Fig. 2: Dynamics of kinetochore proximal phosphosites during kinetochore assembly revealed by spatially referenced proximity phosphoproteomics.

a Schematic of spatially referenced proximity phosphoproteomics workflow to quantify proximal proteins and proximal phosphosites from the same sample. Proximity biotinylation and DSP protein-crosslinking capture proximal proteins in the protein of interest sample and spatial reference sample. Proteins are on-bead digested into peptides for protein and phosphopeptide identification. Proteins or phosphosites significantly enriched (red dots) compared to the spatial reference sample are classed as proximal. b Using KKT3::mT and spatially referenced proximity phosphoproteomics to follow kinetochore assembly during the cell cycle in synchronised parasites. A 30 min biotinylation ‘snapshot’ was taken at three timepoints after release from hydroxyurea synchronisation. BDF5::mT was used as a compartment-matched spatial reference. Histograms of parasite DNA content are shown at each timepoint. The dotted line represents early S (ES) phase c Radial plots of proteins enriched in KKT3::mT samples compared to spatial reference BDF5::mT samples at each timepoint. Log2 fold enrichment increases in a clockwise direction, log10 p values increase from the centre outwards. The dashed line indicates a 1% FDR cutoff for distinguishing proximal (red points) and non-proximal (grey points) proteins. Label-free quantification was performed on five independent biological replicates. d Radial plots of phosphosites enriched in KKT3::mT samples compared to spatial reference BDF5::mT samples at each timepoint. The dashed line indicates a 5% FDR cutoff for distinguishing proximal (coloured points) and non-proximal (grey points) phosphosites. Label-free quantification was performed on five independent biological replicates. Where a phosphosite could not be confidently localised, the possible phosphorylated region is indicated.