Sheathless Capillary Electrophoresis-mass Spectrometry Research Articles

Titanium dioxide-coated core-shell silica microspheres-based solid-phase extraction combined with sheathless capillary electrophoresis-mass spectrometry for analysis of glyphosate, glufosinate and their metabolites in baby foods

Because of their extremely low amount in complex samples, it is quite challenging to accurate determine residues of phosphorus-containing amino-acid-like herbicides (PAAHs) in food products. Here we develop novel core-shell mesoporous silica (CSMS) microspheres coated by titanium dioxide (CSMS@TiO2) for extraction and enrichment of PAAHs in baby foods. After the dispersive solid phase extraction (d-SPE), sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) is utilized to achieve efficient separation and sensitive detection. The synthesized CSMS@TiO2 composites are characterized by various spectroscopic techniques, proving TiO2 is uniformly distributed onto the channel surface of CSMS. The composites have essential features that are favorable for adsorption of the analytes on the material for d-SPE, including uniform diameter (1.0 μm with a shell thickness of 133 nm), large perpendicular mesopores (15.6 nm), high surface area (101.1 m2/g) and large pore volume (0.4 cm3/g). Taking glyphosate, glufosinate and their main metabolites (aminomethylphosphonic acid and 3-methylphosphinicopropionic acid) as analytes, selective and efficient enrichment is achieved by CSMS@TiO2-based d-SPE through the affinity interaction between titanium dioxide and phosphate groups. Sensitive detection of target compounds is achieved with low limits of quantitation (LOQs) between 0.3–1.6 ng/mL and excellent inter/intra-day repeatability. The compounds in nine different commercial baby foods from local markets are analyzed using the proposed method. Good recoveries of 82.3–102.6% are achieved with low RSDs (n = 5) of 2.1–8.3%. Our study indicates that the proposed CSMS@TiO2-based d-SPE combined with sheathless CE-MS is an accurate and reliable approach for sensitive determination of trace-amount PAAHs and their metabolites in complex samples.

Separation, identification and quantification of associated impurities in cobratide using sheathless CE-MS and CE-UV.

Cobratide is a peptide drug extracted from the venom of Chinese cobra, and has been widely used in the clinical treatment of chronic, intractable and persistent pain. In a recent study, it was reported that it has the potential to treat COVID-19. In order to control the quality of commercial cobratide drugs, a protocol was established for the separation, identification and quantification of cobratide and its associated impurities, in which sheathless capillary electrophoresis-mass spectrometry (CE-MS) was used for identification and a rapid capillary electrophoresis-ultraviolet-visible detector (CE-UV) method was developed for accurate quantification. Separation conditions that affect the resolution and MS intensities of cobratide and its associated impurities were investigated, including pH value, concentration of background electrolyte (BGE), ratio of organic additive and sample solution. The optimized CE conditions (BGE: 50 mM NH4Ac, pH 4.0; sample solution: deionized water) were used for both sheathless CE-MS and CE-UV methods. Three associated impurities were separated and identified for the first time by sheathless CE-MS. Then, a rapid CE-UV method was validated and used for accurate quantification of cobratide and its associated impurities. The CE-UV method showed good linearity between concentration and corrected peak area of cobratide in the concentration range of 5.36-536.30 μg mL-1. The limit of quantification of the CE-UV method was 4.16 μg mL-1. The relative standard deviations of migration time were less than 1% for both intra-day and inter-day experiments, and those of corrected peak area were less than 5%. Finally, different cobratide drugs were analyzed to evaluate the batch-to-batch consistency. This established protocol combining sheathless CE-MS and CE-UV methods would provide useful information for both quality control and process analysis of peptide drugs.

Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS

Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be observed contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new analytical strategies are needed for a more accurate and in-depth analysis.Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a positively coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the separation permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond reduction. The six different subunits (Lc1, Lc2, Fd’1, Fd’2, (Fc/2)1 and (Fc/2)2) were separated using the same positively-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resolution (resolving power 140000 at m/z 1100) for a more confident analysis of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately determined in the subunits showing the complementarity of both approaches.

