A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed for the quantification of bioactive peptides in biological fluids. The method employs protein precipitation with 4% trichloroacetic acid (TCA) and selected reaction monitoring (SRM) using an immonium ion as the product ion. This method was applied to determine the synthetic parathyroid hormone (PTH) analog (MW 1721) in rat plasma and human hepcidin-25 (MW 2789) in human serum. TCA clean-up showed a sufficient recovery for peptides with a MW of less than 3000, and would be useful as a simple and rapid method because of direct injection of the supernatant without evaporation or dilution. In addition, TCA clean-up allowed us not only to reduce sample preparation time, but also to select an immonium ion as a product ion of SRM, which led to detection more sensitive than SRM using other types of product ions. The lower limits of quantitation (LLOQs) of the PTH analog and the human hepcidin-25 were 0.2 ng/mL and 5 ng/mL, respectively. This method was fully validated with acceptable linearity, intra- and inter-assay precisions, and accuracy. Furthermore, this simple and rapid method is applicable to pharmacokinetic studies.
Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Furthermore, we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in a mouse model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, to elucidate whether or not the systemic administration of CNP is a novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by iv administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.
Bile acids, synthesized from cholesterol by hepatic enzymes, are excreted into the duodenum via the bile duct as their glycine and taurine conjugates. After assisting in the lipolysis and absorption of fats in the intestinal lumen, bile acids are then returned to the liver. According to this enterohepatic circulation, very small quantities of bile acids are present in blood of healthy subjects. However, in hepatobiliary diseases, disturbances in their synthesis and clearance by the liver, as well as absorption by the intestines cause changes in the level of not only bile acids but also their sulfates and glucuronides in the blood and urine. 1 Since the first report of the occurrence of the bile acid glucuronide in human urine, it has been believed that bile acids are conjugated with glucuronic acid through the inherent 3α -hydroxy group on the steroid nucleus to form their ether type glucuronides. 2-6 Recently, we have disclosed the formation of ester-type acyl glucuronides of bile acid conjugated through the 24-carboxy group in an incubation mixture with hepatic microsomal fractions from rats 7 , employing liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS); here the acyl glucuronides were characterized as their acetate-methyl ester derivatives. In addition, we have synthesized the acyl glucuronides of cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and lithocholic acids as authentic specimens. 8 The present paper deals with the development of the simultaneous determination of the bile acid 24-glucuronides in human urine using LC/electrospray ionization (ESI)-MS without derivatization. Experimental LC/ESI-MSThe liquid chromatographic separation was performed with a Nano-space SI-1 (Shiseido, Ltd., Tokyo, Japan) on a Develosil ODS-HG-5 column (5 µm, 150×2.0 mm i.d.) (Nomura Chemical, Aichi, Japan) with a flow rate of 150 µl/min. The mass spectrometric detection was carried out using a JMS-LCmate doublefocusing mass spectrometer (JEOL, Tokyo, Japan) equipped with an ESI system in the negative ion mode. The resolution of the mass spectrometer was set at 750 and 5000 for the low-resolution and high-resolution analyses, respectively. For the rock-mass signal at m/z 602.9125, the standard solution containing sodium trifluoroacetic acid and sodium formic acid (each 50 µg/ml) in MeOH was added to the postcolumn effluent using a "T-type" connector at a flow rate of 50 µl/min. MaterialsBile acid 3-9 and 24-glucuronides 8 (Fig. 1) A method for the separation and determination of bile acid 24-glucuronides using liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. In the ESI mode, 24-glucuronides were characterized by an intense deprotonated molecule [M-H] -with a fragment ion [M-H-176] -formed by elimination of the glucuronic acid moiety. The ratio of these negative ions was significantly influenced by the orifice-1 and the ring lens voltages. Bile acid 24-glucuronides in human urine were extracted with an OASIS HLB car...
Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method's high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.
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