Reliable Reagent Research Articles

Multifunctional Sensors for Successive Detection of Endogenous ONOO- and Mitochondrial Viscosity: Discriminating Normal to Cancer Models.

Diagnosing cancer in its early stages can play an important role in prolonging the lifespan of patients, which demands the use of powerful tools to detect biomarkers accurately. However, since most fluorescent probes described for cancer diagnosis are limited to recognizing a single molecule, achieving the high accuracy criteria remains difficult. Here, sensor 1 is constructed for the sequential detection of D, ONOO-, and viscosity. Initially, sensor 1 detected D and underwent an intramolecular charge transfer mechanism, resulting in the formation of 2 and fluorescence quenching at 587 nm. Subsequently, the intermediate (2) monitored ONOO- and reproduced sensor 1 reversibly with fluorescence enhancement at 496 nm, showing concentration-related quantitative analysis. Similar sensing processes were observed in monitoring ONOO- and viscosity by synthetically developed sensor 2. The proposed mechanisms of sensors 1 and 2 are verified through various characterizations (1H NMR, HR-MS, and HPLC) and DFT calculations. Investigations on endogenous ONOO- and mitochondrial viscosity in cancer (HeLa) and normal (NCM460) cells were conducted to distinguish cancerous cells from normal cells. We anticipated that sensor 2 could effectively serve as a reliable bioanalytical reagent for cancer diagnosis at an earlier stage through sequential detection of two cancer markers, ONOO- and mitochondrial viscosity, in living cells. Importantly, sensor 2 has been employed for imaging ONOO- in normal and liver injury mouse models and tissues, achieving outstanding results.

Chemo- and regioselective demethylation of 2-bromo-α-resorcylic acid derivatives using alkylthiolate salts

We report here the chemo- and regioselective demethylation of 3,5-dimethoxy-2-bromo-α-resorcylic acid esters by a thiolate nucleophilic displacement reaction. Optimized conditions facilitate yields up to 93% for o-bromo selective demethylation of diverse ester derivatives of dimethoxy 2-bromo-α-resorcylic acid. Our results highlight a new, efficient, and reliable demethylation reagent, as well as a useful steric bias directing strategy.

Bioinspired Fluorine Labeling for 19F NMR-Based Plasma Amine Profiling.

Metabolite profiling serves as a powerful tool that advances our understanding of biological systems, disease mechanisms, and environmental interactions. In this study, we present an approach employing 19F-nuclear magnetic resonance (19F NMR) spectroscopy for plasma amine profiling. This method utilizes a highly efficient and reliable fluorine-labeling reagent, 3,5-difluorosalicylaldehyde, which effectively emulates pyridoxal phosphate, facilitating the formation of Schiff base compounds with primary amines. The fluorine labeling allows for distinct resolution of 19F NMR signals from amine mixtures, leading to precise identification and quantification of amine metabolites in human plasma. This advancement offers valuable tools for furthering metabolomics research.

Recombinant T7 RNA polymerase production using ClearColi BL21(DE3) and animal-free media for in vitro transcription.

In vitro transcription (IVT) using T7 RNA polymerase (RNAP) is integral to RNA research, yet producing this enzyme in E. coli presents challenges regarding endotoxins and animal-sourced toxins. This study demonstrates the viable production and characterization of T7 RNAP using ClearColi BL21(DE3) (an endotoxin-free E. coli strain) and animal-free media. Compared to BL21(DE3) with animal-free medium, soluble T7 RNAP expression is ~50% lower in ClearColi BL21(DE3). Optimal soluble T7 RNAP expression in flask fermentation is achieved through the design of experiments (DoE). Specification and functional testing showed that the endotoxin-free T7 RNAP has comparable activity to conventional T7 RNAP. After Ni-NTA purification, endotoxin levels were approximately 109-fold lower than T7 RNAP from BL21(DE3) with animal-free medium. Furthermore, a full factorial DoE created an optimal IVT system that maximized mRNA yield from the endotoxin-free and animal-free T7 RNAP. This work addresses critical challenges in recombinant T7 RNAP production through innovative host and medium combinations, avoided endotoxin risks and animal-derived toxins. Together with an optimized IVT reaction system, this study represents a significant advance for safe and reliable reagent manufacturing and RNA therapeutics. KEY POINTS: • Optimized IVT system maximizes mRNA yields, enabling the synthesis of long RNAs. • Novel production method yields endotoxin-free and animal-free T7 RNAP. • The T7 RNAP has equivalent specifications and function to conventional T7 RNAP.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis

Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryptococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-defining condition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test (LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. The aim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supply the NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performance and its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY) (Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLIS and LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictive values of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effective reagent, especially useful in countries where the commercial kit is not generally available and must be obtained at a high cost. National production of reagents is the best choice for a reliable access to the rapid diagnosis of CM in Argentina.

A Water-Stable Terbium(III)-Organic Framework as a Chemosensor for Inorganic Ions, Nitro-Containing Compounds and Antibiotics in Aqueous Solutions.

Effective detection of organic/inorganic pollutants, such as antibiotics, nitro-compounds, excessive Fe3+ and MnO4 - , is crucial for human health and environmental protection. Here, a new terbium(III)-organic framework, namely [Tb(TATAB)(H2 O)]⋅2H2 O (Tb-MOF, H3 TATAB=4,4',4''-s-triazine-1,3,5-triyltri-m-aminobenzoic acid), was assembled and characterized. The Tb-MOF exhibits a water-stable 3D bnn framework. Due to the existence of competitive absorption, Tb-MOF has a high selectivity for detecting Fe3+ , MnO4 - , 4-nirophenol and nitroimidazole (ronidazole, metronidazole, dimetridazole, ornidazole) in aqueous through luminescent quenching. The results suggest that Tb-MOF is a simple and reliable reagent with multiple sensor responses in practical applications. To the best of our knowledge, this work represents the first TbIII -based MOF as an efficient fluorescent sensor for detecting metal ions, inorganic anions, nitro-compounds, and antibiotics simultaneously.

I2-Mediated Iodobenzannulation of Yne-Allenones toward 1-Naphthols and Their Synthetic Application.

A new I2-mediated iodobenzannulation of yne-allenones has been established, enabling breaking/rearranging of C≡C bonds to selectively access 4-iodonaphthalen-1-ols with generally good yields. The resulting 4-iodonaphthalen-1-ols could serve as a new and reliable coupling reagent, which further reacted with H2O under the oxygen conditions to generate unexpected 1,2-carbonyls with good yields through Pd-catalyzed deiodinated carbonylation, whereas employment of benzene-1,2-diamine as the nucleophile rendered 3-(quinoxalin-2-yl)naphthalen-1-ols through Pd-catalyzed [4 + 2] heterocyclization. On the basis of the controlled experiments, the mechanism for forming 1,2-carbonyls was proposed, including an oxidative addition, 1,3-palladium migration, reductive elimination, and oxidative dehydrogenation sequence.

Affinity purification of the avidin protein family, based on crystal structures of avidin-HABA complexes

Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10−16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10−6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

Experiment to Enhance Catalytic Activity of α-FeOOH in Heterogeneous UV-Fenton System by Addition of Oxalate

