Acid N-succinimidyl Ester Research Articles

A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N′,N′-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

MS Identification of Blood Plasma Proteins Concentrated on a Photocrosslinker-Modified Surface.

This work demonstrates the use of a modified mica to concentrate proteins, which is required for proteomic profiling of blood plasma by mass spectrometry (MS). The surface of mica substrates, which are routinely used in atomic force microscopy (AFM), was modified with a photocrosslinker to allow "irreversible" binding of proteins via covalent bond formation. This modified substrate was called the AFM chip. This study aimed to determine the role of the surface and crosslinker in the efficient concentration of various types of proteins in plasma over a wide concentration range. The substrate surface was modified with a 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) photocrosslinker, activated by UV irradiation. AFM chips were incubated with plasma samples from a healthy volunteer at various dilution ratios (102X, 104X, and 106X). Control experiments were performed without UV irradiation to evaluate the contribution of physical protein adsorption to the concentration efficiency. AFM imaging confirmed the presence of protein layers on the chip surface after incubation with the samples. MS analysis of different samples indicated that the proteomic profile of the AFM-visualized layers contained common and unique proteins. In the working series of experiments, 228 proteins were identified on the chip surface for all samples, and 21 proteins were not identified in the control series. In the control series, a total of 220 proteins were identified on the chip surface, seven of which were not found in the working series. In plasma samples at various dilution ratios, a total of 146 proteins were identified without the concentration step, while 17 proteins were not detected in the series using AFM chips. The introduction of a concentration step using AFM chips allowed us to identify more proteins than in plasma samples without this step. We found that AFM chips with a modified surface facilitate the efficient concentration of proteins owing to the adsorption factor and the formation of covalent bonds between the proteins and the chip surface. The results of our study can be applied in the development of highly sensitive analytical systems for determining the complete composition of the plasma proteome.

Mass Spectrometric Identification of BSA Covalently Captured onto a Chip for Atomic Force Microscopy.

Mass spectrometry (MS) is one of the main techniques for protein identification. Herein, MS has been employed for the identification of bovine serum albumin (BSA), which was covalently immobilized on the surface of a mica chip intended for investigation by atomic force microscopy (AFM). For the immobilization, two different types of crosslinkers have been used: 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) and dithiobis(succinimidyl propionate) (DSP). According to the data obtained by using an AFM-based molecular detector, the SuccBB crosslinker was more efficient in BSA immobilization than the DSP. The type of crosslinker used for protein capturing has been found to affect the results of MS identification. The results obtained herein can be applied in the development of novel systems intended for the highly sensitive analysis of proteins with molecular detectors.

Application of 4-Diethylaminobenzoic Acid <i>N</i>-Succinimidyl Ester and Its Deuterated Isotopologue as Derivatization Reagents to Quantitative Analysis of γ-Aminobutyric Acid in Serum by LC/ESI-MS/MS

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter having anti-anxiety, stress-reducing and sleep-enhancing effects. The blood GABA concentration has the potential to become a diagnostic index for depression and schizophrenia. In this study, we developed a method for the quantification of GABA in human serum by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using a pair of reagents, 4-diethylaminobenzoic acid N-succinimidyl ester (DEABAS) and its deuterated isotopologue (dDEABAS). The deproteinized serum samples were derivatized with DEABAS, to which the dDEABAS-derivatized standard GABA of known amount was then added. The DEABAS-derivatization enhanced not only the ESI-MS/MS sensitivity but also the LC retention, which enabled the separation of GABA from the interfering isomers. Satisfactory assay precision and accuracy were also acquired by adding dDEABAS-derivatized GABA, which served as the internal standard and worked well for correcting the matrix effect and instrument drift. The developed method had satisfactory applicability to the real sample analysis because it was successfully used to quantify the serum GABA of the healthy subjects.

