Certification and stability assessment of recombinant human growth hormone as a certified reference material for protein quantification.

A certified reference material (CRM) for the quantification of protein, essential to manage quality control and quality assurance in protein-related works, has been developed. Amino acid analysis with conventional acid hydrolysis and isotope dilution HPLC-MS was used to establish an SI-traceable absolute protein quantification method using recombinant human growth hormone (hGH) as a model protein. The certification method was verified by comparative studies between 1) different methods of protein quantification based on microwave-assisted hydrolysis, and 2) different labs as part of the Asian Collaboration on Reference Material project with Japan, China, and Korea. Certification, evaluation of measurement uncertainty, homogeneity testing, and stability testing were carried out, after which the candidate CRM for hGH quantification was successfully certified with excellent agreement within the certified value in the two comparative studies. Although the quantification value of hGH by amino acid analysis showed good robustness in various conditions, results of intact protein analysis showed degradation profiles in temperatures higher than 4 °C. Consequently, storage and dissemination conditions should be set in accordance with stability tests. Based on the results, this method is believed to be suitable for accurate quantification of hGH. Additionally, it can also be used as a guide to preparation of CRM, and instructions for quality management of protein work for other similar proteins.

ß-cell serotonin production is associated with female sex, old age, and diabetes-free condition.

Serotonin is known to be present in pancreatic ß-cells and to play several physiological roles, including insulin secretion, ß-cell proliferation, and paracrine inhibition of α-cells. However, the serotonin production of different cell lines and islets has not been compared based on age, sex, and diabetes related conditions. Here, we directly compared the serotonin concentrations in ßTC and MIN6 cell lines, as well as in islets from mice using ultra-performance liquid chromatography tandem mass spectrometry. The average serotonin concentration was 5-10 ng/mg protein in the islets of male and non-pregnant female mice. The serotonin level was higher in females than males at 8 weeks, although there was no difference at 1 year. Furthermore, we observed serotonin by immunofluorescence staining in the pancreatic tissues of mice and human. Serotonin was detected by immunofluorescence staining in a portion of ß-cells from islets of old female mice, but not of male or young female mice. A similar pattern was observed in human pancreas as well. In humans, serotonin production in ß-cells was associated with a diabetes-free condition. Thus, serotonin production in ß-cells was associated with old age, female sex, and diabetes-free condition.

Fully international system of units-traceable glycated hemoglobin quantification using two stages of isotope-dilution high-performance liquid chromatography-tandem mass spectrometry.

Glycated hemoglobin (HbA1c), defined as hemoglobin (Hb) molecules having a stable adduct of glucose on the N-terminal of the ß-chains, has been endorsed as a diagnostic tool for diabetes mellitus and a prediction indicator for the development of diabetes complications. Here we describe an accurate procedure using two stages of isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the quantification of HbA1c that provides full traceability to International System of Units (SI). First, synthetic peptides representing specific markers of HbA1c (G-hexa) and hemoglobin A0 (Hexa) were certified by amino acid analysis via acid hydrolysis as reference materials (RMs) for the next step. For this peptide certification, three amino acids (proline, valine, and leucine) were determined by hydrolysis with 10M hydrochloric acid at 130°C for 48h followed by ID-LC-MS/MS. Then, HbA1c content in blood was quantified with the ratio of specific proteolytic peptides from HbA1c and HbA0 via enzyme digestion using ID-LC-MS/MS with the certified peptides as RMs and isotope-labeled peptides as internal standards. Results demonstrate complete traceability to SI-units throughout this procedure. Reliability was confirmed through comparative studies with commercially available RMs for HbA1c, and other routine HbA1c diagnostic methods as well. Following full method validation, we applied this procedure to the certification of candidate hemolysate-certified RMs for HbA1c content, as well as 52 real clinical samples. All of the results showed the suitability of this method to act as a primary reference measurement procedure for HbA1c in complex biological samples.

A reference measurement procedure for amino acids in blood using isotope dilution ultra-performance liquid chromatography-tandem mass spectrometry.

We described a reference measurement procedure for amino acid (AA) quantification in blood samples based on deproteinization with 5-sulfosalicylic acid (SSA) and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (LC-MS) method. The serum was deproteinized with 15% v/v SSA and the supernatant was injected directly into the LC-MS system without further processing. Compared with the use of other precipitants and water as a control, five model AAs-valine, isoleucine, leucine, tyrosine, and phenylalanine-in the SSA-treated samples showed ionization enhancement as well as stable background signals without significant ion suppression effects. Five analytes were clearly separated within 3min using gradient elution and ion-pair chromatography of water and acetonitrile containing 0.1% v/v trifluoroacetic acid. The limit of detection range of this method was 2-52fmol, and the RSDs of accuracy and precision from intra- and inter-day assays were within 2.7%. The method was applied to various blood samples including serum, whole blood and plasma, with no reasonable measurement bias revealed. The quantification accuracy of this method was then assessed using commercially available plasma certified reference material (CRM) for AA, and the results agreed well within certified values. We finally applied this method to the determination of candidate serum CRM. The optimized protocol was found to be suitable for the accurate quantification of five AAs in serum, and may satisfactorily serve as a primary method for AA measurement in various blood matrices.

