In collaboration with Dr. Justin Walley (PLPM), we have recently characterized a large-scale catalog of ubiquitinated proteins from Arabidopsis roots using diGly affinity purification followed by mass spectrometry.
Ubiquitination is a key posttranslational modification that can impact protein function and stability. Proteomic identification of ubiquitinated proteins as well as specific sites of ubiquitination in plant model species have lagged behind other eukaryotic models and thus represent a knowledge gap within the plant science community. In this project, we generated an improved workflow for identification of ubiquitinated peptides from Arabidopsis using an immunoprecipitation followed by mass spectrometry (IP-MS/MS) approach, which is often termed “diGly” enrichment. We also established an isobaric labelling method for diGly enriched peptides from plant samples using Tandem Mass Tags (TMT), which enables relative quantification of up to 16 samples in a single mass spectrometry analysis. Critically, TMT multiplexing improves sample throughput and significantly minimizes the number of missing peptides quantified across all experimental conditions. Finally, this method provides an accessible protocol with commercially available reagents.
We report a deep catalogue comprised of 7,130 ubiquitin sites arising from 3,178 proteins detected in primary Arabidopsis roots. This is significant because it represents ~140% increase in identified ubiquitin sites compared to previous reports in Arabidopsis. Furthermore, we quantified changes in both protein abundance and ubiquitin site levels following auxin (indole-3-acetic acid, or IAA) and 26S proteosome inhibition (using the chemical bortezomib). Gene Ontology analyses determined that ubiquitinated proteins fall into numerous biological processes including hormone signaling, protein homeostasis, and root morphogenesis. We uncovered a novel enriched QKUb motif among proteins regulated by bortezomib. Finally, we observed widespread modifications on transcription factors. Thus, we hypothesized that ubiquitination of transcription factors may impact protein stability and tested this idea using in vivo degradation assays. In doing so, we identified specific lysine residues on the transcription factors CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) that mediate their stability.
This project provides a wealth of quantitative proteomics data for a key organ in Arabidopsis which can be integrated with existing databases such as the Plant PTM viewer and queried for functional genomics studies. The datasets generated in this project are an important proteomics resource that is of high value and interest to the community.
Duration: 05/14/2021