Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Yeast background with high-concentration spikes

ABSTRACT: Microarray measurements are susceptible to error caused by cross-hybridization and bulk hybridization. To characterize the effect of these off-target interactions, we analyzed samples made from baseline (S. cerevisiae) alone or mixed with a high-concentration 1kb spike. The spike is a 1 kb sequence amplified from the Schizosaccharomyces pombe gene SPAC27D7.10c, with an added oligo(dT)-T7 promoter. The baseline and spike samples were mixed at the double-stranded cDNA stage. Three arrays: 100% baseline, 99% baseline + 1% spike, 90% baseline + 10% spike. There are no replicates.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Zoltan Szallasi

PROVIDER: E-GEOD-16980 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization.

Eklund Aron C AC Friis Pia P Wernersson Rasmus R Szallasi Zoltan Z

Nucleic acids research 20091206 4

DNA microarray measurements are susceptible to error caused by non-specific hybridization between a probe and a target (cross-hybridization), or between two targets (bulk-hybridization). Search algorithms such as BLASTN can quickly identify potentially hybridizing sequences. We set out to improve BLASTN accuracy by modifying the substitution matrix and gap penalties. We generated gene expression microarray data for samples in which 1 or 10% of the target mass was an exogenous spike of known sequ ...[more]

PMID: 19969549

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Project description:Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached nearly comprehensive coverage, and rapid, targeted approaches have been developed for this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M. (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we couple an ultra high pressure liquid chromatography system to a novel bench top Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid, and robust analysis of the yeast proteome. Single runs of filter-aided sample preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an average of 3923 proteins. Combined analysis of six single runs improved these values to more than 4000 identified proteins/run, close to the total number of proteins expressed under standard conditions, with median sequence coverage of 23%. Because of the absence of fractionation steps, only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity. Median coverage of proteins in Kyoto Encyclopedia of Genes and Genomes pathways with at least 10 members was 88%, and pathways not covered were not expected to be active under the conditions used. To study perturbations of the yeast proteome, we developed an external, heavy lysine-labeled SILAC yeast standard representing different proteome states. This spike-in standard was employed to measure the heat shock response of the yeast proteome. Bioinformatic analysis of the heat shock response revealed that translation-related functions were down-regulated prominently, including nucleolar processes. Conversely, stress-related pathways were up-regulated. The proteomic technology described here is straightforward, rapid, and robust, potentially enabling widespread use in the yeast and other biological research communities. Data analysis: The raw files were processed using MaxQuant version 1.2.0.34. The fragmentation spectra were searched against the yeast ORF database (release date of February 3, 2011; 6752 entries) using the Andromeda search engine with the initial precursor and fragment mass tolerances set to 7 and 20 ppm, respectively, and with up to two missed cleavages. Carabamidomethlyation of cysteine was set as a fixed modification, and oxidation of methionine and protein N-terminal acetylation were chosen as variable modifications for database searching. Both peptide and protein identifications were filtered at 1% false discovery rate and thus were not dependent on the peptide score. Bioinformatics analysis was performed using the Perseus tools available in the MaxQuant environment.

Dataset Information

Yeast background with high-concentration spikes

Publications

Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization.

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