Q exactive mass spectrometry system

Rat brain lysate was incubated with glutathione Sepharose beads coated with GST-CaMKI-cat (500 pmol). The proteins bound to CaMKI-cat were incubated with or without ATP and Mg²⁺ and then denatured with guanidine hydrochloride and digested with trypsin for 16 h at 37 °C following reduction, alkylation, demineralization, and concentration. The phosphopeptides were concentrated using a Titansphere Phos-TiO Kit (GL Sciences) according to the manufacturer’s instructions. Nanoelectrospray tandem mass analysis was performed using a Q Exactive mass spectrometry system (Thermo Fisher Scientific Inc.) combined with an HTC-PAL autosampler and a Paradigm MS4 HPLC System (Michrom BioResources Inc.) using a C18 reverse-phase column and a Michrom ADVANCE Plug-and-Play NanoSpray Source. A peak list was generated and calibrated using MaxQuant software. Database searches were performed against the complete proteome set of Rattus norvegicus in UniProt KB 2014_07 concatenated with reverse copies of all sequences. The ion intensities of the identified peptides in the phosphorylated sample were compared with those of the nonphosphorylated sample, and the phosphorylation sites exhibiting greater than fivefold increases in ion intensity were regarded as candidate substrates^{36 (link)}.

Biotin-labeled AS-tDR-007333 probe and control probe were transcribed in vitro using the transcript Aid T7 High Yield Transcription Kit (Thermo Scientific, Shanghai, China) according to the manufacture’s guidelines. PC9 cells were cultured in RIPA Lysis and Extraction Buffer (Thermo Scientific), and the supernatant was incubated with biotinylated probes and then mixed with Dynabeads MyOne Streptavidin C1 beads (Thermo Scientific, Shanghai, China). Then, the RNA–protein mixture was boiled in SDS buffer, subjected to SDS-PAGE and silver staining. Protein bands with significant differences were cut and subjected to protein mass spectrometry on the Q Exactive Mass spectrometry System (Thermo Scientific). Raw mass spectrometry data were processed using MM File Conversion, and protein identifications were analyzed using Mascot v2.6.0 against the Human UniProt database.