Integrating Internal Fragments in the Interpretation of Top-Down Sequencing Data of Larger Oligonucleotides.

In the context of direct top-down analysis or concerted bottom-up characterization of nucleic acid samples, the waning yield of terminal fragments as a function of precursor ion size poses a significant challenge to the gas-phase sequencing of progressively larger oligonucleotides. In this report, we examined the behavior of oligoribonucleotide samples ranging from 20 to 364 nt upon collision-induced dissociation (CID). The experimental data showed a progressive shift from terminal to internal fragments as a function of size. The systematic evaluation of experimental factors, such as collision energy, precursor charge, sample temperature, and the presence of chaotropic agents, showed that this trend could be modestly alleviated but not suppressed. This inexorable effect, which has been reported also for other activation techniques, prompted a re-examination of the features that have traditionally discouraged the utilization of internal fragments as a source of sequence information in data interpretation procedures. Our simulations highlighted the ability of internal fragments to produce self-consistent ladders with either end corresponding to each nucleotide in the sequence, which enables both proper alignment and correct recognition of intervening nucleotides. In turn, contiguous ladders display extensive overlaps with one another and with the ladders formed by terminal fragments, which unambiguously constrain their mutual placement within the analyte sequence. The experimental data borne out the predictions by showing ladders with extensive overlaps, which translated into uninterrupted "walks" covering the entire sequence with no gaps from end to end. More significantly, the results showed that combining the information afforded by internal and terminal ladders resulted in much a greater sequence coverage and nucleotide coverage depth than those achievable when either type of information was considered separately. The examination of a series of 58-mer oligonucleotides with high sequence homology showed that the assignment ambiguities engendered by internal fragments did not significantly exceed those afforded by the terminal ones. Therefore, the balance between potential benefits and perils of including the former makes a compelling argument for the development of integrated data interpretation strategies, which are better equipped for dealing with the changing fragmentation patterns obtained from progressively larger oligonucleotides.

Web2.0 paves new ways for collaborative and exploratory analysis of chemical compounds in spectrometry data.

In nowadays life science projects, sharing data and data interpretation is becoming increasingly important. This considerably calls for novel information technology approaches, which enable the integration of expert knowledge from different disciplines in combination with advanced data analysis facilities in a collaborative manner. Since the recent development of web technologies offers scientific communities new ways for cooperation and communication, we propose a fully web-based software approach for the collaborative analysis of bioimage data and demonstrate the applicability of Web2.0 techniques to ion mobility spectrometry image data. Our approach allows collaborating experts to easily share, explore and discuss complex image data without any installation of software packages. Scientists only need a username and a password to get access to our system and can directly start exploring and analyzing their data.

Diffractive beam splitter characterization via a power-recycled interferometer.

We used the high-precision laser interferometer technique of power recycling to characterize the optical loss of an all-reflective grating beam splitter. This beam splitter was used to set up a Michelson interferometer with a power-recycling resonator with a finesse of 883. Analyzing the results obtained, we determined the beam splitter's total optical loss to be (0.193+/-0.019)%. Low loss all-reflective beam splitters might find application in future high-power laser interferometers for the detection of gravitational waves.