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.
Highlights
From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China; the ¶School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; the Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China; and the **Departments of Structural Biology and Developmental Neurobiology, St
To address the bias in ubiquitin-binding domains (UBDs) and efficiently recover the ubiquitinome during purification, we systematically evaluated the affinity of a number of UBDs, and we constructed an artificial tandem hybrid UBD (ThUBD) that exhibited no strong bias toward any of the seven tested polyubiquitin chains
Binding Affinity of the Dimer of ThUBDs Reaches to Saturation—To optimize UBDs for the ubiquitinated proteomics study, we investigated the effects of the tandem units on ubiquitin binding affinity
Summary
Protein Purification, and Immobilization—The UBDs used in this study were DSK2p-derived UBA (DSK2, 327–371 amino acids), ubiquilin 1-derived UBA (UQ1, 541–586 amino acids), ubiquilin 2-derived UBA (UQ2, 575– 624 amino acids), RABGEF1-derived A20-ZnF (A20, 9 –73 amino acids), and HDAC6-derived ZnF-UBP (HDAC6, 985–1152 amino acids). Targeted Quantitative MS of Polyubiquitin Linkages Using the Stable Isotope Labeling by Amino Acids in Cell Culture-Absolute Quantification (SILAC-AQUA) Approach—To accurately quantify the abundance of the seven polyubiquitin chains from each ubiquitin conjugate purified with different UBDs, ubiquitin conjugates labeled with heavy isotope 13C6-lysine (Lys-6) and 15N413C6-arginine (Arg-10) were purified with nickel beads as a quantification standard to mix with each sample [18]. Eluted peptides were detected on the Orbitrap mass spectrometer in a survey scan (300 –1600 m/z, resolution 30,000) followed by selective reaction monitoring scans for seven ubiquitin chains ions in the LTQ. Purified GST-UBD proteins were resuspended in PBS buffer and immobilized onto NanoCapture GoldTM SPR-active slides following the standard amine coupling chemistry, as described previously [54]. The reconstructed images revealed the different purification methods with regard to molecular weight and abundance
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