To identify PLK-1-driven changes in SPD-5 interactions, we found it was necessary to start with completely dephosphorylated SPD-5. Purification of dephosphorylated SPD-5 was achieved through modification of our standard protocol. In short, insect cell lysates were passed through 4 ml of Ni-NTA beads twice, washed five times with buffer 1 (25 mM HEPES, 500 mM NaCl, 30 mM imidazole, 1% glycerol, 0.1% CHAPS, pH 7.4), then twice with buffer 2 (150 mM KCl, 25 mM HEPES, pH 7.4) at 4°C. Ni-NTA-bound SPD-5 was incubated for 1 h at room temperature in dephosphorylation buffer (1× PMP buffer (NEB) + 1 mM MnCl2), + 40,000 U lambda phosphatase (400,000 U/ml, NEB). Beads were then washed twice with buffer 1 at 4°C. Dephosphorylated SPD-5 was eluted from the Ni-NTA beads using 15 ml of Buffer 3 (25 mM HEPES, 500 mM NaCl, 250 mM imidazole, 1% glycerol, 0.1% CHAPS, pH 7.4). SPD-5 was then bound to 500 μl MBP-trap beads (Chromotek) and the column was washed 3× with buffer 4 (25 mM HEPES, 500 mM NaCl, 1% glycerol, 0.1% CHAPS, pH 7.4). Dephosphorylated SPD-5 was eluted from the MBP-trap beads by overnight incubation in buffer 4 + 100 μl of PreScission protease (1 mg/ml; Acro Biosystems) at 4°C. Eluted protein was further purified and stored as described above. Dephosphorylation of SPD-5 was confirmed by PTM identification using mass spectrometry showing effective removal of 99.8–100% of phosphates.

Crosslinking reactions were prepared at room temperature with 1 μM dephosphorylated SPD-5, 1 μM PLK-1 (KD/CA), 0.2 mM ATP, 10 mM MgCl2, 150 mM KCl, 25 mM HEPES, pH 7.4 and 0.5 mM DTT. To analyze SPD-5 multimers, samples were incubated for 2 h at room temperature, followed by the addition of 8 mM DMTMM for 45 min at room temperature (shaking at 300 rpm). To enrich for monomers, samples were incubated at room temperature for 2 h, then chilled on ice and gently pipetted for 10 min before adding DMTMM. To quench the reaction, we added 50 mM ammonium bicarbonate for 15 min at room temperature (shaking at 300 rpm). Samples without crosslinker were used for mass spectrometry PTM analysis to identify phosphorylated sites (Data S1).

Samples were run on an SDS-PAGE gel to separate crosslinked species. Bands corresponding to monomeric or multimeric protein were excised from the gel, then digested overnight with trypsin (Pierce), reduced with DTT, and alkylated with iodoacetamide (Sigma-Aldrich). Samples were cleaned using solid-phase extraction with an Oasis HLB plate (Waters), then injected into an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Peptides were separated using a 75-μm i.d., 75-cm long EasySpray column (Thermo Fisher Scientific) and eluted with a gradient at a flow rate of 250 nl/min from 0 to 5% buffer B over 1 min, 5%–40% B over 60 min, 40%–99% over 25 min, and held at 99% B for 5 min before returning to 0% B for column equilibration. Buffer A contained 2% (vol/vol) ACN and 0.1% formic acid in water, and buffer B contained 80% (vol/vol) ACN, 10% (vol/vol) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 1.5–2.4 kV and an ion transfer tube temperature of 275°C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using collision-induced dissociation (CID) for ions with charges 3–7. Dynamic exclusion was set for 25 s after an ion was selected for fragmentation.

For data analysis, each Thermo.raw file was converted to.mzXML format for analysis using an in-house installation of xQuest (Leitner et al., 2014). Score thresholds were set through xProphet (Leitner et al., 2014), which uses a target/decoy model. The search parameters were set as follows. For zero-length crosslink search with DMTMM: maximum number of missed cleavages = 2, peptide length = 5–50 residues, fixed modifications carbamidomethyl-Cys (mass shift = 57.02146 Da), mass shift of crosslinker = −18.010595 Da, no monolink mass specified, MS1 tolerance = 15 ppm, and MS2 tolerance = 0.2 Da for common ions and 0.3 Da for crosslink ions; search in enumeration mode. The false discovery rates (FDR) were <20% (SPD-5(FL) or <5% (SPD-5[541–677]) at the link level (see Datas S3, S4, and S5 for further information about crosslinked peptide pairs). Samples were also reanalyzed accounting for mass shifts due to the presence of phospho-serines and phospho-threonines. A cumulative list of crosslinked pairs can be found in Data S2.