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.
- Klíčová slova
- CE-MS interface, Capillary electrophoresis, Liquid junction, Mass spectrometry,
- MeSH
- elektroforéza kapilární * metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací * MeSH
- hmotnostní spektrometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Klíčová slova
- HILIC, Mass spectrometry, Oligosaccharide, Saccharide/glycan labeling, Selectivity,
- MeSH
- acetonitrily chemie MeSH
- amidy chemie MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- chromatografie kapalinová * MeSH
- formiáty chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- isomerie MeSH
- oligosacharidy izolace a purifikace MeSH
- ortoaminobenzoáty chemie MeSH
- rozpouštědla chemie MeSH
- sacharidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetonitrile MeSH Prohlížeč
- acetonitrily MeSH
- amidy MeSH
- anthranilamide MeSH Prohlížeč
- anthranilic acid MeSH Prohlížeč
- formiáty MeSH
- formic acid MeSH Prohlížeč
- oligosacharidy MeSH
- ortoaminobenzoáty MeSH
- rozpouštědla MeSH
- sacharidy MeSH
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.
- Klíčová slova
- Capillary electrophoresis, Fluorescence, Labeling, Oligosaccharides, Reductive amination,
- MeSH
- elektroforéza kapilární * MeSH
- kationty MeSH
- lasery MeSH
- oligosacharidy MeSH
- pyreny MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kationty MeSH
- oligosacharidy MeSH
- pyreny MeSH
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.
- Klíčová slova
- Biomarkers, Glycans, Glycosylation, Prostate cancer, Prostate-specific antigen,
- MeSH
- biologické markery MeSH
- glykosylace MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádory prostaty * diagnóza MeSH
- prostatický specifický antigen * metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- biologické markery MeSH
- nádorové biomarkery MeSH
- prostatický specifický antigen * MeSH
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.
- Klíčová slova
- quaternary ammonium salts, saccharide labeling, salting-out liquid-liquid extraction, sample preparation,
- Publikační typ
- časopisecké články MeSH
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.
- Klíčová slova
- Hydrazone formation, Labeling, Oligosaccharides, Reductive amination,
- MeSH
- aminace MeSH
- chromatografie kapalinová metody MeSH
- elektroforéza kapilární metody MeSH
- fluorescenční barviva chemie MeSH
- hmotnostní spektrometrie metody MeSH
- hydrazony chemie MeSH
- oligosacharidy analýza chemie MeSH
- pyreny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-aminopyrene-3,6,8-trisulfonic acid MeSH Prohlížeč
- fluorescenční barviva MeSH
- hydrazony MeSH
- oligosacharidy MeSH
- pyreny MeSH
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.
- Klíčová slova
- Capillary electrophoresis, Fluorescence, Glycans, Labeling, Mass spectrometry, Oligosaccharides,
- Publikační typ
- časopisecké články MeSH
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.
- Klíčová slova
- CE-MS interface, Capillary electrophoresis, Liquid junction, Microfabrication,
- MeSH
- chemické modely MeSH
- elektroforéza kapilární přístrojové vybavení MeSH
- hmotnostní spektrometrie přístrojové vybavení MeSH
- laboratorní automatizace MeSH
- mikrofluidní analytické techniky přístrojové vybavení metody MeSH
- proteiny analýza izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny MeSH
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.
- Klíčová slova
- CE-MS, Cationic tags, Labeling, N-Linked glycans,
- MeSH
- aminace MeSH
- aminy chemie MeSH
- elektroforéza kapilární metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- kationty chemie MeSH
- polysacharidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminy MeSH
- kationty MeSH
- polysacharidy MeSH
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.
- Klíčová slova
- Capillary electrophoresis, Hexahistidine, Labeling, Mass spectrometry, N-linked glycans, Oligosaccharides,
- MeSH
- elektroforéza kapilární metody MeSH
- fetuin A chemie MeSH
- histidin chemie MeSH
- hmotnostní spektrometrie metody MeSH
- oligopeptidy chemie MeSH
- oligosacharidy analýza chemie izolace a purifikace MeSH
- polysacharidy analýza chemie izolace a purifikace MeSH
- ribonukleasy chemie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fetuin A MeSH
- His-His-His-His-His-His MeSH Prohlížeč
- histidin MeSH
- oligopeptidy MeSH
- oligosacharidy MeSH
- polysacharidy MeSH
- ribonuclease B MeSH Prohlížeč
- ribonukleasy MeSH