Inclusion of a coumarin derivative inside the macrocyclic hosts: A spectroscopic, thermodynamic and theoretical investigation

In aqueous medium, the guest-host interaction of 7-(diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester (7-DCCAE) with cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8) are very interesting. The interaction of 7-DCCAE with CB7 and CB8 have been investigated in a systematic way by different types of spectroscopic techniques such as UV–Vis spectroscopy, steady state and time resolved fluorescence emission spectroscopy, 1H-NMR along with thermodynamic approach by isothermal titration calorimetry measurement. We observed that the photophysical properties of the aqueous solution of 7-DCCAE are modulated in the presence of CB7 and CB8. We observed that 1:1 complex is formed between 7-DCCAE with CB7 and CB8. From ITC measurement, we obtained different thermodynamic parameters associated with host-guest complexation. We have drawn up the driving forces involved for the host-guest complexation from ITC measurement. By using density functional theory geometry and frequency of the 7-DCCAE-CBn complexes were optimized. We observed that both the complexation processes were exothermic in nature from ITC as well as from computational study.

A coumarin-based fluorescence resonance energy transfer probe targeting matrix metalloproteinase-2 for the detection of cervical cancer.

Cervical cancer is one of the most common gynecological malignancies worldwide. At present, the methods used for cervical cancer screening have many disadvantages. Matrix metalloproteinase-2 (MMP-2) is low or absent in normal cervical epithelial cells while overexpressed in cervical intraepithelial neoplasia and cervical cancer, which provides the possibility for the detection of cervical cancer based on MMP-2. The development of a strategy with high sensitivity and specificity for cervical cancer detection would be expected to improve the cure rate. Connecting a fluorophore [7-(diethylamino)-2-oxo-2H-chromene-3-succinimidyl ester] with a quencher {4-[4-(dimethylamino)phenylazo]benzoic acid N-succinimidyl ester} via an MMP-2 substrate peptide GPLGVRGKGG, a new coumarin-based fluorescence resonance energy transfer probe targeting MMP-2 was prepared to examine cervical cancer by cell imaging. The cervical cancer cell lines showed green fluorescence with intensities in the order of CaSki>SiHa>C33A>HeLa, which indicated that the cervical cancer cell lines possessed significantly differential expression levels of MMP-2. Quantitative(real‑time) PCR, RT-PCR, and western blot experiments were consistent with such results. The fluorescence detection limit of the probe for MMP-2 was estimated to be 0.05ng/ml. Therefore, this MMP-2 probe potentially provides a sensitive and specific visual method for cervical cancer screening, diagnosis and prognostic judgment.

Modified carbon black as label in a colorimetric on-chip immunoassay for histamine

The present study demonstrates the modification of carbon black and its usage as a detection label in colorimetric immunoassays on a microarray format. We show the feasibility using a binding inhibition assay for histamine, which is a marker of food freshness and quality. The modified carbon nanoparticles (CNPs) were characterized by determining functional surface groups, elemental composition, size, surface charge and dispersion stability to investigate the influence of modification on the assay performance with the overall goal to optimize assay conditions and enhance signal strength. Carbon nanoparticles were oxidized and further modified with silanes with different functional end groups (thiol, epoxy, amine) 3-glycidoxypropyltrimethoxysilane being the most effective modification. Additionally, 1-pyrenebutanoic acid N-succinimidyl ester (PSE) was tested as conjugation agent allowing semi-quantitative read-out by naked eye over a broad histamine range (0-600 μg/mL), similar to just oxidized CNPs. The dispersion stability was enhanced with acrylic polymer Atlox and dispersion agent xanthan. However, no concentration dependent response towards histamine was observed. With oxidized CNPs read-out could be done by naked eye; otherwise an office scanner was needed for detection. Lower zeta potential was recorded after oxidation, resulting in better colloidal stability. Electrostatic stabilization was enough to create stable suspensions, and further modification was not necessary for the antibody coupling and stability in the assay.

Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay.

Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principal regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. Developed as tools to investigate MMP function and as potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. This chapter focuses on the use of enzyme kinetics to characterize inhibitors of MMPs. MMP activity is measured via fluorescence spectroscopy using a fluorogenic substrate that contains a 7-methoxycoumarin-4-acetic acid N-succinimidyl ester (Mca) fluorophore and a 2,4-dinitrophenyl (Dpa) quencher separated by a scissile bond. MMP inhibitor (MMPI) potency can be determined from the reduction in fluorescent intensity when compared to the absence of the inhibitor. This chapter describes a technique to characterize a variety of MMPs through enzyme inhibition assays.

