title: Ubiquitin ligase substrate identification through quantitative proteomics at both the protein and peptide levels.
abstract: Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.
authors: Department of Molecular Sciences, Amgen, Seattle, Washington 98119.
authors: Kimberly A
authors: KA
authors: Lee
authors: Department of Inflammation, Amgen, Seattle, Washington 98119.
authors: Lisa P
authors: LP
authors: Hammerle
authors: Department of Lead Discovery, Amgen, Cambridge, Massachusetts 02142.
authors: Paul S
authors: PS
authors: Andrews
authors: Cell Signaling Technology Inc., Danvers, Massachusetts 01923.
authors: Matthew P
authors: MP
authors: Stokes
authors: Department of Inflammation, Amgen, Seattle, Washington 98119.
authors: Tomas
authors: T
authors: Mustelin
authors: Cell Signaling Technology Inc., Danvers, Massachusetts 01923.
authors: Jeffrey C
authors: JC
authors: Silva
authors: Department of Inflammation, Amgen, Seattle, Washington 98119.
authors: Roy A
authors: RA
authors: Black
authors: Department of Inflammation, Amgen, Seattle, Washington 98119. Electronic address: jdoedens@yahoo.com.
authors: John R
authors: JR
authors: Doedens
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D020543:Proteome; D025801:Ubiquitin; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases