Methods for peptide identification by spectral comparison

Background  

Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies.     

Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors

Background

Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment.

Results

In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways.

Conclusions

We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.     

Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction

Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (. Biophys. J. 77:000-000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca(2+) concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca(2+) concentrations. At all Ca(2+) concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca(2+) is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca(2+)-dependent equilibration among different states of the thin filament can fully account for the Ca(2+) dependence of force redevelopment and the fluorescence changes described in this study.     

Integrated conodont and radiolarian biostratigraphy of the upper Norian in Baoshan Block,Southwestern China

Two significant stratigraphical microfossils, conodonts and radiolarians, are usually used for the Upper Triassic chronostratigraphy. The Baoshan Block was located in eastern Tethys during the Late Triassic where the biostratigraphical data of Upper Triassic are still poorly known. We collected new samples from the Hongyan section (HY) for biostratigraphical study. This 24-m-thick section in Dabaozi Village, Baoshan City, is mainly composed of thin-layered limestones, sandstone and siltstone. The conodont fauna is referred to Sevatian 1 (late Norian), in which the species Mockina englandi, Mockina carinata and Mockina mosheri morphotype B are first recognized in the Baoshan Block, and thus eastern Tethys. The Norian radiolarian associations are first reported in the Baoshan Block, which correlate with the biozonation of North America and also that proposed for Central Japan. The radiolarian assemblages found in the analysed samples in HY section can be referred to the Sevatian Betraccium deweveri Zone. The Baoshan Block is a key area for conodont and radiolarian-based correlations between the Tethys, Japan and North American domains.     

Enzyme mimics complexing Cu(II) ion: structure-function relationships.

Five peptides containing (His-X2)-His or (His-X3)-His motifs have been designed and synthesized to coordinate Cu(II). Structural information was obtained by various spectroscopic techniques and was used as constraint to search for local conformational energy minima by molecular mechanics. Thermodynamic stability constants of the Cu(II) chelates was obtained by 19F-NMR. The synthesized Cu(II)-peptide chelates were tested as catalysts of some important red-ox processes occuring in biological systems, in particular oxidation of ascorbate and dismutation of superoxide ion. The catalytic efficiency of the five chelates was much lower than that of ascorbate oxidase. On the contrary, two of them showed kinetic constants for superoxide dismutation about one order of magnitude lower than that of the enzyme Cu,Zn superoxide dismutase. In both cases, the catalytic properties were dependent on the peptide sequence. The relationships between structure and activity are discussed to find the structural parameters crucial for catalytic activity that can be modulated by appropriate design and synthesis of the peptides.