CMRI Synapse Proteomics - SLK

Activity-dependent regulation of SLK phosphorylation
Data License: CC BY 4.0 | ProteomeXchange: PXD036547 | doi:
  • Organism: Mus musculus
  • Instrument: Q Exactive
  • SpikeIn: No
  • Keywords: parallel reaction monitoring, Ste20-like kinase, neurons
  • Lab head: Mark Graham Submitter: Mark Graham
Maintaining an appropriate balance between excitation and inhibition is critical for information processing in cortical neurons. It is known that cortical neurons receive widely disparate levels of excitation. To ensure efficient coding, they are capable of cell-autonomously adjusting the inhibition they receive to the individual levels of excitatory input, but the underlying mechanisms are not understood. The article associated with this data shows that Ste20-like kinase (SLK) in cortical neurons mediates cell-autonomous regulation of excitation-inhibition balance in the thalamocortical feed-forward circuit, but not in the feed-back circuit. The parallel reaction monitoring data here supports the link between activity, SLK phosphorylation and function.
Experiment Description
Each phosphopeptide sample in 5 μL was injected into a Dionex UltiMate 3000 RSLC nano system and analysed by a Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific). The column was 300 × 0.075 mm fused silica with a pulled tip and packed with ReproSil Pur C18 AQ 1.9 μm resin (Dr Maisch, Germany). The electrospray conditions were as described previously (Müller et al., 2022, PMID:35443170 }. The sample was loaded in 99% buffer A (0.1% formic acid in water) and 1% buffer B (0.1% formic acid, 90% acetonitrile, 9.9% water) for 25 min at 300 nL/min, then the gradient was to 5% B in 1 min at 250 nL/min, to 25% B in 74 min, to 35% B in 8 min, to 99% B in 1 min, held at 99% B for 2 min before returning to 1% B in 1 min and held at 99% B for 8 min whilst the flow rate was increased to 300 nL/min for equilibration. Parallel reaction monitoring MS was performed for 120 min. The MS/MS resolution was 70,000, the automatic gain control target was 3,000,000, the maximum ion time was 220 ms, the isolation window was 0.7 m/z and the normalised collision energy was 30. Approximately 2500 ng of non-phosphopeptide in 3.5 μL was injected using a different gradient for analysis. The sample loading was in 99% A for 17.5 min at 300 nL/min, then the gradient was to 6% B in 1 min at 250 nL/min, to 28% B in 101.5 min, and then the gradient continued the same as for phosphopeptides to a total length 140 min. The parallel reaction monitoring MS settings were the same as for phosphopeptides. Phosphopeptides and non-phosphopeptides were selected from previous analyses in our laboratory and from public databases. Three phosphopeptides that have stable levels of phosphorylation in a selection of mouse phosphoproteomic studies were includes for normalisation. Two peptides from tubulin alpha-4A chain and four peptides from cytoplasmic dynein 1 heavy chain 1 were included as loading controls. Each peptide was targeted within a 12 min or wider retention time window. All MS files were searched using MaxQuant to create a library of search results as evidence of correct identification and retention time. Within MaxQuant, the Mus musculus reference proteome with 55,315 entries, downloaded from UniProt on August 11 2022, was used, as well as the in-built contaminants fasta file. Oxidation of Met, acetylation of N-terminal residues, phosphorylation of Ser/Thr/Tyr and deamidation of Asn/Gln were variable modifications and carbamidomethylation of Cys was a fixed modification. Minimum peptide length was 6, trypsin/P was used for enzyme specificity with a maximum of 2 missed cleavages. All other MaxQuant settings were default. The MaxQuant msms.txt output file and the raw files were examined in Skyline Five or more fragment ions and a maximum of 2 ppm mass accuracy was required for each peptide. The quantitative values for each peptide were exported to Excel and the peptide intensities were normalised to the loading controls. The data was then exported to Prism 9.0.2 for statistical analysis. Multiple unpaired t-tests were performed and the P value was adjusted with a false discovery rate of 5%. All data with an adjusted P value > 0.05 was considered significant.
Sample Description
Male C57Bl6/N mice were injected with 1 mg/kg scopolamine methyl nitrate (Sigma). Twenty minutes later, status epilepticus was induced by subcutaneous injection with 335 mg/kg pilocarpine hydrochloride (Sigma). At 4 and 24 h after injection of pilocarpine, both hemispheres of the hippocampus were removed and frozen using dry ice. Tissue was homogenized in 500 µl PBS (45 sec), before 125 µl lysis buffer was added [2% SDS, 50 mM trisaminomethane/HCl pH 7.4, 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N0, N0-tetraacetic acid, 2 mM ethylenediaminetetraacetic acid, Complete Protease Inhibitor Cocktail (Roche), 2 mM phenylmethylsulfonyl (Sigma-Aldrich), 5 mM NaF (Sigma-Aldrich), 2 mM beta-glycerophosphate (Sigma-Aldrich), Phosphatase Inhibitor Cocktail 2 (1:1000) and PhosSTOP (Roche)]. Lysates were incubated at 85°C for 10 min and subsequently sonicated for 3 x 10 sec. Samples were frozen on dry ice before being lyophilized. samples were resuspended in a 100 μL solution of 10 mM tris(2-carboxyethyl) phosphine, reduced for 10 minutes at 85°C and subsequently alkylated in 25 mM iodoacetamide at 22°C in the dark. Proteins were precipitated from the samples using chloroform-methanol extraction (Wessel, 1984, PMID:6731838) and air dried. The precipitates were dissolved in 20 µL solution containing 7.8 M urea, 50 mM triethylammonium bicarbonate (TEAB) and 5 µg of Lys-C (FUJIFILM Wako Pure Chemical Corporation) for an 8-hour digestion at 25°C. The samples were diluted with a solution containing 170 µL 100 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid (HEPES) (pH 8) and 5 µg TrypZean trypsin (Sigma) for subsequent digestion at 37°C for 4 hours. The trypsin digestion was repeated with an additional 5 µg for 4 hours under the same conditions. Approximately 600 ug of each sample was purified by solid phase extraction (Oasis 30 mg sorbent, Waters) and enriched for phosphopeptides using titanium dioxide enrichment as previously described {Engholm-Keller, 2016, PMID:26584925 }. One third of purified phosphopeptide material was analysed by MS.
Created on 9/5/22, 10:33 PM
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