Dynamics of the Arabidopsis diurnal proteome and phosphoproteome
Using proteotypic peptides and label-free quantitative proteomics analysis we identified 7060 unique proteins, of which we were able to quantify 4762 proteins with two and more proteotypic peptides over the 24 h photoperiod time-course (Supplemental Figure 1; Table 1; Supplemental Table 1). Statistical analysis showed that 288 of these proteins were significantly changing in abundance (ANOVA P value ≤ 0.05, FC > 1.5); Table 1; Supplemental Table 2), suggesting that a portion (~6%) of the identified and tracked proteome is dynamically regulated during the diurnal cycle. Additionally, using a dimethyl-labeling approach we identified a total of 2298 phosphopeptides (Supplemental Figure 1; Supplemental Table 3), of which 1776 had a phosphorylation site probability score ≥ 0.8. We were able to quantify 1056 of these phosphopeptides (present in at least 2 biological replicates and in 3 out of 3 time points for each transition; Table 1). This corresponded to a total of 1803 identified phosphorylation sites, of which 253 (14%) represented newly identified phosphorylation sites when compared to the compendium of 79,334 known phosphorylation sites (PhosPhat 4.0; Heazlewood et al., 2008). A total of 271 phosphopeptides on 226 proteins (~26% of all quantified phosphopeptides) significantly changed in abundance (ANOVA P value ≤ 0.05) at either the D-L, L-D or both transitions (Table 1; Supplemental Table 4).