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The development of reliable single-cell dispensers and substantial sensitivity improvement in mass spectrometry made proteomic profiling of individual cells achievable. Yet, there are no established methods for single-cell glycome analysis due to the inability to amplify glycans and sample losses associated with sample processing and glycan labeling. In this work, we present an integrated platform coupling online in-capillary sample processing with high-sensitivity label-free capillary electrophoresis-mass spectrometry for N-glycan profiling of single mammalian cells. Direct and unbiased quantitative characterization of single-cell surface N-glycomes are demonstrated for HeLa and U87 cells, with the detection of up to 100 N-glycans per single cell. Interestingly, N-glycome alterations are unequivocally detected at the single-cell level in HeLa and U87 cells stimulated with lipopolysaccharide. The developed workflow is also applied to the profiling of ng-level amounts (5-500 ng) of blood-derived protein, extracellular vesicle, and total plasma isolates, resulting in over 170, 220, and 370 quantitated N-glycans, respectively., (© 2024. The Author(s).)

We developed a highly sensitive method for profiling of N-glycans released from proteins based on capillary zone electrophoresis coupled to electrospray ionization mass spectrometry (CZE-ESI-MS) and applied the technique to glycan analysis of plasma and blood-derived isolates. The combination of dopant-enriched nitrogen (DEN)-gas introduced into the nanoelectrospray microenvironment with optimized ionization, desolvation, and CZE-MS conditions improved the detection sensitivity up to ∼100-fold, as directly compared to the conventional mode of instrument operation through peak intensity measurements. Analyses without supplemental pressure increased the resolution ∼7-fold in the separation of closely related and isobaric glycans. The developed method was evaluated for qualitative and quantitative glycan profiling of three types of blood isolates: plasma, total serum immunoglobulin G (IgG), and total plasma extracellular vesicles (EVs). The comparative glycan analysis of IgG and EV isolates and total plasma was conducted for the first time and resulted in detection of >200, >400, and >500 N-glycans for injected sample amounts equivalent to <500 nL of blood. Structural CZE-MS 2 analysis resulted in the identification of highly diverse glycans, assignment of α-2,6-linked sialic acids, and differentiation of positional isomers. Unmatched depth of N-glycan profiling was achieved compared to previously reported methods for the analysis of minute amounts of similar complexity blood isolates.

High-resolution capillary zone electrophoresis - mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the F C /2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS 2 . Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography - mass spectrometry-based approaches., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). Graphical Abstract ᅟ.

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.

A new high-performance capillary electrophoresis assay for aminothiols in human blood, including homocysteine, a marker of several human metabolism disorders, has been developed. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fused-silica capillary (65 cm x 50 microm I.D.) at 30 degrees C. With the electric field of 250 V cm(-1), separation of homocysteine, glutathione and cysteine occurred at less than 10 min. Detection at 250 or 234 nm was used to confirm the monobimane-thiols peaks. The detection limit was approximately 5 nmol/ml for all labeled aminothiols. The proposed method for these compounds' analysis included simple sample preparation, high selectivity, good linearity (r2>0.999), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<5% for three times consequently injected sample); high reliability and the small volumes required for analysis made it suitable for clinical studies.

This is an initial report to propose a novel approach in high-performance capillary electrophoresis (HPCE) for the direct detection of compounds without natural absorbance in the UV and visible spectral range, such as amino acids and carbohydrates. A refractometry detector with the 2 nl cell (Applied Systems, Minsk, Belarus) was employed to identify amino acids and carbohydrates without derivatization. The first results are provided on separation of seven free amino acids in the phosphate running buffer and three free carbohydrates in the borate-sodium dodecyl sulfate running buffer and detection by refractometer. Fused capillaries of 50 or 75 microm internal diameter and separation voltage (10-23 kV) were applied. Detection limits ranged typically from 10 to 100 fmol and the response was linear over two orders of magnitude for most of the amino acids and carbohydrates. The HPCE system demonstrated good long-term stability and reproducibility with a relative standard deviation, less than 5% for the migration time (n=10).

The techniques for micro-level analysis of some widespread unusual amino acids (phosphorylated and hydroxylated ones) as well as of some genetically non-encoded amino acids were developed for their subsequent identification in the peptide and protein amino acid sequence by narrow-bore column high-performance liquid chromatography (approximately 10 pmol of the sample), high-performance capillary electrophoresis (approximately 1-10 pmol), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (approximately 1-10 pmol), and automatic protein gas phase sequencing (approximately 1-50 pmol).