Photophysics of a Coumarin in Different Solvents: Use of Different Solvatochromic Models

This study reported the photophysics of 7-(diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester (7-DCCAE) in different neat solvents of varying polarity using steady-state absorption, fluorescence emission and picosecond time-resolved spectroscopy. In nonpolar solvents, the dye molecule predominantly exists in nonpolar structure and exhibits very low value of nonradiative decay rate constant (k(nr)), demonstrating the emission takes place from S(1) -LE to S(0) ground state. The fluorescence quantum yields, lifetime values of 7-DCCAE in different solvents are rationalized on the basis of intramolecular charge transfer (ICT) followed by twisted intramolecular charge transfer state formation (TICT) as well as specific solute-solvent interactions. Several solvatochromic models (such as Lippert, Dimroth, Kamlet-Taft, Catalán 3P and Catalán 4P models) were used to analyze the solvatochromic shift of 7-DCCAE in different solvents. The different empirical models show that the observed results are better correlate for nonchlorinated solvents and provide statistically significant best-fit result. A comparison was done between comparatively new solvatochromic model (Catalán 3P and Catalán 4P model) with Kamlet-Taft model. The ground state structure of the said molecule was optimized by using Density Functional Theory (DFT).

The photophysics of 7-(diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester in reverse micelle: excitation wavelength dependent dynamics

This paper focuses on the photophysical studies of 7-(diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester (7-DCCAE) in aqueous reverse micelles using steady state absorption, fluorescence emission and picosecond time resolved emission spectroscopy. We used sodium dioctylsulfosuccinate (AOT) as surfactant to prepare reverse micelles. We have observed excitation wavelength dependent photophysics of 7-DCCAE in the reverse micelles. The red edge excitation shift (REES) was found in the reverse micelles, at different w0 values. The REES gradually decreases with increase in size of the water pool. The rotational relaxation time of the dye molecule decreases with increasing the mole ratio of water to surfactant (w0) inside the reverse micelles. The fluorescence anisotropy decays were found biexponential in nature with fast and slow reorientation times, which is explained by two steps and “wobbling-in-cone model”. We have observed the excitation wavelength dependent dynamics of 7-DCCAE in the reverse micelles. We have observed the difference in the photophysics 7-DCCAE with its acid form.

MALDI mass spectrometry‐based sequence analysis of arginine‐containing glycopeptides: improved fragmentation of glycan and peptide chains by modifying arginine residue

This paper describes an improved method for the sequence analysis of Arg-containing glycopeptide by MALDI mass spectrometry (MS). The method uses amino group derivatization (4-aza-6-(2,6-dimethyl-1-piperidinyl)-5-oxohexanoic acid N-succinimidyl ester) and removal (carboxypeptidase B) or modification (peptidylarginine deiminase 4) of the arginine residue of the peptide. The derivatization attaches a basic tertiary amine moiety onto the peptides, and the enzymatic treatment removes or modifies the arginine residue. Fragmentation of the resulting glycopeptide under low-energy collision-induced dissociation yielded a simplified ion series of both the glycan and the peptide that can facilitate their sequencing. The feasibility of the method was studied using α1 -acid glycoprotein-derived N-linked glycopeptides, and glycan and peptide in each glycopeptide were successfully sequenced by MALDI tandem MS (MS/MS).

Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.

Identification of Amino Acid Residues Responsible for the Release of Free Drug from an Antibody–Drug Conjugate Utilizing Lysine–Succinimidyl Ester Chemistry

Unexpected release of free drug was observed during the stability testing of an antibody-drug conjugate (ADC). The ADC was designed to use lysine-succinimidyl ester chemistry to conjugate small molecule cytotoxic drugs to the antibody. To elucidate the mechanism of the release of free drug, a succinimidyl ester analog, 7-hydroxy-4-methyl-3-coumarinylacetic acid N-succinimidyl ester, and a series of peptides were used to probe the potential side reaction of succinimidyl ester with other amino acid residues. Cysteine and tyrosine residues were found to be reactive to succinimidyl ester, and the bonds formed through these reactions were found to be labile. Combining the fluorescent property of the succinimidyl ester analog and mass spectroscopy analysis, specific cysteine and tyrosine residues of the antibody were found to be reactive to succinimidyl ester and the bonds formed through this reaction were susceptible to hydrolysis.