Snake venom characteristic peptides: novel fingerprints for species identification by sheathless capillary electrophoresis-electrospray ionization-mass spectrometry.

Snake venom is a complex mixture mainly consisting of proteins and peptides which varies with different species. These variations lead to different toxic mechanisms and different anti-venom serums for treatment and the determination of their use as drugs. Hence, it is important to develop a sensitive and reliable method to identify the species of snakes from venoms. Herein, we present a novel strategy based on the sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CESI-MS) system to characterize snake venom proteins. Through the determination of peptides, we found the characteristic peptides of 8 different snakes with high sensitivity (1 μg mL-1) and high selectivity, which provided a reliable method for the species identification and purity detection of snake venom samples.

Utility of sheathless capillary electrophoresis-mass spectrometry for metabolic profiling of limited sample amounts

Metabolomics studies using a small amount of cells may save time and money, while in some cases (e.g., profiling pathogenic cells in an early-stage tissue), only a small number of cells are accessible for analysis. The analysis of small amounts of biological samples challenges the analytical toolbox used in present-day metabolomics studies, and a significant number of crucial biological questions cannot be properly addressed. To allow metabolic profiling of limited sample amounts, the potential of capillary electrophoresis-mass spectrometry (CE–MS) using a sheathless porous tip interface has been assessed using HepG2 cells in starting amounts of 500 and 10,000 cells as a model system in this work. It is shown that highly efficient and information-rich metabolic profiles for cationic metabolites at low-pH separation conditions could be obtained by sheathless CE–MS using an injection volume of only circa 42 nL, which equals the content/aliquot of circa 0.25 and 5 HepG2 cells, respectively. With as little as the content of 0.25 cell injected, more than 24 cationic metabolites could be identified. A further improvement of sample preparation and/or the injection part is required in order to effectively analyze the compounds of interest in very low sample amounts by sheathless CE–MS. However, the results obtained so far clearly indicate the strong potential of the proposed method for metabolic profiling of limited sample amounts.

Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry

Antibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have explored the capabilities of low-flow sheathless capillary electrophoresis-mass spectrometry (CE-MS) for the high-resolution and sensitive profiling of antibody therapeutics. Near-zero electroosmotic flow was achieved by employing a novel neutral capillary coating that also prevents protein adsorption. CE-MS analysis of intact model proteins using an acidic background electrolyte demonstrated satisfactory performance, with overall migration-time RSDs below 2.2% from three different capillaries tested. For system evaluation, three nanobody preparations, including mono- and bivalent forms, and three monoclonal antibodies (mAbs) were analyzed. Intact nanobodies were resolved from their degradation products, which could be assigned to deamidated, cleaved, and truncated forms at the C-terminal tag. Excellent resolution of isomeric deamidated products was obtained. The mAbs were analyzed intact and after digestion by the endoproteinase IdeS (middle-up approach). CE-MS of intact mAbs provided resolution of clipped species (e.g. light chain and light chain-heavy chain fragments) from the native protein. Moreover, glycoforms containing sialic acids were resolved from their non-sialylated counterparts. For IdeS-digested, F (ab)2 and Fc/2 portions where efficiently resolved for the three mAbs. Whereas the migration time of the Fc/2 fragments was fairly similar, the migration time of the F (ab)2 part was strongly varied among the mAbs. For all mAbs, separation of Fc/2 charge variants - including sialylated glycoforms and other post-translational modifications, such as loss of C-terminal lysine or asparagine deamidation - was achieved. This allowed a detailed and reliable assessment of the Fc/2 heterogeneity (18–33 proteoforms) of the three analyzed mAbs.

Sheathless Capillary Electrophoresis-Mass Spectrometry for theProfiling of Charged Metabolites in Biological Samples.

