Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)

Abstract

Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community. Molecular & Cellular

A rapid and easy protein N‐terminal profiling strategy using (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip

“…When preparing samples before MS analysis, LC or SDS‐PAGE can be utilized to aid in the reduction of sample complexity and improve proteome coverage . Herein, StageTip was chosen as a fast and easy sample preparation method to achieve depth of coverage.…”

Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics

“…Sample Preparation for LC-MS/MS Analysis-The samples for the LC-MS/MS analysis were either in-gel or in-solution digested, as previously described (36,37). The samples were reduced using 5 mM DTT at 60°C for 15 min and were fully alkylated using 15 mM IAA at RT in the darkness for 30 min.…”

“…For LysargiNase digestion, the samples were solubilized in buffer (20 mM HEPES, 0.05% RapiGest, and 10 mM CaCl 2 , pH 8.3) with a final ratio of 1:100 (weight ratio of protease to protein) and incubated at 37°C overnight. The digested peptides were desalted using StageTip (36,38). The MS analysis was performed using LTQ-Orbitrap Velos (Thermo Electron, San Jose, CA) as previously described (36,38).…”

“…The digested peptides were desalted using StageTip (36,38). The MS analysis was performed using LTQ-Orbitrap Velos (Thermo Electron, San Jose, CA) as previously described (36,38). The resulting peptides were resolved in buffer A (2% acetonitrile in 0.2% formic acid) and loaded onto an in-house-packed capillary column (75 m i.d.…”

Enzyme–substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases

“…Only two candidates were chosen for validation; these proteins had the highest spectral counts, and they were subsequently validated as substrates. With a focus on further optimizing the TUBES-type pull-down approach, Xu and colleagues systematically tested the binding capabilities of a number of different Ubiquitin Binding Domains (UBDs) [100]. By testing the binding capacity of five different UBAs, both alone and in combination, for ubiquitin, the authors designed Tandem hybrid UBDs (ThUBDs) that had unbiased binding to all seven types of polyubiquitin chains.…”