Preparing to read the ubiquitin code: a middle‐out strategy for characterization of all lysine‐linked diubiquitins

Abstract

Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis, and many other cellular functions. There is great interest in, and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry-based studies of the ubiquitin code we have assembled seven isomeric diubiquitins with all-native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new mass spectrometry-based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here we report the use of Asp-selective acid cleavage, separation by reverse phase HPLC, and characterization by tandem mass spectrometry to distinguish and characterize all seven isomeric lysine-linked ubiquitin dimers.

Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top‐down mass spectrometry

“…Ubiquitin trimers were diluted to 0.15 mg/ml in 12.5% acetic acid and digested for 60 s at 140 °C using 300 W of power in a CEM Discover microwave (Matthews, NC). These conditions have been previously determined to produce partial cleavage of polyubiquitins at Asp residues . Digested trimers were lyophilized and resuspended in solvent A (97.5% water, 2.5% ACN, and 0.1% formic acid) at 0.1 mg/ml for chromatography performed using the liquid chromatography–tandem mass spectrometry system specified in the preceding texts.…”

“…These conditions have been previously determined to produce partial cleavage of polyubiquitins at Asp residues. [10,11] Digested trimers were lyophilized and resuspended in solvent A (97.5% water, 2.5% ACN, and 0.1% formic acid) at 0.1 mg/ml for chromatography performed using the liquid chromatography-tandem mass spectrometry system specified in the preceding texts. Five microliters of each digested sample were injected, concentrated, and desalted on a Zorbax C8 trap (0.5 × 3 mm; Agilent Technologies) for 5 min before being separated on a Zorbax C8 column (3.5 μm, 150 mm × 75 μm; Agilent Technologies, Santa Clara, CA) with a 500 nL/min flow rate and a gradient of 30% to 37% solvent B (solvent B: 75% ACN, 25% water, and 0.1% formic acid) over 45 min.…”

“…Two strategies have been evaluated to shorten the branches in polyubiquitins and still retain a connectivity backbone: timelimited acid cleavage and limited trypsinolysis. [10,11,15,22] Both of these middle-out approaches have been demonstrated successfully on tri-Ubs, the focus of the present work. In time-limited studies a large mixture of peptides is created, reflecting many potential cleavage sites.…”

“…This strategy has been used very successfully in identifying ubiquitinated proteins; however, it has the drawback that the tag (GG) does not retain information on the length or connectivity of polyubiquitin modifications. Two strategies have been evaluated to shorten the branches in polyubiquitins and still retain a connectivity backbone: time‐limited acid cleavage and limited trypsinolysis . Both of these middle‐out approaches have been demonstrated successfully on tri‐Ubs, the focus of the present work.…”

Prostate cancer-associated mutation in SPOP impairs its ability to target Cdc20 for poly-ubiquitination and degradation

“…Based on Cullin scaffold proteins (Cullin1, 2, 3, 4A, 4B, 5 and 7), CRLs can be categorized into seven subfamilies (termed CRL1 through CRL7, respectively) [2, 20]. Among these CRLs, the CRL1 complex (also named SCF), is well studied at both biochemical and physiological levels [21, 22]. Recently, the emerging CRL3 subfamily complex is identified as major regulators for different cellular processes and disruption of this degradation pathway has been linked to various human diseases, including nerve degeneration and cancers [23].…”