Ubiquitin diGLY Proteomics as an Approach to Identify and Quantify the Ubiquitin-Modified Proteome

Abstract

Protein ubiquitylation is one of the most prevalent post-translational modifications (PTM) within cells. Ubiquitin modification of target lysine residues typically marks substrates for proteasome-dependent degradation. However, ubiquitylation can also alter protein function through modulation of protein complexes, localization or activity, without impacting protein turnover. Taken together, ubiquitylation imparts critical regulatory control over nearly every cellular, physiological, and pathophysiological process. Affinity purification techniques coupled with quantitative mass spectrometry have been robust tools to identify PTMs on endogenous proteins. A peptide antibody-based affinity approach has been successfully utilized to enrich for and identify endogenously ubiquitylated proteins. These antibodies recognize the Lys-ϵ-Gly-Gly (diGLY) remnant that is generated following trypsin digestion of ubiquitylated proteins, and these peptides can then be identified by standard mass spectrometry approaches. This technique has led to the identification of >50,000 ubiquitylation sites in human cells and quantitative information about how many of these sites are altered upon exposure to diverse proteotoxic stressors. In addition, the diGLY proteomics approach has led to the identification of specific ubiquitin ligase targets. Here we provide a detailed method to interrogate the ubiquitin-modified proteome from any eukaryotic organism or tissue.

Proteomics Approaches for Biomarker and Drug Target Discovery in ALS and FTD

“…The covalent attachment of ubiquitin on lysine residues on proteins allows trypsin to cleave both the C-terminus of ubiquitin and the C-terminus of the lysine amino acid on the ubiquitinated protein exposing two glycine residues. Ubiquitin remnant motif (K-𝜀-GG) antibodies are used to enrich peptides containing the di-Gly-Gly motif, followed by elution and analysis by LC-MS/MS in which the presence of the GlyGly residue on lysine (+m/z 114.04) in the MS spectra is an indication of a ubiquitinated peptide (Peng et al, 2003; Xu et al, 2010; Fulzele and Bennett, 2018). However, ubiquitin-like proteins also exist that may be identified using this method such as SUMO (sumoylation), NEDD8 (neddylation) and interferon-stimulated gene 15 (ISG15; isg15ylation) also known as ubiquitin cross-reactive protein (UCRP) (Hemelaar et al, 2004).…”

Strategy for Development of Site-Specific Ubiquitin Antibodies

“…It may also be beneficial to include independent peptide-Ub conjugates in the ELISA screens for selection and monitoring of hybridoma clones. Antibodies recognizing lysine-Ub conjugates provide powerful reagents for detecting poly-and mono-ubiquitinated proteins in cells and for affinity purification (Fujimuro and Yokosawa, 2005;Fulzele and Bennett, 2018;van Wijk et al, 2019). The clones we describe here potentially expand the toolbox for detecting protein ubiquitination but it will be important to determine their binding properties to the different types of linkages in more detail (Fujimuro et al, 1994;Newton et al, 2008Newton et al, , 2012Matsumoto et al, 2010Matsumoto et al, , 2012, and to do so in the assays of interest and not just on immunoblots.…”

“…Different ubiquitination patterns involve the attachment of one ubiquitin molecule (Ub1) to a specific lysine residue of a target protein or the attachment of a poly-ubiquitin chain involving homogeneous or mixed chains by forming isopeptide bonds between the Nterminal methionine (Met1-linked ubiquitination) or any of the other internal lysines on ubiquitin (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) (Chen and Sun, 2009;Husnjak and Dikic, 2012;Ramanathan and Ye, 2012;Oh et al, 2018;Rape, 2018;Clague et al, 2019;Mattern et al, 2019;Spit et al, 2019). Efficient monitoring of these types of events requires reagents that specifically recognize the attachment of ubiquitin to one particular lysine residue of a protein or of ubiquitin itself (poly-Ub) (Fujimuro et al, 1994;Fujimuro and Yokosawa, 2005;Newton et al, 2008Newton et al, , 2012Matsumoto et al, 2010Matsumoto et al, , 2012Fulzele and Bennett, 2018;Mattern et al, 2019;van Wijk et al, 2019). To date, very few reagents exist that fulfill these criteria.…”

Targeted Degradation of Glucose Transporters Protects against Arsenic Toxicity

“…After trypsin digestion of an extract, proteins that had been previously modified by ubiquitin remain modified by the GG remnant of the ubiquitin that had been attached to the substrate's acceptor lysine residue. These so-called KGG peptides can be enriched by affinity purification with an anti-GG antibody and then quantitated by isobaric-labeling mass spectrometry (38,39). We used this approach to determine the ubiquitinated proteome at 0, 1, and 4 h after arsenic treatment.…”