Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing

A new method to determine N-terminal amino acid sequences of multiple proteins at low pmol level by a parallel processing has been developed. The method contains the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling with sulfosucccimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate(sulfo-NHS-SS-biotin) to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease; (4) specific isolation of N-terminal fragments of proteins by affinity capture using the biotin-avidin system; (5) de novo sequence analysis of peptides by MALDI-TOF-/MALDI-TOF-PSD mass spectrometry with effective utilization of the CAF (chemically assisted fragmentation) method.1 This method is also effective for N-terminal sequencing of each protein in a mixture of several proteins, and for sequencing components of a multiprotein complex. It is expected to become an essential proteomics tool for identifying proteins, especially when used in combination with a C-terminal sequencing method.     

Simultaneous detection of N-terminal fragment ions in a protein mixture using a ruthenium(II) complex

Use of a bis(terpyridine)ruthenium(II) derivative as an N-terminal labeling reagent resulted in the simultaneous detection and individual determination of all the N-terminal fragments of the proteins in a mixture without requiring any separation. All of the N-termini of the guanidinated proteins were labeled selectively by the ruthenium complex (-CO-labeling). After chymotryptic digestion, the fragments were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and post-source decay (PSD). The -CO moiety exclusively enhanced N-terminal fragment ions in mass spectra and enabled easy N-terminal sequencing. In a mixture containing three different proteins (lysozyme, ubiquitin, and insulin), all of the N-terminal fragment ions labeled with the ruthenium complex were found to produce uniformly intense peaks without the detection of the other unlabeled fragments. The N-terminal sequences of these ions were determined individually by PSD analysis. Application to unknown proteins from Thermus thermophilus HB8 with two-dimensional electrophoretic separation resulted in the successful determination of the N-terminal sequence and easy identification of the target protein.     

Dalitz plot analysis of Coulomb exploding O3 in ultrashort intense laser fields

The three-body Coulomb explosion of O3, O3(3+)-->O++O++O+, in ultrashort intense laser fields (2x10(15) W/cm2) is studied with two different pulse durations (9 and 40 fs) by the coincidence momentum imaging method. In addition to a decrease in the total kinetic energy release, a broadening in the Dalitz plot distribution [Philos. Mag. 44, 1068 (1953)] is observed when the pulse duration is increased from 9 to 40 fs. The analysis based on a simple Coulomb explosion model shows that the geometrical structure of O3 remains almost unchanged during the interaction with the few-cycle intense laser fields, while a significant structural deformation along all the three vibrational coordinates, including the antisymmetric stretching coordinate, is identified in the 40 fs intense laser fields. The observed nuclear dynamics are discussed in terms of the population transfer to the excited states of O3.     

Throughput Measurement of a Multilayer-Coated Schwarzschild Objective Using Synchrotron Radiation

The throughput of a Schwarzschild objective using undulator synchrotron radiation was measured. Conventionally, the throughput was estimated from the squared reflectivity of one multilayer mirror and from the obstruction ratio. However, we evaluated the transmission ratio from the input and output photon flux using a precisely calibrated monochromatic beam from an undulator light source. It was found that the objective has a maximum throughput of 8.5% at a wavelength of 13.9 nm.     

Teaching-Learning of Evaluative Criteria for Mathematical Arguments Through Classroom Discourse: A Cross-National Study

Videotaped lessons of 5th graders on equivalent fractions from 7 American and 6 Japanese classrooms were analyzed in terms of a recurrent pattern in public discourse among a teacher and students. This pattern—called inquiry, response, feedback—occurs when a teacher initiates discourse (mostly with an inquiry), a student or students respond (often with an answer to the teacher inquiry), and the teacher provides feedback to the student's response. We found2 approaches to the teaching-learning of the criteria for evaluating mathematical arguments. In the Japanese classroom, students were encouraged to offer their own argument to the whole class and evaluate arguments proposed by other students. They seldom were given direct evaluation by their teacher. In contrast, American teachers often gave individual elaboration as well as direct evaluation to the student's responses, and some of the teachers offered their own opinions about mathematics, about valid ways of argumentation, or about both. The Japanese approach would help students acquire evaluative criteria indirectly through participating in mathematical discourse, whereas the American approach would help students learn modes of arguments through direct instruction.     

Further test of the isolated pentagon rule: Thermodynamic and kinetic stabilities of C84 fullerene isomers

Thermodynamic and kinetic stabilities of 73 C₈₄ fullerene isomers were estimated from the MM3 heats of formation and the recently defined bond resonance energies (BREs), respectively. The BRE represents the contribution of a given π bond in a molecule to the topological resonance energy (TRE). All π bonds shared by two pentagons turned out to be highly reactive without exceptions. C₈₄ fullerene isomers with such π bonds must be incapable of survival during harsh synthetic processes. Thus, the isolated pentagon rule (IPR) proved to be applicable to such large fullerene cages. For sufficiently large fullerenes like C₈₄, some isolated-pentagon isomers are also predicted to be very unstable with highly antiaromatic π bonds. © 1996 by John Wiley & Sons, Inc.