BiPS, a Photocleavable, Isotopically Coded, Fluorescent Cross-linker for Structural Proteomics

Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic acid N-succinimidyl ester (BiPS), that overcomes many of the challenges associated with other cross-linking reagents. BiPS is distinguished from other cross-linkers by a unique combination of properties: it is photocleavable, fluorescent, homobifunctional, amine-reactive, and isotopically coded. As demonstrated with a model protein complex, RNase S, the fluorescent moiety of BiPS allows for sensitive and specific monitoring of the different cross-linking steps, including detection and isolation of cross-linked proteins by gel electrophoresis, determination of in-gel digestion completion, and fluorescence-based separation of cross-linked peptides by HPLC. The isotopic coding of BiPS results in characteristic ion signal "doublets" in mass spectra, thereby permitting ready detection of cross-linker-containing peptides. Under MALDI-MS conditions, partial photocleavage of the cross-linker occurs, releasing the cross-linked peptides. This allows differentiation between dead-end, intra-, and interpeptide cross-links based on losses of specific mass fragments. It also allows the use of the isotope doublets as mass spectrometric "signatures." A software program was developed that permits automatic cross-link identification and assignment of the cross-link type. Furthermore photocleavage of BiPS assists in cross-link identification by allowing separate tandem mass spectrometry sequencing of each peptide comprising the original cross-link. By combining the use of BiPS with MS, we have provided the first direct evidence for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein beta-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.

Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging

To take full advantage of the unique optical properties of quantum dots (QDs) and expedite future near-infrared fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably directed to a specific organ or disease site after systemic administration. Recently, we reported the use of peptide-conjugated QDs for non-invasive NIRF imaging of tumor vasculature markers in small animal models. In this protocol, we describe the detailed procedure for the preparation of such peptide-conjugated QDs using commercially available PEG-coated QDs and arginine-glycine-aspartic acid (RGD) peptides. Conjugation of the thiolated RGD peptide to the QDs was achieved through a heterobifunctional linker, 4-maleimidobutyric acid N-succinimidyl ester. Competitive cell binding assay, using (125)I-echistatin as the radioligand, and live cell staining were carried out to confirm the successful attachment of the RGD peptides to the QD surface before in vivo imaging of tumor-bearing mice. In general, QD conjugation and in vitro validation of the peptide-conjugated QDs can be accomplished within 1-2 d; in vivo imaging will take another 1-2 d depending on the experimental design.

An ELISA for apolipoprotein M reveals a strong correlation to total cholesterol in human plasma

Apolipoprotein M (apoM) is a 188 amino acid, 25 kDa protein belonging to the lipocalin protein superfamily. Although predominantly associated with high density lipoprotein, apoM is found in all major lipoprotein classes. To facilitate clinical studies of apoM, we have developed a sandwich ELISA for the measurement of apoM in human plasma. This method has been used to investigate normal apoM variation and to establish reference values for healthy individuals through the measurement of 598 samples from the Nordic Reference Interval Project Bio-bank and Database (NOBIDA) biobank. For women 18-49 years old, the reference interval for apoM was 0.58-1.18 micromol/l, whereas for women 50+ years and for men, the reference range was 0.61-1.30 micromol/l. Correlation studies of apoM with 26 common clinical chemical analytes from the NOBIDA database revealed a marked positive correlation with plasma total cholesterol (r = 0.52) and LDL and HDL cholesterol (r = 0.43 and 0.36, respectively). There was no statistically significant correlation with HDL/total cholesterol ratio or body mass index. In conclusion, a sandwich ELISA for the measurement of apoM in human plasma shows that apoM concentration is strongly correlated to total cholesterol in healthy individuals.

