Intermolecular interactions stabilising the CAL1–CENP-A/H4 complex were analysed using chemical Cross-Linking Mass Spectrometry (CLMS). Purified recombinant CAL11-160–CENP-A101-225–H4 complex was crosslinked using EDC and BS3. The crosslinked peptides were analysed by mass spectrometry to identify intra- and intermolecular contacts. Notably, the data revealed several intramolecular crosslinks between the N- and C-terminal regions of CAL11-160, suggesting a direct interaction between these regions.
Crosslinking was performed on gel filtered complexes dialysied into PBS. 30 ug EDC (Thermo Fisher Scientific) and 66 ug sulfo-NHS (Thermo Fisher Scientific) were used to crosslink 10 ug of protein for 1.5 h at RT. 30 ug of BS3 was used to crosslink 10 ug of protein for 2 h at RT. The reactions were quenched with Tris-HCl or ammonium bicarbonate respectively before separation on 4-12% Bis-Tris plus gels (Invitrogen). Following previously established protocol (Maiolica, Cittaro et al., 2007), the bands were excised and proteins were digested with trypsin (Pierce) overnight at 37˚C after being reduced and alkylated. The digested peptides were loaded onto C18-Stage-tips (Rappsilber, Mann et al., 2007) for LC-MS/MS analysis. LC-MS/MS analysis was performed using an Orbitrap Fusion Lumos (Thermo Fisher Scientific) with a “high/high” acquisition strategy. The peptide separation was carried out on an EASY-Spray column (50 cm × 75 μm i.d., PepMap C18, 2 μm particles, 100 Å pore size, Thermo Fisher Scientific). Mobile phase A consisted of water and 0.1% v/v formic acid. Mobile phase B consisted of 80% v/v acetonitrile and 0.1% v/v formic acid. Peptides were loaded at a flow rate of 0.3 μl/min and eluted at 0.2 μl/min using a linear gradient going from 2% mobile phase B to 40% mobile phase B over 109 followed by a linear increase from 40% to 95% mobile phase B in 11 min. The eluted peptides were directly introduced into the mass spectrometer. MS data were acquired in the data-dependent mode with a 3 s acquisition cycle. Precursor spectra were recorded in the Orbitrap with a resolution of 120,000. The ions with a precursor charge state between 3+ and 8+ were isolated with a window size of 1.6 m/z and fragmented using high-energy collision dissociation (HCD) with a collision energy of 30. The fragmentation spectra were recorded in the Orbitrap with a resolution of 15,000. Dynamic exclusion was enabled with single repeat count and 60 s exclusion duration.
Medina-Pritchard B, Lazou V, Zou J, Byron O, Abad MA, Rappsilber J, Heun P, Jeyaprakash AA. Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1. EMBO J. 2020:e103234, PubMed: 32134144