Immune Complexome Analysis

Immune complexes (ICs) are produced during an immune response and may reflect some aspects of an ongoing immune response. Therefore, the identity of antigens incorporated into ICs provides the information that in the future may aid in the development of diagnosis and treatment strategies for autoimmune diseases, infection, cancer, and transplantation therapy, and this information might be more relevant than the information on free antigens. Because ICs may contain many antigens, comprehensive identification and profiling of such antigens are more effective than immunoblotting detection. Here, we introduced mass spectrometry (MS)-based two approaches (immunoproteomics and immune complexome analysis) to comprehensively identify the antigens. Immunoproteomics is a concept to identify disease-associated antigens that elicit immune responses by combining protein separation (two-dimensional electrophoresis, gel-free separation), immunological detection (Western blotting), and MS or by combining immunocapture and MS. Immune complexome analysis is designed for identifying antigens in circulating ICs and consists of ICs separation from serum and direct tryptic digestion followed by nano-liquid chromatography-tandem MS

Poly(l-lactic acid)-modified silica stationary phase for reversed-phase and hydrophilic interaction liquid chromatography

Poly(L-lactic acid) is a linear aliphatic thermoplastic polyester that can be produced from renewable resources. A poly(L-lactic acid)-modified silica stationary phase was newly prepared by amide bond reaction between amino groups on aminopropyl silica and carboxylic acid groups at the end of the poly(L-lactic acid) chain. The poly(L-lactic acid)-silica column was characterized in reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with the use of different mobile phase compositions. The poly(L-lactic acid)-silica column was found to work in both modes, and the retention of test compounds depending on acetonitrile content exhibited "U-shaped" curves, which was an indicator of reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention behavior. In addition, carbonyl groups included into the poly(L-lactic acid) backbone work as an electron-accepting group toward a polycyclic aromatic hydrocarbon and provide π- π interactions

Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography?fluorescence detection

A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2′-furil to form fluorescent difurylimidazole derivatives that were separated on a C 18 column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, respectively. The method showed good linearity over the concentration ranges of 0.100-5.00, 0.200-10.0, 0.200-40.0, and 0.400-10.0 nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions

Study on the Timing of Degassing for Reproducible Preparation of Polymer-Based Monolithic Columns

The influence of timing of degassing during the preparation procedure on reproducible preparation of polymer-based monolithic columns was investigated. The degassing of each solvent before preparing the polymerization mixture exhibited good reproducibility and was found to be appropriate for the monolithic column without any change in the composition of the polymerization mixture

Quinone-based antibody labeling reagent for enzyme-free chemiluminescent immunoassays. Application to avidin and biotinylated anti-rabbit IgG labeling

Chemiluminescence-enzyme immunoassays make it possible to measure trace components with high sensitivity and selectivity due to the high specificity of the antigen-antibody reaction and the high sensitivity of chemiluminescence assays. However, using an enzyme-labeled antibody suffers from many problems such as low reproducibility due to the instability of the enzyme and inhibition of antigen-antibody reaction due to its steric effect. Therefore, herein we report an innovative non-enzymatic chemiluminescence immunoassays labeling reagent through using quinone as a signal-generating tag coupled with biotin as a binder, to overcome enzymatic labeling problems. Biotinylated-1,4-naphthoquinone (biotin-NQ) was synthesized and characterized and it could produce long-lasting chemiluminescence upon mixing with dithiothreitol and luminol based on the redox cycle of quinone. Biotin-NQ showed exceptional stability towards different stress factors that may be encountered during performing the immunoassay such as high temperatures, highly acidic and alkaline conditions, and repeated freeze-thaw cycles. On the other hand, all these conditions lead to decreased labeling enzyme reactivity due to possible denaturation of its protein structure. Finally, the measurement of the biotin-labeled antibody was successfully performed using biotin-NQ and avidin. As a result, the antibody could be detected down to 25.7 nM which is 2.5 times sensitive than biotin-HRP chemiluminescence-enzyme immunoassays. Moreover, our method was applied successfully for determination of avidin using immobilized biotinylated antibody and biotin-NQ, which simulates immunoassays. Avidin could be detected down to 23.4 nM with excellent linearity (r = 0.996). Accordingly, our developed reagent, biotin-NQ, could be used as a universal highly stable, cost-effective, and steric free non-enzymatic label for immunoassays