Capillary electrophoresis (CE) is well suited for the profiling of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. The protocol presented here is based on using a recently developed sheathless interfacing design, i.e., a porous tip interface, for coupling CE to electrospray ionization mass spectrometry (MS). It is demonstrated that sheathless CE-MS employing a bare fused-silica capillary at low-pH separation conditions can be used for the profiling of both cationic and anionic metabolites by only switching the MS detection and electrophoretic separation voltage polarity. The proposed sheathless CE-MS protocol allows efficient and sensitive profiles to be obtainedfor a broad array of charged metabolites, including amino acids, organic acids, nucleotides, and sugar phosphates, in various biological samples, such as urine and extracts of the glioblastoma cell line.

Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis – Tandem mass spectrometry as nanoESI infusion platform and separation method

Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent complexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical techniques for complete structure assessment. We report the development of complementary approaches implementing sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the different aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to antibody ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(ab')2 subunits incorporating 1, 0 to 4 and 0 to 8 payloads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hydrophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multilevel characterization of these complex biomolecules.

Sheathless capillary electrophoresis-mass spectrometry for anionic metabolic profiling.

The performance of CE coupled on‐line to MS via a sheathless porous tip sprayer was evaluated for anionic metabolic profiling. A representative metabolite mixture and biological samples were used for the evaluation of various analytical parameters, such as peak efficiency (plate numbers), migration time and peak area repeatability, and LODs. The BGE, i.e. 10% acetic acid (pH 2.2), previously used for cationic metabolic profiling was now assessed for anionic metabolic profiling by using MS detection in negative ion mode. For test compounds, RSDs for migration times and peak areas were below 2 and 11%, respectively, and plate numbers ranged from 60 000 to 40 0000 demonstrating a high separation efficiency. Critical metabolites with low or no retention on reversed‐phase LC could be efficiently separated and selectively analyzed by the sheathless CE‐MS method. An injection volume of only circa 20 nL resulted in LODs between 10 and 200 nM (corresponding to an amount of 0.4–4 fmol), which was an at least tenfold improvement as compared to LODs obtained by conventional CE‐MS approaches for these analytes. The methodology was applied to anionic metabolic profiling of glioblastoma cell line extracts. Overall, a sheathless CE‐MS method has been developed for highly efficient and sensitive anionic metabolic profiling studies, which can also be used for cationic metabolic profiling studies by only switching the MS detection and separation voltage polarity.

Low-picomolar analysis of peptides by on-line coupling of fritless solid-phase extraction to sheathless capillary electrophoresis-mass spectrometry

A novel fritless solid-phase extraction (SPE) microcartridge was designed for combination with sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) employing a prototype porous-tip capillary for nanoelectrospray ionization (nanoESI). The inlet of the separation capillary (30μm inner diameter (id), 150μm outer diameter (od)) was inserted in a 4mm long SPE microcartridge (150μm id, 365μm od) packed with a C18 sorbent of 55–105μm particle size. Performance of the SPE-CE-MS system was evaluated using diluted solutions of the three opioid peptides dynorphin A (1–7) (DynA), endomorphin 1 (End1) and met-enkephalin (Met). Sample volumes of 1.5μL were loaded on the SPE microcartridge and the retained peptides were eluted with 22nL of an acidic methanol/water (60:40, v/v) solution. Using a pressure of 50mbar during separation to speed up the analysis, good peptide resolution was obtained with acceptable plate numbers (between 53,000 and 92,000). Intraday relative standard deviations (% RSD) for peptide migration times and peak areas were below 4% and 9%, respectively. The SPE-CE-MS method showed good linearity in the 0.05–5ngmL−1 range and limits of detection (LODs) were 10pgmL−1. However, loading a larger volume of sample (8μL), LODs could be decreased down to 2pgmL−1 (2.2–3.5pM). This represents an improvement of up to 5000-fold with respect to the LODs achieved by sheathless CE-MS without on-line preconcentration demonstrating the potential of on-line SPE for further enhancing sensitivity.

Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes

Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the β ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. β ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MDa.