α-FeOOH was prepared by a symmetrical precipitation method and characterized by XRD, FT-IR, SEM, BET, and EDS techniques. The oxalate enhanced experiment for the catalytic activity of α-FeOOH during the degradation of Orange Ⅱ by the heterogeneous UV-Fenton process was evaluated. The results showed that the rate of Orange Ⅱ degradation in this heterogeneous UV-Fenton system could increase significantly with oxalate as an enhancing reagent, with the peak values of enhanced efficiency reaching 116.9% when the concentration of additional oxalate was 0.4 mmol·L-1. Under this condition, more than 99% of Orange Ⅱ (0.2 mmol·L-1) was degraded in 15 min. However, under the same condition, the inhibition ratios of malonate, acetate, EDTA and citrate were 5.2%, 8.1%, 23.2%, and 25.7%, respectively. Compared with the basic system, the enhanced efficiency of the mineralization rate constant can reach 69.9% when the concentration of additional oxalate was 0.4 mmol·L-1. As a result, the time required for mineralization could be reduced greatly. In the heterogeneous UV-Fenton system, the additional oxalate could provide a new photo-reduction pathway for Fe3+ to Fe2+, enhancing the ratio of the homogeneous UV-Fenton process by increasing the Fe leaching of α-FeOOH and the concentration of hydroxyl radicals, leading to higher degradation efficiency of Orange Ⅱ. As a result, an enhancement effect of 101.5% could be obtained even after three cycles. The iron ions leached from catalysts could be re-adsorbed after treatment, avoiding the loss of active species from catalysts and additional pollution caused by iron ions. The results indicated that additional oxalate was a stable and reliable enhancing reagent on the catalytic activity of α-FeOOH in the heterogeneous UV-Fenton system.

Genetic fusion protein 3×STa-ovalbumin is an effective coating antigen in ELISA to titrate anti-STa antibodies.

Heat-stable toxin type I (STa)-ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti-STa antibodies for ETEC vaccine candidates. STa-ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti-STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa-ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti-STa antibody titration ELISA. Data showed fusion protein 3×STa-ovalbumin was effectively expressed and extracted, and anti-STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa-ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti-STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa-ovalbumin is an effective ELISA coating antigen for anti-STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.

False high level in total bilirubin estimation in nonicteric serum

In day to day clinical biochemistry laboratory practices, occasionally abnormal levels of individual parameters are noted. These reports cannot be explained immediately with certainty always. Inquisitiveness with in-depth analysis might reveal the possible cause sometimes. To find out the possible cause of false elevation in total bilirubin estimation in nonicteric serum from one patient was the objective. Routine estimation of total bilirubin in serum/plasma by the decade old practice of using very reliable and stable reagent kit from Roche in the biochemistry laboratory of a general hospital revealed a falsely very high value of total bilirubin in a patient’s blood sample for the first time. Subsequently, the same sample was subjected to estimation of total bilirubin by DiaSys and Randox reagents along with simultaneous re-estimation by Roche reagents in next three days. Repeated estimations by Roche reagents showed falsely very high level of total bilirubin. However, estimation by DiaSys and Randox reagents showed acceptable normal levels as per visual estimation. There was interference, most probably due to paraprotein in the estimation of total bilirubin by Roche reagents but not by DiaSys and Randox reagents in that particular sample.Keywords: False, high, positive, total bilirubin, nonicteric, serum.

Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels.

Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, β-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, β-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. β-Glucosidase, β-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.

Generation of Pax1/PAX1-Specific Monoclonal Antibodies.

Pax genes encode an evolutionary conserved group of transcription factors with multiple roles during embryonic development and for cell type specification in normal and malignant tissues of the adult organism. In mice, Pax1 is required for the formation of specific skeletal structures as well as for the development of a fully functional thymus. In humans, the PAX1 locus has been linked to otofaciocervical syndrome, idiopathic scoliosis, and to a higher susceptibility for androgenic alopecia. In addition, the methylation status of PAX1 has recently emerged as a sensitive marker for predictive screening of cervical cancer. To provide a reagent for reproducible detection of Pax1 expression, we have generated rat monoclonal antibodies (MAbs) against the murine Pax1 protein. MAbs of one clone (clone 5A2) specifically detect mouse Pax1 protein in Western blot analyses. Moreover, the anti-Pax1 MAbs cross-react with human PAX1 protein and are applicable in immunohistochemical detection procedures using paraformaldehyde/formalin-fixed tissues embedded in paraffin. The anti-Pax1 MAbs provide a reliable reagent for reproducible Pax1/PAX1 protein expression analyses and, therefore, may help to improve diagnostic protocols in clinical settings involving deregulated expression of Pax1/PAX1.

