Computational protocol: NLK-mediated phosphorylation of HDAC1 negatively regulates Wnt signaling

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Protocol publication

[…] Protein samples were separated by SDS–PAGE, and selected gel bands were excised for analysis. After reduction and alkylation using DTT and IAA, respectively, in-gel digestion was performed by using porcine trypsin (Promega) as endoprotease. After extraction, peptides were phosphoenriched by using TiO2 Mag Sepharose beads (GE Healthcare Life Sciences). Dry peptides were resuspended in 0.1% formic acid and separated by using an Eksigent NanoLC 2D system (Eksigent, Dublin, CA) that was coupled to an LTQ-Orbitrap XL ETD mass spectrometer (Thermo Fisher Scientific, Germany) operated in data-dependent acquisition mode. The peptides were loaded and washed for 15 min on a precolumn (Acclaim PepMap 100, C18, 3-μm particle size, 50-μm diameter; Thermo Fisher Scientific, Germany) at a constant flow of 5 μl/min solvent B (0.1% formic acid [FA] in acetonitrile [ACM]) before separation on an analytical column (10-μm fused silica emitter, 75-μm × 16-cm Pico Tip Emitter; New Objective) packed in-house with C18 material ReproSil-Pur. A linear 60-min gradient in 0.1% FA buffer from 3–90% ACN at a flow rate of 300 nl/min was used. The MS/MS analysis was performed by using multistage activation and top 10 collision-induced dissociation and detection in the linear ion trap, rejecting molecules with an unassigned charge state or a charged state. MS/MS data were converted into Mascot Generic Format (MGF) by using Proteo­Wizard (www.nature.com/nbt/journal/v30/n10/full/nbt.2377.html) and searched against the SwissProt part of the UniProt database. Mascot 2.4.1 (www.matrixscience.com) searches were performed from Proteios Software Environment (http://pubs.acs.org/doi/abs/10.1021/pr900189c). Complementary searches in X!Tandem and MS-GF+ (www.nature.com/ncomms/2014/141031/ncomms6277/full/ncomms6277.html) were performed by using SearchGUI (www.ncbi.nlm.nih.gov/pubmed/21337703), and annotated spectra were visualized in PeptideShaker (www.nature.com/nbt/journal/v33/n1/full/nbt.3109.html). For all searches, 10-ppm precursor tolerance and 0.5-Da fragment tolerance, fixed carbamidomethylation of C, variable oxidation of M and variable phosphorylation of ST, and up to one missed cleavage were used as search settings. […]

Pipeline specifications

Software tools ProteoWizard, ProSE, MS-GF+, SearchGUI, PeptideShaker
Application MS-based untargeted proteomics
Organisms Mus musculus