Leaves were homogenized, centrifuged, and mixed with 25 µL RFP-Trap Agarose or GFP-Trap Agarose (ChromoTek). Beads were spun down, washed, and stored at −80 until mass spectrometry analysis. The washed beads were incubated for 30 min with elution buffer. Tryptic peptide mixtures were loaded on Evotips (Evosep) and peptides were separated and injected via a CaptiveSpray source and 10-μm emitter into a timsTOF pro mass spectrometer (Bruker) ran in PASEF mode (89 (link)).
Raw mass spectrometry data were analyzed with MaxQuant against the Arabidopsis Uniprot FASTA database. Statistical analyses of LFQ-derived protein expression data were performed using the automated analysis pipeline of the Clinical Knowledge Graph (90 (link)). Differentially expressed proteins in each group comparison were identified by SAMR multiclass test with permutation-based FDR correction for multiple hypothesis, followed by post hoc pairwise comparison unpaired t tests using the same parameters and permutation-based FDR correction (91 ). Significantly regulated proteins are colored in red and blue in the volcano plots for up and down-regulated proteins, respectively.
Raw mass spectrometry data were analyzed with MaxQuant against the Arabidopsis Uniprot FASTA database. Statistical analyses of LFQ-derived protein expression data were performed using the automated analysis pipeline of the Clinical Knowledge Graph (90 (link)). Differentially expressed proteins in each group comparison were identified by SAMR multiclass test with permutation-based FDR correction for multiple hypothesis, followed by post hoc pairwise comparison unpaired t tests using the same parameters and permutation-based FDR correction (91 ). Significantly regulated proteins are colored in red and blue in the volcano plots for up and down-regulated proteins, respectively.
