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EvoSep-timsTOF proximity dependent biotinylation optimization

ABSTRACT: This dataset involved the optimization of proximity dependent biotinylation experiments across the available EvoSep gradients on a Bruker timsTOF Pro 2 MS. Various different sample loads are compared and statistically significant preys are used to select optimal settings for these kinds of experiments.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Anne-Claude Gingras

PROVIDER: MSV000095270 | MassIVE | Tue Jul 09 05:57:00 BST 2024

SECONDARY ACCESSION(S): PXD053794

REPOSITORIES: MassIVE

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Project description:Background: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. Results: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays. For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirm an unbiased determination of generally optimal experimental conditions. Conclusions: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive profiling of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks. Supporting material, including source code and data, is available at http://bioinf.boku.ac.at/pub/optMA2010/. Optimization of hybridization temperature via an assessment of differential expression between two samples (male vs female Drosophila melanogaster) in 6 technical replicates (3 regular + 3 dye-swaps) for hybridizations at different temperatures (in two batches of 50, 52, 54, and 56; and 47, 49, 50, and 51 degree Celsius, with the repeated hybridization at 50 degree Celsius serving to demonstrate batch-to-batch stability).

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EvoSep-timsTOF proximity dependent biotinylation optimization

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