Efficiency of Collisionally-activated dissociation and 193-nm photodissociation of peptide ions in fourier transform mass spectrometry

AbstractFor tandem mass spectrometry, the Fourier transform instrument exhibits advantages for the use of collisionally-activated dissociation (CAD). The CAD energy deposited in larger ions can be greatly increased by extending the collision time to as much as 120 s, and the efficiency of trapping and measuring CAD product ions in many times greater than the found for triple-quadrupole or magnetic sector instruments, although the increased pressure from the collision gas is an offsetting disadvantage. A novel system that uses the same laser for photodesorption of ions and their subsequent photodissociation can produce complete dissociation of larger oligopeptide ions and unusually abundant fragment ions. In comparison to CAD, much more internal energy can be deposited in the primary ions using 193-nm photons, sufficient to dissociate peptide ions of m/z > 2000. Mass spectra closely resembling ion photodissociation spectra can also be obtained by neutral photodissociation (193-nm laser irradiation of the sample) followed by ion photodesorption

An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

<p>Abstract</p> <p>Background</p> <p>Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches.</p> <p>Results</p> <p>In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA) will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay.</p> <p>Conclusions</p> <p>By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at <url>http://www.laurenzi.net</url>.</p

Sequence-Dependent Fluorescence of Cyanine Dyes on Microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5′-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5′ guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5′-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling

Molecular Characterization of the Region 7q22.1 in Splenic Marginal Zone Lymphomas

Splenic marginal zone lymphomas (SMZL) are an uncommon type of B-cell non-Hodgkin's lymphoma (NHL-B) in which no specific chromosomal translocations have been described. In contrast, the most frequent cytogenetic abnormality is the loss of the long arm of chromosome 7 (7q). Previous reports have located this loss in the 7q32 region. In order to better characterize the genomic imbalances in SMZL, molecular studies were carried out in 73 patients with SMZL. To gain insight into the mapping at 7q a tiling array was also used. The results confirmed the loss of 7q as the most frequent change. In addition, several abnormalities, including 4q22.1, 1q21.3–q22, 6q25.3, 20q13.33, 3q28, 2q23.3–q24.1 and 17p13, were also present. A loss of 7q22.1 at 99925039–101348479 bp was observed in half of the cases. The region of 7q22.1 has not previously been characterised in SMZL. Our results confirmed the presence of a new region of loss on chromosome 7 in these NHL

Position dependent mismatch discrimination on DNA microarrays – experiments and model

<p>Abstract</p> <p>Background</p> <p>The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.</p> <p>Results</p> <p>We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends) as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results.</p> <p>Conclusion</p> <p>Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN) can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.</p

Hybridization thermodynamics of NimbleGen Microarrays

Background While microarrays are the predominant method for gene expression profiling, probe signal variation is still an area of active research. Probe signal is sequence dependent and affected by probe-target binding strength and the competing formation of probe-probe dimers and secondary structures in probes and targets. Results We demonstrate the benefits of an improved model for microarray hybridization and assess the relative contributions of the probe-target binding strength and the different competing structures. Remarkably, specific and unspecific hybridization were apparently driven by different energetic contributions: For unspecific hybridization, the melting temperature Tm was the best predictor of signal variation. For specific hybridization, however, the effective interaction energy that fully considered competing structures was twice as powerful a predictor of probe signal variation. We show that this was largely due to the effects of secondary structures in the probe and target molecules. The predictive power of the strength of these intramolecular structures was already comparable to that of the melting temperature or the free energy of the probe-target duplex. Conclusions This analysis illustrates the importance of considering both the effects of probe-target binding strength and the different competing structures. For specific hybridization, the secondary structures of probe and target molecules turn out to be at least as important as the probe-target binding strength for an understanding of the observed microarray signal intensities. Besides their relevance for the design of new arrays, our results demonstrate the value of improving thermodynamic models for the read-out and interpretation of microarray signals

Design, Validation and Annotation of Transcriptome-Wide Oligonucleotide Probes for the Oligochaete Annelid Eisenia fetida

High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs). Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59%) probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1) does not require a major genomics core facility; (2) allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3) is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4) if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is particularly applicable to organisms with a wealth of EST sequences but unfinished genome