Aqueous-Soluble, Non-Reversible Lipid Conjugate of Salmon Calcitonin: Synthesis, Characterization and In Vivo Activity

A novel, non-reversible, aqueous-based lipidization strategy with palmitic acid as a model lipid was evaluated for conjugation with salmon calcitonin (sCT). A water-soluble epsilon-maleimido lysine derivative of palmitic acid was synthesized from reaction of palmitic acid N-succinimidyl ester and epsilon-maleimido lysine. The latter was generated from reaction of alpha-Boc-lysine and methylpyrrolecarboxylate, with subsequent deprotection of the Boc group. The palmitic derivative was further conjugated with sCT via a thio-ether bond to produce Mal-sCT in aqueous solution. The identity and purity of Mal-sCT was confirmed by Electrospray Ionisation Mass spectrometry (ESI-MS) and HPLC. Yield of Mal-sCT was 83%. Dynamic light scattering and circular dichroism data suggested that Mal-sCT presented as a stable helical structure in aqueous solutions of varying polarity, with a propensity to aggregate at concentrations above 11 microM. Cellular uptake of Mal-sCT was twice that of sCT in the Caco-2 cell model, and the conjugate was more resistant to liver enzyme degradation. Mal-sCT exhibited comparable hypocalcemic activity to sCT when administered subcutaneously in the rat model at sCT equivalent dose of 0.114 mg/kg. Peroral Mal-sCT, however, produced variability in therapeutic outcome. While four out of six rats did not respond following intragastric gavage with Mal-sCT, two rats showed significantly suppressed plasma calcium levels (approximately 60% of baseline) for up to 10 h. A novel non-reversible, water-soluble lipid conjugate of sCT was successfully synthesized that showed (1) different aggregation behavior and secondary structure, (2) improved enzymatic stability and cellular uptake, and (3) comparable hypocalcemic activity in vivo compared to sCT.

Synthesis of fluorescent and radioactive analogues of two lactosylceramides and glucosylceramide containing β-thioglycosidic bonds that are resistant to enzymatic degradation

Condensation of 2-S-(2,3,4,6- tetra-O- acetyl-β- d-galactopyranosyl)-2-thiopseudourea hydrobromide with 2,3,6- tri-O- benzoyl-4-O- trifluoromethylsulfonyl-β- d-galactopyranosyl -(1 → 1)-(2S,3R,4E)-3-O- benzoyl-2-dichloroacetamido-4-octadecen-1,3-diol afforded S-(2,3,4,6- tetra-O- acetyl-β- d-galactopyranosyl)-(1 → 4)-2,3,6-tri -O- benzoyl-4-thio-β- d-glucopyranosyl -(1 → 1)-(2S, 3R,4E)-3-O- benzoyl-2-dichloroacetamido-4-octadecen-1,3-diol in good yield. Removal of the protecting groups, followed by selective N-acylation of the sphingosine amino group with either a fluorescent or a radioactive fatty acid, gave labeled lactosylceramide analogues in good yield. Since these products contained a β-thioglycosidic bond between the two sugar moieties, they were totally resistant to the action of acid lysosomal glycosidases. Likewise, condensation of 2-S-(2,3,4,6- tetra-O- acetyl-β- d-glucopyranosyl)-2-thiopseudourea hydrobromide and 2,3,4,6- tetra-O- acetyl-β- d-galactopyranosyl- (1 → 4)-2,3,6- tri-O- acetyl-1-S- acetyl-1-thio-β- d-glucopyranose with (2 R,3 R,4 E)-3- O-benzoyl-2-dichloroacetamido-1-iodo-4-octadecen-3-ol in methanolic sodium acetate afforded the corresponding β-thioglycosides 14 and 16, respectively, in good yield. These β-thioglycosides were converted into glucosylceramide and lactosylceramide analogues following removal of the protecting groups and by subsequent selective N-acylation using either a fluorescent or radioactive fatty acid N-succinimidyl ester. Whereas the glucosylthioceramides thus obtained proved to be completely undegradable by lysosomal glucocerebrosidase, the lactosylceramides containing the β-thioglycosidic bond between the lactose and the ceramide residues could be degraded by lysosomal GM1-β-galactosidase to give the corresponding glucosylthioceramides. These compounds did not yield to any further enzymatic degradation.