Quantitation of Lan antigen in Lan+, Lan+(w) and Lan- phenotypes.

The Lan antigen (LAN1) is a clinically significant, high-frequency red blood cell (RBC) antigen. Lan was originally described in 1961 and assigned to the LAN blood group system in 20121,2. Lan is currently the only antigen within the LAN blood group system and Lan+, Lan−, Lan+w and Lan+w/− phenotypes have been defined1–4. The Lan− phenotype is very rare worldwide with a frequency of less than 1% in all populations tested to date2,5,6. For example, a Japanese screening study identified 14 Lan− individuals among 713,384 blood donors, giving a frequency of 0.002%2. Lan− individuals are usually identified due to the detection of anti-Lan during investigations into haemolytic disease of the foetus and newborn7,8 or when serological testing is performed to find compatible blood units for a patient5,9. Transfusion support for Lan− individuals is highly challenging due to the scarcity of both compatible blood and suitable anti-Lan reagents for screening for compatible blood. The first monoclonal anti-Lan antibody (OSK43) was produced in 2012 by Helias et al. and this antibody has proven to be of huge benefit as a reliable reagent for screening for Lan− individuals2. The carrier of the Lan antigen is the ABCB6 protein encoded by the ABCB6 gene at chromosome 2q36, containing 19 exons2. Numerous genetic variants have been identified as encoding for the Lan− phenotype2,4,10–12. The initial sequencing study identified ten novel alleles, including frameshift, nonsense and splice-site mutations, within 12 unrelated Lan− individuals and found that each individual was heterozygous for two ABCB6 null alleles2. Subsequently, Saison et al. characterised yet another ABCB6 null allele, a single nucleotide variant missense mutation (c.574C>T)10. To date this single nucleotide variant is the most common mutation causing the Lan− phenotype4,10. It has been suggested that the frequency of ABCB6 null alleles differs between populations as sequencing of 27 Japanese Lan− individuals identified a further ten novel alleles and revealed that none of the donors carried the c.574C>T variant12. The Lan+w phenotype has been described in individuals heterozygous for an ABCB6 null allele and a wild-type allele, and these individuals typically express 50% of the normal level of Lan antigen4,10. The fourth Lan phenotype is referred to as Lan+w/− as cells serologically type as either Lan+w or Lan− depending on the anti-Lan utilised3. A comprehensive study involving serological and molecular characterisation of Lan phenotypes was recently performed by Reid et al. and for the first time alleles encoding Lan+w/− phenotypes were defined4. Lan+w/− individuals are heterozygous for an ABCB6 null allele and a variant allele encoding for weakened Lan expression. Antigens can be quantified by flow cytometry by converting the fluorescent intensity of staining into an antibody-binding capacity (ABC), relating to the number of monoclonal antibody molecules bound to a cell. This is performed utilising populations of calibrated microspheres coated with defined amounts of a capture antibody, to create a calibration curve used for quantitation of ABC. The usefulness of determining ABC values has been well established, particularly for lymphocyte antigens13. In previous studies, calibrated microsphere-based assays have been validated to aid in the diagnosis, prognosis, and treatment monitoring of diseases including chronic lymphocytic leukaemia14,15 and human immunodeficiency virus infection16. In the context of RBCs, glycophorin A and RhD antigen expression has been investigated utilising traditional flow cytometric approaches17,18 and, more recently, calibrated microspheres19,20. The variability of Lan antigen expression has never been quantitatively investigated. In this study, we investigated the expression of Lan antigen by developing a novel indirect staining protocol capable of quantitating the number of Lan sites per RBC in samples reported as Lan+, Lan+w, and Lan−.

