Dataset Information

Protease-resistant streptavidin for interaction proteomics

ABSTRACT: Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners by mass spectrometry. Since contamination by streptavidin limits protein identification, we here present protease-resistant streptavidin beads (prS). Their application to different biotinylated biomolecules (DNA, protein and lipid) result in >100 streptavidin contamination and better coverage of both the target and its protein interactors without the need for sample pre-fractionation.

INSTRUMENT(S): LTQ Orbitrap, Orbitrap Fusion, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Diploid Cell

SUBMITTER: Gianluca Sigismondo

LAB HEAD: Jeroen Krijgsveld

PROVIDER: PXD016576 | Pride | 2020-05-27

REPOSITORIES: Pride

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Protease-resistant streptavidin for interaction proteomics.

Rafiee Mahmoud-Reza MR Sigismondo Gianluca G Kalxdorf Mathias M Förster Laura L Brügger Britta B Béthune Julien J Krijgsveld Jeroen J

Molecular systems biology 20200501 5

Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biot ...[more]

PMID: 32400114

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Project description:To gain insight into the molecular changes of sleep need, this study addresses gene expression changes in a subpopulation of neurons selectively activated by sleep deprivation. Whole brain expression analyses after 6h sleep deprivation clearly indicate that Homer1a is the best index of sleep need, consistently in all mouse strains analyzed. Transgenic mice expressing a FLAG-tagged poly(A)-binding protein (PABP) under the control of Homer1a promoter were generated. Because PABP binds the poly(A) tails of mRNA, affinity purification of FLAG-tagged PABP proteins from whole brain lysates, is expected to co-precipitate all mRNAs from neurons expressing Homer1a. Three other activity-induced genes (Ptgs2, Jph3, and Nptx2) were identified by this technique to be over-expressed after sleep loss. All four genes play a role in recovery from glutamate-induced neuronal hyperactivity. The consistent activation of Homer1a suggests a role for sleep in intracellular calcium homeostasis for protecting and recovering from the neuronal activation imposed by wakefulness. Experiment Overall Design: Experiments were performed on male mice, 12 weeks of age +/- 1 week. Animals were housed in polycarbonate cages (31x18x18cm) in an experimental room with an ambient temperature varying from 23Â° to 25Â°C under a 12:12 hrs light/dark cycle. Food and water were available ad libitum. At light onset mice were either sleep deprived by gentle handling (n=10) or left undisturbed (n=10) for 6 hrs. Animals were then randomly sacrificed by cervical dislocation. Total RNA from the whole brain was isolated for control (n=4) and sleep deprived (n=4) using a commercial RNA extraction kit (RNeasy Lipid Tissue Kit, Quiagen). Specific Homer1a-expressing cells polyA RNAs were immunoprecipitated following the total brain crosslinking (1% formaldehyde perfusion) for sleep deprived (n=6) and control (n=6) animals. The total RNA from the pull-down supernatants were also harvested (n=4). To test for transcriptional changes after sleep deprivation Homer1a-expressing cells, we proceeded in 2 steps: (1) identify probe sets enriched in the pull-down extracts, (2) among those probe sets compare sleep deprivation to control condition in both pull-down (6 vs. 6 chip comparison) and whole-brain (4 vs. 4 chip comparison) extracts. 4728 probe sets were significantly enriched at 5% FDR when pull-downs were compared to both supernatant and whole-brain extracts.

Dataset Information

Protease-resistant streptavidin for interaction proteomics

Publications

Protease-resistant streptavidin for interaction proteomics.

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