Sheathless CE-MS Research Articles

Metabolome Analysis by Sheathless CE-MS

Metabolome Analysis by Sheathless CE-MS

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.

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.

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.

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.

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.

Analytics of the therapeutic peptide aviptadil by sheathless CE-MS and comparison with nanoRP-HPLC–MS

Purification and quality control of therapeutic peptides is often performed by one single method, RP-HPLC. As usage of an orthogonal technique is highly advisable for quality assurance, capillary electrophoresis (CE) employing a coated capillary coupled via a sheathless interface to a mass spectrometer was applied in parallel. The basic therapeutic peptide aviptadil served as a model substance to study the impurity profiles revealing 15 detectable impurities using CE-MS, two were detected by an appropriate nanoRP-HPLC–MS method. None of the impurities detected by CE were observed in LC and vice versa. The LOD in CE-MS was determined in the base peak electropherogram at ∼1fmol, a value 2500 times smaller than the LOD found in nanoRP-HPLC–MS (3pmol). In nanoRP-HPLC–MS only 0.2% of the extrapolated CE-MS signal for a 25ng aviptadil load was observed. We conclude that both, the LOD as well as the impurity profile of aviptadil, as analyzed by nanoRP-HPLC are influenced by both, the ligand-derivatized silica matrix and the flow-rate. Peptides may disappear completely and their variable emergence may lead to the determination of incorrect ratios as present in the sample.

Low-Flow Sheathless Capillary Electrophoresis–Mass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-β allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.

Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS

The need for sensitive analytical technologies applicable to metabolic profiling of volume-restricted biological samples is high. Here, we demonstrate feasibility of capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (MS) with sheathless nano-electrospray interface for non-targeted profiling of ionogenic metabolites in body fluids of experimental animals. A representative mixture of the metabolites and body fluids of mice such as cerebrospinal fluid (CSF), urine and plasma were used as examples of low-volume biological samples for method evaluation. An injection volume of only 9 nL resulted in limits of detection between 0.7 and 12 nM for the metabolite mixture. The method allowed the detection of ~350 molecular features in mouse CSF (an injection volume of ca. 45 nL), while ~400 features were observed in mouse plasma and ~3,500 features in mouse urine (an injection volume of ca. 9 nL). The low-volume body fluid samples were analyzed directly after only 1:1 dilution with water, thereby fully retaining sample integrity, which is of crucial importance for non-targeted metabolic profiling. As little is known about the metabolic composition of mouse CSF, we identified a fraction of the molecular features in mouse CSF using accurate mass information, migration times, MS/MS data, and comparison with authentic standards. We conclude that sheathless CE-MS can be used for sensitive metabolic profiling of volume-restricted biological samples.

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

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