Microfabricated Liquid Junction Capillary Electrophoresis-Mass Spectrometry Interface.

Coupling of capillary electrophoresis (CE) with mass spectrometry (MS) represents a powerful combination for performing rapid, efficient, and sensitive analysis of a variety of compounds. Here we describe a construction, operation, and application of a microfabricated liquid junction CE-MS interface. The interface is designed as a microfabricated unit with an integrated liquid junction and electrospray tip made from polyimide, which is positioned in a plastic connection block securing the separation CE capillary and attachable to the CE instrument. The application was demonstrated by CE-MS analysis of dextran oligomers labeled by (2-aminoethyl)trimethylammonium (AETMA) salt.

Separation of labeled isomeric oligosaccharides by hydrophilic interaction liquid chromatography - the role of organic solvent in manipulating separation selectivity of the amide stationary phase.

The advantages of using mixtures of organic solvents for the separation of labeled oligosaccharides on the amide stationary phase under hydrophilic interaction liquid chromatography conditions are presented. The effect of the type of buffer as well as solvent or their mixtures on retention of uracil, saccharide labeling reagents (2-aminobenzoic acid, 2-aminobenzamide, ethyl 4-aminobenzoate, procainamide), and corresponding labeled saccharides were evaluated. The successful isocratic separation of labeled isomeric trisaccharides (maltotriose, panose, and isomaltotriose) was achieved in the mobile phase consisting of a 90% (v/v) mixture of organic solvents (methanol/acetonitrile 60:40) and 10% (v/v) 30 mM ammonium formate, pH 3.3. Changing the volume ratio between methanol/acetonitrile from 60:40 to 50:50 (v/v) allowed to obtain the separation of di-, tri-, and tetrasaccharides labeled by ethyl 4-aminobenzoate in less than 10.5 min.

Characterization of multi-cationic aminopyrene-based tag for oligosaccharide labeling by capillary electrophoresis with laser-induced fluorescence detection.

In this work, we characterize a previously synthesized multi-cationic aminopyrene-based labeling tag for oligosaccharide analysis by capillary electrophoresis with laser-induced fluorescence detection (CE/LIF). The fluorescent tag, 4,4',4''-(8-aminopyrene-1,3,6-trisulfonyl)tris(1-methylpiperazine) (APTMP), was characterized by reaction with standard maltooligosaccharides and the labeling parameters such as fluorescent tag concentration, labeling temperature, and time as well as influence of a reducing agent and its solvent were investigated in terms of labeling efficiency. The nanomolar limit of detection of CE/LIF analysis of APTMP labeled maltopentaose was determined. However, significant amount of the oligosaccharides was reduced to alditols, which negatively affects the yield and rate of the labeling reaction. Under optimized conditions, a highly reproducible labeling by multi-cationic APTMP was obtained; however, the most commonly used labeling by multi-anionic 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) is superior compared to APTMP labeling. Lower reactivity of APTMP compared to APTS can be explained by the loss of nucleophilicity induced by substitution of the sulfonate groups with more electron-withdrawing aminosulfonyl ones. On contrary, APTMP is still a promising tag for oligosaccharide labeling followed by CE-MS in a positive ion mode, which is considered to be more sensitive than MS detection of APTS in a negative ion mode.

Separation based characterization methods for the N-glycosylation analysis of prostate-specific antigen.

Prostate cancer has the highest malignancy rate diagnosed in men worldwide. Albeit, the gold standard serum prostate-specific antigen (PSA) assays reduced the mortality rate of the disease, the number of false positive diagnoses steeply increased. Therefore, there is an urgent need for complementary biomarkers to enhance the specificity and selectivity of current diagnostic methods. Information about PSA glycosylation can help to fulfill this gap as alterations of its carbohydrate moieties due to cancerous transformation may represent additional markers to distinguish malignant from benign tumors. However, development of suitable methods and instrumentations to investigate the N-glycosylation profile of PSA represents a challenge. In this paper, we critically review the current bioanalytical trends and strategies in the field of PSA glycobiomarker research focusing on separation based characterization methods.

No-additive salting-out liquid-liquid extraction-A tool for purification of positively charged compounds from highly salted reaction mixtures.

