Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs

Abstract

ORCID ID: 0000-0001-9536-0487 (K.J.v.W.)Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry-based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts.

Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways

“…Using this strategy, we identified a total of 2104 unique phosphosites, spanning 1586 phosphopeptides on 1080 proteins from unfractionated Arabidopsis seedling lysates (supplemental Table S1). These numbers are similar in size to previously reported phosphoproteome data sets (40,59). When compared with other global phosphoproteome studies we found that 159 phosphopeptides are newly reported (supplemental Table S2) (59).…”

“…These numbers are similar in size to previously reported phosphoproteome data sets (40,59). When compared with other global phosphoproteome studies we found that 159 phosphopeptides are newly reported (supplemental Table S2) (59). The distribution of phosphorylation events was Ser (89%), Thr (10%), and Tyr (1%) (supplemental Fig.…”

Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms

“…Light-dependent phosphorylation modulates the activity of proteins involved in photosynthesis (Bellafiore et al, 2005;Bonardi et al, 2005;Pesaresi et al, 2009;Rochaix et al, 2012). Analysis of 27 phosphoproteomic data sets of Arabidopsis (Arabidopsis thaliana) protein extracts identified over 60,000 phosphorylation sites in 8,141 proteins (van Wijk et al, 2014) with over 300 identified to be plastid localized (Schönberg et al, 2014). Important chloroplast processes, including metabolism, photosynthesis, gene expression, and signaling, are often controlled by protein phosphorylation in acclimation to environmental changes (Bellafiore et al, 2005;Bonardi et al, 2005;Salinas et al, 2006;Baginsky and Gruissem, 2009).…”

On the Extent of Tyrosine Phosphorylation in Chloroplasts

“…Many of the phospho-Tyr-containing peptides were identified in analyses that applied multistage activation to elevate fragment ion intensity in spectra dominated by the neutral loss of phosphoric acid from phosphoSer and/or phospho-Thr, sometimes in combination with searches for the phospho-Tyr-specific immonium ion at mass-to-charge ratio 216.0426 (see table 1 in van Wijk et al, 2014). In one instance, phospho-Tyr-specific antibodies were used to enrich Tyr phosphorylated proteins from Arabidopsis full cell extracts (Mithoe et al, 2012).…”

“…However, mass spectrometry-based phosphoproteomics experiments with plant cell extracts reported phospho-Tyrcontaining peptides in chloroplasts but surprisingly not in abundant thylakoid membrane proteins or Calvin cycle enzymes. A recent meta-analysis collated data from 27 published studies and several internal data sets, resulting in a cumulative data set with 5% Tyrphosphorylated peptides in the entire data set and 12% to 19% in the mitochondria (van Wijk et al, 2014). In this data set, almost 30% of the plastid phosphoproteins are flagged as Tyr phosphorylated (90 proteins from around 300; see supplemental table 5B in van Wijk et al, 2014), standing in stark contrast to dedicated plastid phosphoproteome analyses that identified less than 1% Tyr phosphorylation in the cellular phosphoproteome and none in chloroplast proteins (Reiland et al, 2009).…”