques. Theoretical calibration curves were generated for both CE- and FA-IA and results indicated that CE-IA provided higher sensitivity than FA-IA. These results were confirmed in an experiment where both assays showed limits of detection (LOD) of 30 nM, but the CE-IA had ∼300-fold larger sensitivity from 0 to 200 nM glucagon. However, in online experiments where reagents were mixed within the device, the sensitivity of the CE-IA was reduced ∼3-fold resulting in a higher LOD of 70 nM, whereas the FA-IA remained essentially unchanged. This lowered sensitivity in the online CE-IA was likely due to poor sampling by electroosmotic flow from the high salt solution necessary in online experiments, whereas pressure-based sampling used in FA-IA was not affected. We conclude that FA-IA, despite lowered sensitivity, is more suitable for online mixing scenarios due to the ability to use pressure-driven flow and other practical advantages such as the use of larger channels.

Miniaturized nanoelectrospray interface for coupling capillary electrophoresis with mass spectrometry detection.

A miniaturized electrospray interface consisting of a microfluidic nanosprayer and nanospray module is reported in the presented short communication. The nanosprayer was fabricated using silicon (Si) technology suitable for cost-efficient high-volume mass production. The nanospray module enabled the positioning of the nanosprayer in front of a mass spectrometry entrance and its coupling with capillary electrophoresis based on the liquid junction principle. A case study of top-down and bottom-up proteomic analyses of intact cytochrome c and its tryptic digest demonstrates the practical applicability of the developed interface.

A bioinspired approach for the modulation of electroosmotic flow and protein-surface interactions in capillary electrophoresis using silylated amino-amides blocks and covalent grafting.

We explore a bioinspired approach to design tailored functionalized capillary electrophoresis (CE) surfaces based on covalent grafting for biomolecules analysis. First, the approach aims to overcome well-known common obstacles in CE protein analysis affecting considerably the CE performance (asymmetry, resolution, and repeatability) such as the unspecific adsorption on fused silica surface and the lack of control of electroosmotic flow (EOF). Then, our approach, which relies on new amino-amide mimic hybrid precursors synthesized by silylation of amino-amides (Si-AA) derivatives with 3-isocyanatopropyltriethoxysilane, aims to recapitulate the diversity of protein-protein interactions (π-π stacking, ionic, Van der Waals…) found in physiological condition (bioinspired approach) to improve the performance of CE protein analysis (electrochromatography). As a proof of concept, these silylated Si-AA (tyrosinamide silylation, serinamide silylation, argininamide silylation, leucinamide silylation, and isoglutamine silylation acid) have been covalently grafted in physiological conditions in different amount on bare fused silica capillary giving rise to a biomimetic coating and allowing both the modulation of EOF and protein-surface interactions. The analytical performances of amino-amide functionalized capillaries were assessed using lysozyme, cytochrome C and ribonuclease A and compared to traditional capillary coatings poly(ethylene oxide), poly(diallyldimethylammonium chloride), and sodium poly(styrenesulfonate). EOF, protein adsorption rate, protein retention factor k, and selectivity were determined for each coating. All results obtained showed this approach allowed to modulate the EOF, reduce unspecific adsorption, and generate specific interactions with proteins by varying the nature and the amount of Si-AA in the functionalization mixture.

Capillary electrophoresis in the analysis of therapeutic peptides-A review.

Therapeutic peptides are a growing class of innovative drugs with high efficiency and a low risk of adverse effects. These biomolecules fall within the molecular mass range between that of small molecules and proteins. However, their inherent instability and potential for degradation underscore the importance of reliable and effective analytical methods for pharmaceutical quality control, therapeutic drug monitoring, and compliance testing. Liquid chromatography-mass spectrometry (LC-MS) has long time been the "gold standard" conventional method for peptide analysis, but capillary electrophoresis (CE) is increasingly being recognized as a complementary and, in some cases, superior, highly efficient, green, and cost-effective alternative technique. CE can separate peptides composed of different amino acids owing to differences in their net charge and size, determining their migration behavior in an electric field. This review provides a comprehensive overview of therapeutic peptides that have been used in the clinical environment for the last 25 years. It describes the properties, classification, current trends in development, and clinical use of therapeutic peptides. From the analytical point of view, it discusses the challenges associated with the analysis of therapeutic peptides in pharmaceutical and biological matrices, as well as the evaluation of CE as a whole and the comparison with LC methods. The article also highlights the use of microchip electrophoresis, nonaqueous CE, and nonconventional hydrodynamically closed CE systems and their applications. Overall, the article emphasizes the importance of developing new CE-based analytical methods to ensure the high quality, safety, and efficacy of therapeutic peptides in clinical practice.