A novel and original application of salting-out assisted liquid-liquid extraction is presented. This technique was used to purify the final reaction products (quaternary ammonium salts) from unreacted components and by-products present in multiple excesses. The partition of two structurally related compounds as (2-aminoethyl)trimethylammonium salt (a labeling reagent) and a derivative of [2-(imidazoline-1-yl)ethyl]trimethylammonium salt (a final reaction product of N-acetylglucosamine labeling by (2-aminoethyl)trimethylammonium salt) between acetonitrile-rich and water-rich layers was monitored by hydrophilic interaction chromatography with electrospray ionization mass spectrometry. Despite the poor solubility of both highly polar substances in solutions containing a high concentration of acetonitrile, the main portion of the labeling reagent (72%) can be removed from the crude reaction mixture in the first extraction step using 95% acetonitrile/5% water as an extraction solvent. The purified final reaction product contained only 2% of the labeling reagent, and it was suitable for analysis by direct infusion mass spectrometry to confirm its identity. The capability of the suggested purification protocol to process small-volume highly salted reaction mixtures was also proven by analysis of saccharide mixture containing glucose, maltose, and maltotriose labeled by the positively charged tag.

Comparison of oligosaccharide labeling employing reductive amination and hydrazone formation chemistries.

In this work, we compare labeling by two negatively charged fluorescent labels, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and 8-(2-hydrazino-2-oxoethoxy)pyrene-1,3,6-trisulfonic acid (Cascade Blue hydrazide [CBH]). Effectiveness of the labeling chemistries were investigated by 4-hydroxybenzaldehyde and maltoheptaose followed by LC/UV-MS and CE/LIF analysis, respectively. The reaction yield of APTS labeling was determined to be only ∼10%. This is due to reduction of almost 90% of the analyte by sodium cyanoborohydride to alcohol, which cannot be further labeled via reductive amination. However, the CBH labeling provides ∼90% reaction yield based on the LC/UV-MS measurements. The significantly higher labeling yield was also confirmed by CE/LIF measurements. Finally, the more effective hydrazone formation technique of CBH was characterized and applied for N-linked glycan analysis by CE/LIF.

Multi-cationic aminopyrene-based labeling tags for oligosaccharide analysis by capillary electrophoresis-mass spectrometry.

In this work, new multi-cationic aminopyrene-based labeling tags were designed and synthesized for oligosaccharide analysis by capillary electrophoresis-mass spectrometry (CE-MS). The starting compound, 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt, was modified in order to form a sulfonamide derivative having three tertiary amines in the label structure. The sulfonamide derivative was further methylated to generate three permanently charged quaternary ammonium moieties on the label. The synthesized labels were characterized by NMR, IR, UV/Vis, fluorescence spectroscopy and mass spectrometry. Furthermore, the labels were applied for maltooligosaccharide standards as well as N-linked glycans labeling via reductive amination and followed by CE-MS analysis. The CE-MS analysis of maltooligosaccharides labeled by these newly synthesized labels provided the sub-micromolar limit of detection based on the extracted ion electropherogram signals.

Microfabricated liquid junction hybrid capillary electrophoresis-mass spectrometry interface for fully automated operation.

One of the challenging instrumental aspects in coupling an automated CE instrument with ESI mass spectrometry (CE-MS) is finding the balance between the stability, reproducibility and sensitivity of the analysis and compatibility with the standard CE instrumentation. Here, we present a development of a new liquid junction based electrospray interface for automated CE-MS, with a focus on the technical design followed by computer modeling of transport conditions as well as characterization of basic performance of the interface. This hybrid arrangement designed as a microfabricated unit attachable to the automated CE instrument allows using of a wide range of separation capillaries with respect to their diameter, length or internal coating (e.g., for suppressed electroosmotic flow). Different compositions of the ESI liquid and background electrolyte solutions can be used if needed. The microfabricated part, prepared by laser machining from polyimide, includes a self-aligning liquid junction, a short transport channel, and a pointed sprayer for the electrospray ionization. This microfabricated part is positioned in a plastic connection block securing the separation capillary and flushing ports. Transport conditions were modelled using computer simulation and the real life performance of the interface was compared to that of a commercial sheath liquid interface. The basic performance of the interface was demonstrated by separations of peptides, proteins, and oligosaccharides.

Investigation of a side reaction occurring during N-linked glycan labeling by cationic tags.