Determination of Anthraquinone-Tagged Amines Using High-Performance Liquid Chromatography with Online UV Irradiation and Luminol Chemiluminescence Detection

Quinones are frequently used as derivatization reagents in HPLC analysis to improve detection sensitivity. In the present study, a simple, sensitive, and selective chemiluminescence (CL) derivatization strategy for biogenic amines, prior to their HPLC-CL analysis, was developed. The novel CL derivatization strategy was established based on using anthraquinone-2-carbonyl chloride as derivatizing agent for amines and then using the unique property of the quinones’ moiety to generate reactive oxygen species (ROS) in response to UV irradiation. Typical amines such as tryptamine and phenethylamine were derivatized with anthraquinone-2-carbonyl chloride and then injected into an HPLC system equipped with an online photoreactor. The anthraquinone-tagged amines are separated and then UV-irradiated when they pass through a photoreactor to generate ROS from the quinone moiety of the derivative. Tryptamine and phenethylamine can be determined by measuring the chemiluminescence intensity produced by the reaction of the generated ROS with luminol. The chemiluminescence disappears when the photoreactor is turned off, suggesting that ROS are no longer generated from the quinone moiety in the absence of UV irradiation. This result indicates that the generation of ROS could be controlled by turning the photoreactor on and off. Under the optimized conditions, the limits of detection for tryptamine and phenethylamine were 124 and 84 nM, respectively. The developed method is successfully applied to determine the concentrations of tryptamine and phenethylamine in wine samples

4-Carbomethoxybenzaldehyde as a highly sensitive pre-column fluorescence derivatization reagent for 9,10-phenanthrenequinone.

9,10-Phenanthrenequinone (PQ) is harmful environmental pollutant that is detected in airborne particulates. The measurement of PQ in the air should be necessary to evaluate the potential adverse effects of PQ on human health. We have recently developed a determination method for PQ based on the fluorescence derivatization of PQ using benzaldehyde and ammonium acetate as a reagent. In this study, in order to obtain more sensitive and selective fluorescence derivatization reaction, we measured the fluorescence of the reaction mixture of PQ with 21 kinds of aromatic aldehydes in the presence of ammonium acetate. Among the tested aldehydes, 4-carbomethoxybenzaldehyde was found to be the best reagent in regard to fluorescence intensity and emission wavelength maximum. Based on the fluorescence derivatization with 4-carbomethoxybenzaldehyde, a highly sensitive chromatographic method was developed for the determination of PQ with the detection limit (S/N=3) of 1.2 fmol/injection

Coronin1C Is a GDP-Specific Rab44 Effector That Controls Osteoclast Formation by Regulating Cell Motility in Macrophages

Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages

A toxicoproteomic study on cardioprotective effects of pre-administration of docetaxel in a mouse model of adriamycin-induced cardiotoxicity.

Studies suggest that pre-administration of docetaxel (DOC) in adriamycin (ADR)-DOC combination anticancer therapy results in stronger antitumor effects and fewer ADR-induced cardiotoxic deaths in mouse model, yet no mechanism explaining this effect has been established. The aim of this study was to identify cellular processes in mouse heart tissue affected by different ADR/DOC dosing protocols using a toxicoproteomic approach. We applied fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) - which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry - to the proteomic analysis of heart tissue from control, intermittent-dosing (DOC-ADR), and simultaneous-dosing (ADR&DOC) groups. In DOC-ADR group, ADR was administered 12h after DOC injection; in ADR&DOC group, both drugs were administered simultaneously; in control group, saline was administered at the same timing as ADR injection of other groups. Heart samples were isolated from all mice 1 week after the treatment. The highly reproducible and sensitive method (FD-LC-MS/MS) identified nine proteins that were differentially expressed in heart tissue of control and the two treatment groups; seven of these nine proteins participate in cellular energy production pathways, including glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain. Significantly higher expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was observed in the DOC-ADR group, the group with the fewer cardiotoxic deaths, than in the ADR&DOC group. Therefore, GAPDH may have potential as a drug target for protective intervention and a biomarker for evaluation of the cardioprotective effects in pre-clinical studies