Sheathless capillary electrophoresis-mass spectrometry with a high-sensitivity porous sprayer for cationic metabolome analysis

Sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a new tool for comprehensive analysis of charged metabolites. However, it needs to be more sensitive. Here, we report a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry method for cationic metabolome analysis. This system used a high-sensitivity porous sprayer interface and 10% (v/v) acetic acid as the background electrolyte (BGE). Under optimized conditions, 53 cationic metabolites, including amino acids and their derivatives, amines, nucleic acids and small peptides, were successfully separated and selectively detected with a time-of-flight mass spectrometer. At a signal-to-noise ratio of three, the concentration detection limits for these compounds were between 0.004 and 0.8 μmol L(-1) (amount detection limit, 0.01 to 2 fmol) with pressure injection at 20.7 kPa for 5 s (2.6 nL). Compared with conventional sheath-flow CE-MS, the detection limit of the present method was increased more than 5-fold for 21 (40%) of the compounds detected. When the method was applied to the analysis of cationic metabolites obtained from human urine, there was a 10-fold increase in the number of detected peaks compared with conventional methods. More than 180 successive runs could be conducted without any problems, and only the BGE needed to be changed.

Enhancing the Coverage of the Urinary Metabolome by Sheathless Capillary Electrophoresis-Mass Spectrometry

Sheathless capillary electrophoresis-mass spectrometry (CE-MS), using a porous tip sprayer, is proposed for the first time for highly sensitive metabolic profiling of human urine. A representative metabolite mixture and human urine were used for evaluation of the sheathless CE-MS platform. For test compounds, relative standard deviations (RSDs) for migration times and peak areas were below 2% and 12%, respectively, and an injection volume of only ∼8 nL resulted in detection limits between 11 and 120 nM. Approximately 900 molecular features were detected in human urine by sheathless CE-MS whereas about 300 molecular features were found with classical sheath-liquid CE-MS. This difference can probably be attributed to an improved ionization efficiency and increased sensitivity at low flow-rate conditions. The integration of transient-isotachophoresis (t-ITP) as an in-capillary preconcentration procedure in sheathless CE-MS further resulted in subnanomolar limits of detection for compounds of the metabolite mixture, and more than 1300 molecular features were observed in urine. Compared to the classical CE-MS approaches, the integration of t-ITP combined with the use of a sheathless interface provides up to 2 orders of magnitude sensitivity improvement. Hence, sheathless CE-MS can be used for in-depth metabolic profiling of biological samples, and we anticipate that this approach will yield unique information in the field of metabolomics.

Design of a sheathless capillary eletrophoresis-mass spectrometry probe for operation with a Z-spray ionization source.

The construction of a sheathless interface for capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS), for operation with a Z-Spray source on a Micromass Quattro-LC triple quadrupole mass spectrometer is described. Designing the interface involved machining a probe compatible with the setup already in place on the mass spectrometer, i.e., MegaFlow-Z ESI. The probe was made of Lexan with the same dimensions as the ESI probe supplied with the instrument. The electrical connection at the electrospray end of the CE capillary was made possible by gold-coating (sheathless CE-ESI-MS). The probe design as well as the electrical and power supply requirements are described in detail. Experiments were performed using this interface, and CE separations of mixtures containing pmole and sub-pmole amounts of peptides were monitored by on-line MS. For a standard peptide mixture (10(-4) M), separation efficiency was typically characterized by N > 10(4) theoretical plates with S/N > 400. Using the same experimental setup, it was also possible to conduct on-line CE-ESI-tandem MS (MS/MS) experiments on the same peptide mixture, and to determine the sequence of the peptides. MS/MS scan functions for different precursor ions were used either alternately or sequentially and the results from both methods were compared. The possibility of peptide mass mapping was explored, and CE-ESI-MS results were obtained for the digestion products of equine myoglobin. Separation efficiencies and S/N values were similar to those obtained for standard peptides. A complete map of the digestion products was obtained.