Capillary electrophoresis-mass spectrometry was applied for the analysis of oligosaccharides and N-linked glycans with an attached charge label facilitating electrophoretic migration and electrospray ionization efficiency. Several different labeling strategies have been tested with different tags and tagging reactions including reductive amination and hydrazone formation. However, a formation of multiple labeled N-linked glycans was observed by CE-MS in a positive ion mode when positively charged labels such as aliphatic amines containing a quaternary ammonium group were attached to N-linked glycans by reductive amination. A reaction mechanism explaining a side reaction occurring during the labeling and the multiple product formation was proposed and confirmed by using isotopically labeled N-acetylglucosamine. Finally, it was confirmed that derivatization of sugars via a hydrazone formation can be a simpler method with a high reaction yield suitable for high sensitive CE-ESI/MS analyses of N-linked glycans.

Multi-charged labeling of oligosaccharides and N-linked glycans by hexahistidine-based tags for capillary electrophoresis-mass spectrometry analysis.

The labeling by amino acids and peptides was investigated for sensitive and fast analyses of oligosaccharides and N-linked glycans by capillary electrophoresis-mass spectrometry (CE-MS). Peptide tags with a various number of histidine residues were tested for maltooligosaccharide labeling in order to investigate the effect of the size of labels and a number of charges on CE-MS analysis. Nevertheless, the reductive amination labeling of N-linked glycans by a hexahistidine tag resulted in a multiple products formation, therefore a peptide tag was modified by hydrazine functionality in order to perform labeling by hydrazone formation chemistry. This labeling approach significantly improved sensitivity with LOD of labeled maltopentaose determined to be 40 nmol/L and also significantly reduced separation time of neutral maltooligosaccharides and N-linked glycans released from bovine ribonuclease B. Furthermore, the labeling by this multi-cationic peptide hydrazine tag also allowed performing analysis of acidic glycans by CE-MS in a positive ion mode as demonstrated by separation of sialylated N-linked glycans released from bovine fetuin.

Bridged polysilsesquioxane-based wide-bore monolithic capillary columns for hydrophilic interaction chromatography.

The synthesis and characterization of large-bore silica-based monolithic capillary columns (0.32mm×150mm) are presented. Columns were prepared by acidic hydrolysis of a mixture containing tetramethoxysilane (TMOS) and 1,2-bis(trimethoxysilyl)ethane (BTME) in different molar ratios in the presence of polyethylene glycol and urea. The monoliths were modified by zwitterionic monomer [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. Prepared stationary phases were evaluated by scanning electron microscopy and chromatographic separation of nucleobases and their derivatives in the HILIC mode. The best chromatographic results were obtained with the column prepared from the reaction mixture containing BTME and TMOS in a 1:4 molar ratio. The permeability of such column reached 1.68×10-14m2 and the efficiency, expressed as a height equivalent of the theoretical plate, did not exceed 10.5µm for the tested compounds. The columns were successfully applied to HILIC separation of native and labeled oligosaccharides and glycans released from bovine ribonuclease B and human immunoglobulin G.

Fraction transfer process in on-line comprehensive two-dimensional liquid-phase separations.

Two-dimensional liquid-phase separations have gained increasing attention for their ability to separate complex sample mixtures. Among the experimental setups used, an on-line approach is preferred to reduce the probability of sample contamination, for easier automation and high-sample throughput. The interfacing of the separation techniques in the on-line mode brings additional demands on proper optimization of the two-dimensional system. In this review, the possibilities of the on-line coupling of liquid chromatography and liquid chromatography with capillary electrophoresis in two-dimensional systems are discussed. Special attention is paid to the fraction transfer process, which includes an overview of interfaces and experimental setups applied, the compatibility issues of separation systems, and instrumental parameters. The benefits and drawbacks of using electromigration separations in combination with liquid chromatography are presented as well.

Comparison of isocratic retention models for hydrophilic interaction liquid chromatographic separation of native and fluorescently labeled oligosaccharides.

In this work, we have investigated retention of maltooligosaccharides and their fluorescent derivatives in hydrophilic interaction liquid chromatography using four different stationary phases. The non-derivatized maltooligosaccharides (maltose to maltoheptaose) and their derivatives with 2-aminobenzoic acid, 2-aminobenzamide, 2-aminopyridine and 8-aminonaphthalene-1,3,6-trisulfonic acid were analyzed on silica gel, aminopropyl silica, amide (carbamoyl-bonded silica) and ZIC-HILIC zwitterionic sulfobetain bonded phase. The partitioning of the analytes between the bulk mobile phase and adsorbed water-rich layer, polar and ionic interactions of analytes with stationary phase have been evaluated and compared. The effects of the mobile phase additives (0.1% (v/v) of acetic acid and ammonium acetate in concentration range 5-30 mmol L(-1)) on retention were described. The suitability of different models for prediction of retention was tested including linear solvent strength model, quadratic model, mixed-mode model, and empirical Neue-Kuss model. The mixed-mode model was extended to the parameter describing the contribution of monomeric glucose unit to the retention of non-derivatized and derivatized maltooligosaccharides, which was used for evaluation of contribution of both, oligosaccharide backbone and end-group to retention.

