Phosphoproteomics of Synchronized BY-2 Cells

For phosphoproteomics, BY–GTRC cells at 7 d after transfer to fresh medium were synchronized at the DNA replication stage (synthesis [S] phase) as described previously (Nagata & Kumagai, 1999 (link); Kumagai-Sano et al, 2006 (link)). The cells were cultured in the presence of PD-180970, PD-173955-Analog1, PP2, or PP3 for 8–9 h. After confirming that most cells started mitosis using an upright microscope (AxioImager A2; Zeiss), total proteins were extracted using cell lysis buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% [vol/vol] Triton X-100, 25 μM MG-132, and cOmplete Mini Protease Inhibitor Cocktail [Roche]). After the extraction of crude proteins and trypsin digestion, peptides were purified using an immobilized metal ion affinity chromatography column or a sequential enrichment of immobilized metal affinity chromatography column, both of which specifically absorb phosphopeptides. Their amino acid sequences were determined using a high-sensitivity nanoLC-MS/MS system, as previously described (Ohkubo et al, 2021 (link)).
The protein sequences of BY-2 cells were predicted based on the transcriptome (RNA-seq) data, which have been previously reported (Kozgunova et al, 2016 (link)). Each of the transcript data was converted to an amino acid sequence, and a total of 50,171 protein sequences (coded from NtBYT000000.000 to NtBYT078147.000 in Supplemental Data 1) were used as a reference database to map the identified phosphopeptides by MASCOT search to determine the corresponding proteins. Identified proteins and their homologous proteins were aligned using Clustal Omega software (https://www.ebi.ac.uk/Tools/msa/clustalo/). Predictions of the MT-binding region in MAP70 proteins have been previously reported (Korolev et al, 2005 (link)). Kinesin motor domains and coiled-coil domains in PAKRP1 and PAKRP1L were predicted using UniProt (https://www.uniprot.org).