Q exactive plus lc ms system

Mouse cerebral cortex was homogenized on ice with RIPA lysis and proteinase inhibitor mixture. After clearing debris by centrifugation, protein concentration was quantified with BCA Protein Assay Reagent. Total extracted proteins were incubated with anti-Pkm2 antibody (1:50) overnight. Rabbit IgG (30000-0-AP, Proteintech) was used as a negative IP control. The mixtures were incubated with beads for 4 h, and washed 4 times. IP sample and input control were boiled with SDS buffer for 10 min. After proteins were separated on 10% SDS-PAGE gels, gels were stained with Coomassie Brilliant Blue R250 (P0017B, Beyotime) for 2 h at room temperature. After washed with Coomassie Blue Staining Destaining Solution (P0017C, Beyotime) for 4 times overnight, protein bands were cut and pre-processed with acetonitrile. Samples were separated with UltiMate 3000 RSLCnano (Thermo Scientific), and the mass spectrometry was performed using Q Exactive Plus LCMS system (Thermo Scientific). The mass spectrum data were retrieved by MaxQuant (V1.6.2.10) using MaxLFQ algorithm. For phosphorylation site identification, a sample of purified GluA1 that was in vitro phosphorylated by Pkm2 was used. Phosphopeptide matches were analyzed by using PhosphoRS implemented in Proteome Discoverer.

The protein samples were digested in-gel (SDS-PAGE) with trypsin according to the method of Shevchenko et al. (2007) , adapted for use with a JANUS automated liquid handling workstation (Perkin Elmer, Beaconsfield, UK). The extracted peptide digestion products were dried in a vacuum centrifuge (SpeedVac) and dissolved in 10 µL LC-MS loading solvent. One-fifth (2 µL) was sampled for LC-MS analysis as described previously by Ni et al. ( 2018) using a Q Exactive Plus LC-MS system (Thermo Scientific, Hemel Hempstead, UK) and a 'Top 10' data-dependent MS2 method. All parameters were as previously described by Ni et al. (2018) . Database searches were carried out against the Capra hircus (Goat) protein sequences, downloaded from www.uniprot.org/uniprot in fasta format. A Mascot significance value of 0.05 was used to filter unreliable peptide matches.