Enzymatic removal of N-glycans by PNGase F coated magnetic microparticles.

Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.

Self-aligning subatmospheric hybrid liquid junction electrospray interface for capillary electrophoresis.

We report a construction of a self-aligning subatmospheric hybrid liquid junction electrospray interface for CE eliminating the need for manual adjustment by guiding the capillaries in a microfabricated liquid junction glass chip at a defined angle. Both the ESI and separation capillaries are inserted into the microfabricated part until their ends touch. The distance between the capillary openings is defined by the angle between the capillaries. The microfabricated part contains channels for placement of the capillaries and connection of the external electrode reservoirs. It was fabricated using standard photolithographic/wet chemical etching techniques followed by thermal bonding. The liquid junction is connected to a subatmospheric electrospray chamber inducing the flow inside the ESI needle and helping the ion transport via aerodynamic focusing.

Phosphopeptide enrichment with inorganic nanofibers prepared by forcespinning technology.

Efficient selective sample enrichment is often a key procedure in protocols for analyses of complex samples. This applies not only to trace sample components but also to species with weak detection response. For example enrichment of phosphopeptides using selective affinity techniques prior to mass spectrometry analysis is necessary to increase detection sensitivity of phosphopeptides from highly complex peptide mixtures. In this work we have developed inorganic nanofibrous materials based on titanium or zirconium dioxides for selective and efficient enrichment of phosphopeptides for MALDI/MS detection. In comparison to the common bead based materials the presented nanofibrous materials exhibit much higher permeability allowing their use not only for batch mode or packed in the column operation, but also in the pipette tip format without the need for high pressure. Both the methods of preparation and characterization of the resulting materials are presented.

Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis.

There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity. The structural diversity of these attached glycans can be manifested in altered monosaccharide composition and linkages/positions among the monosaccharide building blocks. In addition, as more and more biosimilar products hit the market, understanding the effects of their glycosylation modification has become a recent target in efficacy and safety issues. To ensure consistent quality of these products, glycosylation profiles have to be monitored and controlled in all steps of the manufacturing process, i.e., from clone selection to lot release. In this paper, we describe some of the recently introduced and commonly used sample preparation techniques for capillary electrophoresis (CE)-based profiling and structural elucidation of N-glycans. The presented protocols include protein A affinity partitioning of monoclonal antibodies (mAbs), enzymatic release of the N-linked glycans, labeling of the liberated carbohydrates, reaction mixture purification techniques to remove the excess labeling reagent, and high-resolution and rapid capillary electrophoresis-laser-induced fluorescence (CE-LIF)-based profiling of the labeled and purified N-glycans.

Macroporous cryogel based spin column with immobilized concanavalin A for isolation of glycoproteins.

In this work we have developed a hydrophilic poly(hydroxyethyl methacrylate-co-poly(ethylene glycol) diacrylate) cryogel placed in the centrifugal filter device. The composition of the polymerization mixture as well as the polymerization conditions were optimized in order to prepare a material with bimodal pore size distribution with 20-50 µm flow through macropores and submicrometer pores in the polymer walls. The optimized, mechanically stable, highly porous, material was used for spin column lectin chromatography. The surface of the monolithic scaffold was activated by epichlorohydrin and used for immobilization of concanavalin A to provide the affinity supports for selective isolation of glycoproteins containing high mannose glycan structures. The performance of the developed lectin modified cryogels was evaluated by analyses of glycoprotein mixtures. The efficiency and selectivity of the affinity supports were confirmed by MALDI-MS analysis.

Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis.

In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors.

Nanoparticle-modified monolithic pipette tips for phosphopeptide enrichment.

We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 µL monolithic beds were prepared by UV-initiated polymerization in 200 µL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and ß-casein digests followed by off-line MALDI/MS analysis.