Marinobacter halotolerans sp. nov., a halophilic bacterium isolated from a saltern crystallizing pond.

A Gram-stain-negative, moderately halophilic, motile bacterium, designated strain CP12T, was isolated from a crystallizing pond of a saltern of the Yellow Sea in Korea. Cells of strain CP12T were non-spore-forming rods and produced whitish-yellow colonies. Growth was observed at 10-37 °C (optimum 37 °C), at pH 6.0-9.0 (optimum pH 7.0), and in the presence of 0.5-20 % (w/v) NaCl (optimum 3 %). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CP12T was closely related to Marinobacter flavimaris SW-145T (98.4 % 16S rRNA gene sequence similarity), Marinobacter algicola DG893T (98.2 %), Marinobacter adhaerens HP15T (98.2 %), Marinobacter salsuginis SD-14BT (97.9 %), Marinobacter salarius R9SW1T (97.6 %) and Marinobacter lipolyticus SM19T (97.1 %). DNA-DNA hybridization studies showed values lower than 18.6 % between strain CP12T and any of these species. The predominant respiratory isoprenoid quinone was ubiquinone-9 and the major cellular fatty acids of strain CP12T were C16 : 0, C12 : 0 3-OH, C12 : 0, Summed feature 3, C16 : 0 10-methyl and C18 : 1ω9c. On the basis of phenotypic properties, and phylogenetic and chemotaxonomic data, it is evident that strain CP12T represents a novel species of the genus Marinobacter, for which the name Marinobacter halotolerans sp. nov. is proposed. The type strain is CP12T (=KACC 18381T=NBRC 110910T).

Terrimonas rhizosphaerae sp. nov., isolated from ginseng rhizosphere soil.

The novel isolate belonging to the genus Terrimonas, designated CR94T, was isolated from rhizosphere soil of a ginseng field in Geumsan, Korea. Cells of strain CR94T were strictly aerobic, Gram-stain-negative, non-motile, non-filamentous single rods. Growth was observed at 10-37 °C (optimum 28 °C) and at pH 4.0-10.0 (optimum pH 6.0). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain CR94T belonged to the genus Terrimonas, showing highest sequence similarity to Terrimonas lutea DYT (97.3 %), Terrimonas pekingensis QHT (97.1 %), Terrimonas aquatica RIB1-6T (95.6 %), Terrimonas rubra M-8T (94.7 %) and Terrimonas ferruginea ATCC 13524T (93.8 %). DNA-DNA relatedness values between strain CR94T and T. lutea KACC 13047T, T. pekingensis KACC 18795T, T. ferruginea KACC 11310T and T. aquatica LMG 24825T were 30.5, 28.9, 17.8 and 13.5 %, respectively. The DNA G+C content was 46.5 mol% and the major respiratory quinone was menaquinone-7 (MK-7). The major cellular fatty acids of strain CR94T were iso-C15:1 G and iso-C15 : 0. On the basis of the polyphasic analysis, strain CR94T represents a novel species of the genus Terrimonas, for which the name Terrimonas rhizosphaerae sp. nov. is proposed. The type strain is CR94T (=KACC 17564T=NCAIM B 025317T).

Denitratimonas tolerans gen. nov., sp. nov., a denitrifying bacterium isolated from a bioreactor for tannery wastewater treatment.

A denitrifying bacterium, designated strain E4-1(T), was isolated from a bioreactor for tannery wastewater treatment, and its taxonomic position was investigated using a polyphasic approach. Strain E4-1(T), a facultative anaerobic bacterium, was observed to grow between 0 and 12 % (w/v) NaCl, between pH 3.0 and 12.0. Cells were found to be oxidase-positive and catalase-negative. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain E4-1(T) forms a distinct lineage with respect to closely related genera in the family Xanthomonadaceae, and is closely related to Chiayiivirga, Aquimonas and Dokdonella, and the levels of 16S rRNA gene sequence similarity with respect to the type species of related genera are less than 93.9 %. The predominant respiratory quinone was determined to be ubiquinone-8 (Q-8) and the major cellular fatty acids were determined to be iso-C15:0, iso-C17:1 ω9c, iso-C11:0 and iso-C11:0 3OH. Based on physiological, biochemical and chemotaxonomic properties together with results of comparative 16S rRNA gene sequence analysis, strain E4-1(T) is considered to represent a novel species in a new genus, for which the name Denitratimonas tolerans gen. nov., sp. nov. is proposed. The type strain is E4-1(T) (=KACC 17565(T) = NCAIM B 025327(T)).