Cationic amino acid based lipids as effective nonviral gene delivery vectors for primary cultured neurons.

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA-), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.

The Dimethoxyphenylbenzyl Protecting Group: An Alternative to the p-Methoxybenzyl Group for Protection of Carbohydrates

A reliable reagent system for the cleavage of 4-(3,4-dimethoxyphenyl)benzyl (DMPBn) ethers under acidic conditions has been established. Treatment of DMPBn-protected mono- and pseudodisaccharides with TFA in anhydrous CH2Cl2 and 3,4-(methylenedioxy)toluene as a cation scavenger resulted in the selective cleavage of the DMPBn ether giving the corresponding deprotected products in moderate to high yields. Examples are reported which show that allyl, benzyl, and p-bromobenzyl ethers, esters, and glycosidic linkages are stable to these reaction conditions. The selective cleavage of allyl, p-bromobenzyl, and PMB ethers in protected carbohydrates containing DMPBn ethers are also demonstrated. This work establishes the 4-(3,4-dimethoxyphenyl)benzyl ether as an effective and robust alternative to p-methoxybenzyl as a protecting group for alcohols.

Evaluation of O6-methylguanine-DNA methyltransferase by immunohistochemistry: Best clinical and research practices

O6-Methyguanine-DNA methyltransferase (MGMT) repairs DNA damage and acts as a tumor suppressor in normal cells by preventing DNA mutations. Several antibodies against MGMT are used for immunohistochemical assessment of this marker and no universal standard is adopted. We evaluated the immunohistochemical expression of MGMT with 5 commercially available primary antibodies in 59 invasive breast carcinomas. A tissue microarray was constructed using 59 invasive breast carcinoma samples. Five primary antibodies against MGMT were used for immunohistochemistry, including clones MT3.1, SPM287, and MT23.2. Heat-induced antigen retrieval and polymer-based immunohistochemistry were performed. Stains were analyzed by microscopy and automated digital slide technology. qRT-PCR was performed for all tumors. Clone SPM287 had the highest sensitivity ( p < 0.001), and clone MT3.1 had the lowest sensitivity ( p < 0.001). Clone MT23.2 generated higher levels of cytoplasmic staining, which was not observed with the other antibodies ( p < 0.001). Fifty-six samples (94.9%) showed hypoexpression of MGMT compared with normal breast, as measured by qRT-PCR ( p < 0.001). Only clone SPM287 correlated significantly with the qRT-PCR results ( p = 0.027). Antibody clone SPM287 is the most sensitive and specific antibody for the immunohistochemical evaluation of MGMT, rendering it a reliable and effective reagent for research and clinical practice in breast cancer.

Understanding the enthalpic and entropic contributions to solvent effects on osmium tetroxide complexation with pyridine

Solvent effects on the complex formation of osmium tetroxide (which is the most reliable reagent available for the cis-hydroxylation of alkenes) and pyridine were investigated using spectrophotometric techniques in different solvents (benzene, chloroform, acetone, acetonitrile, anisole, tetrahydrofuran, nitrobenzene and nitromethane). The enthalpy and entropy exhibit opposing effects on stability constant with the change in solvent. In these solvents, the entropy and enthalpy of the reaction are found to be mainly influenced by the donor number (DN) of the solvents.

NaIO 4–KI–NaN 3 as a new reagent system for C–H functionalization in hydrocarbons

The NaIO 4–KI–NaN 3 combination has been found to be an efficient, reliable, and inexpensive reagent system for mono- and 1,2-difunctionalization of hydrocarbons via C–H bond activation to afford vicinal azido- and acetoxy iodinations of cyclic hydrocarbons.

Determination of Fluorescence-Labeled Asparaginyl-Oligosaccharide in Glycoprotein by Reversed-Phase Ultraperformance Liquid Chromatography with Electrospray Ionization Time-of-Flight Mass Spectrometry

Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.