High-Energy Electron Transfer Dissociation (HE-ETD) Using Alkali Metal Targets for Sequence Analysis of Post-Translational Peptides

Post-translational modifications (PTMs) of proteins are important in the activation, localization, and regulation of protein function in vivo. The usefulness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) in tandem mass spectrometry (MS/MS) using low-energy (LE) trap type mass spectrometer is associated with no loss of a labile PTM group regarding peptide and protein sequencing. The experimental results of high-energy (HE) collision induced dissociation (CID) using the Xe and Cs targets and LE-ETD were compared for doubly-phosphorylated peptides TGFLT(p)EY(p)VATR (1). Although HE-CID using the Xe target did not provide information on the amino acid sequence, HE-CID using the Cs target provided all the z-type ions without loss of the phosphate groups as a result of HE-ETD process, while LE-ETD using fluoranthene anion gave only z-type ions from z5 to z11. The difference in the results of HE-CID between the Xe and Cs targets demonstrated that HE-ETD process with the Cs target took place much more dominantly than collisional activation. The difference between HE-ETD using Cs targets and LE-ETD using the anion demonstrated that mass discrimination was much weaker in the high-energy process. HE-ETD was also applied to three other phosphopeptides YGGMHRQEX(p)VDC (2: X = S, 3: X = T, 4: X = Y). The HE-CID spectra of the doubly-protonated phosphopeptides (= [M + 2H]2+) of 2, 3, and 4 using the Cs target showed a very similar feature that the c-type ions from c7 to c11 and the z-type ions from z7 to z11 were formed via N–Cα bond cleavage without a loss of the phosphate group

Characterization of Bacillus strains of marine origin

A total of twenty aerobic endospore-forming bacilli, isolated from marine invertebrates and sea water of different areas of the Pacific Ocean, were taxonomically characterized. Most of the bacilli (11 strains) of marine origin belonged to the species Bacillus subtilis, according to their phenotypic characteristics, antibiotic susceptibility profiles, and fatty acids patterns. A group of four alkaliphilic strains formed a separate cluster that was tentatively classified as B. horti. One isolate, KMM 1717, associated with a sponge from the Coral Sea was identified as B. pumilus. Two strains, Bacillus KMM 1916 and KMM 1918, showed antibiotic sensitivity profiles similar to B. licheniformis, but they had a distinct fatty acid composition and peculiar phenotypic traits. The taxonomic affiliation of KMM 1810 and KMM 1763 remained unclear since their fatty acid composition and antibiotic sensitivity patterns were not resembled with none of these obtained for Bacillus strains

An Active C-Terminally Truncated Form of Ca2+/Calmodulin-Dependent Protein Kinase Phosphatase-N (CaMKP-N/PPM1E)

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and its nuclear homolog CaMKP-N (PPM1E) are Ser/Thr protein phosphatases that belong to the PPM family. CaMKP-N is expressed in the brain and undergoes proteolytic processing to yield a C-terminally truncated form. The physiological significance of this processing, however, is not fully understood. Using a wheat-embryo cell-free protein expression system, we prepared human CaMKP-N (hCaMKP-N(WT)) and the truncated form, hCaMKP-N(1–559), to compare their enzymatic properties using a phosphopeptide substrate. The hCaMKP-N(1–559) exhibited a much higher value than the hCaMKP-N(WT) did, suggesting that the processing may be a regulatory mechanism to generate a more active species. The active form, hCaMKP-N(1–559), showed Mn2+ or Mg2+-dependent phosphatase activity with a strong preference for phospho-Thr residues and was severely inhibited by NaF, but not by okadaic acid, calyculin A, or 1-amino-8-naphthol-2,4-disulfonic acid, a specific inhibitor of CaMKP. It could bind to postsynaptic density and dephosphorylate the autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Furthermore, it was inactivated by H2O2 treatment, and the inactivation was completely reversed by treatment with DTT, implying that this process is reversibly regulated by oxidation/reduction. The truncated CaMKP-N may play an important physiological role in neuronal cells.This work was supported, in part, by Grants-in-Aid for Scientific Research (21590334) from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from the Japan Foundation for Applied Enzymology

A light in the dark: ecology, evolution and molecular basis of copepod bioluminescence

Within the calanoid copepods, the bioluminescent species comprise 5-59% of the abundance and 10-15% of the biomass in the world's oceans. Most of the luminous species belong to the superfamily Augaptiloidea. The composition of bioluminescent species within the calanoid copepods shows latitudinal patterns; 5-25% of total calanoid copepods are found in high-latitude oceans, while 34-59% are in low-latitude oceans, reflecting a prey-predator relationship. Bioluminescent species of calanoid copepods are able to produce the light-emitting substrate coelenterazine. It is then transferred to higher predators through the food chain, and might be used for bioluminescence in other luminous organisms. A notable feature of copepod bioluminescence is the secreted-type, and its major function may be as an antipredatory response or a defensive behavior. Identification of more than 20 luciferase genes from calanoid copepods has revealed the highly conserved sequences of those genes. This leads us to the speculation that the genes for luciferase within the group of calanoid copepods have evolved independently of comparable genes outside of this group. We discuss here the ecological and biological functions of copepod bioluminescence, the significant diversity in luminous intensity, which might be evolutionarily relevant to their motility and habitat depth, and the promising future directions of bioluminescence studies

Complete mitochondrial genome sequence of Japanese forest green tree frog (Rhacophorus arboreus)

We determined the complete mitochondrial genome sequence of the Japanese forest green tree frog (Rhacophorus arboreus). The mitochondrial genome is 22,236 bp in length, which encodes 13 protein-coding genes, 2 rRNA, and 22 tRNA genes, and two control regions (D-loops). The whole gene arrangement of R. arboreus was the same as that of Rhacophorus omeimontis and Rhacophorus schlegelii