Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

Abstract

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (Mud-PIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

“…The search parameters were similar to those used by Maor et al (22) and were: mass ϭ average, precursor ion tolerance ϭ Ϯ 2 Da, enzyme ϭ trypsin, allowed missed cleavages ϭ 3, variable modifications of methionine, and lysine (ϩ114.1026 Da) to account for the diglycine tag, and static modifications of cysteine. Spectrum assignments to unambiguous peptides with a probability Ͼ 0.1 were inserted into the pep2pro database.…”

“…The raw mass spectrometry data described in Maor et al (22) were converted into mzXML file format and the data were searched with SEQUEST/PeptideProphet against the TAIR7 decoy database with supplemented contaminants (fgcz_3702_TAIR7_ 20070425decoy.fasta). The search parameters were similar to those used by Maor et al (22) and were: mass ϭ average, precursor ion tolerance ϭ Ϯ 2 Da, enzyme ϭ trypsin, allowed missed cleavages ϭ 3, variable modifications of methionine, and lysine (ϩ114.1026 Da) to account for the diglycine tag, and static modifications of cysteine.…”

“…The identification of ubiquitylated proteins was mainly achieved using ubiquitin binding domain (UBAs) affinity chromatography and mass spectrometry (22)(23)(24)(25)(26). Here we report a similar approach for the identification of ubiquitylated Arabidopsis proteins, but using a different experimental method, which allowed us to detect proteins that until now have been refractory to identification.…”

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

“…Suspension cultures of Arabidopsis thaliana MM1 were basically maintained as described (Maor et al, 2007). Arabidopsis MM1 cells were grown in flasks in 100 mL of liquid Murashige and Skoog (MS) medium containing 3% Suc supplemented with 0.5 mg/L MES, pH 5.7, 0.5 mg/L naphthaleneacetic acid, and 0.05 mg/L 6-benzylaminopurine under longday conditions at 22°C on a shaker (120 rpm).…”

From proteomics toward systems biology: integration of different types of proteomics data into network models

“…However, due to the detection limit of MS, only abundant peptides can be detected (commonly referred to as an undersampling issue) when a sample includes a large number of proteins at comparable concentrations (7). However, several thousands of proteins can be detected using current technologies when additional fractionation methods, such as, 2-D gel electrophoresis (13) or Multidimensional Protein Identification Technology (MudPIT) using Strong Cation Exchange (SCX) (14,15), are used to further resolve sample complexity. MS-based proteomic analysis commonly involves the following steps: 1) sample preparation, 2) LC-MS/MS analysis, and 3) computational analysis of the MS data for protein quantification and identification (16).…”