Recent advances in defining the ubiquitylome

Abstract

Ubiquitin is a small 8.5 kDa protein that is conjugated to a target protein in a concerted three step enzymatic process. Ubiquitin addition can drastically affect function or target the modified protein for degradation. Ubiquitin modifications have important regulatory roles in disease progression, such as in cancer and neurodegenerative diseases to name a few. As a consequence, it is imperative to identify important ubiquitin targets to elucidate the role of the modification. Proteomic studies have sought to understand this role by identifying proteome-wide ubiquitylated proteins. Two central ideas have developed to characterize the ubiquitylome: affinity purification of ubiquitylated proteins and optimization of GG-peptide enrichment. In this review, we will discuss recent advances in both approaches and discuss how these studies are essential to pharmacoproteomics.

Keywords::

• K-ε-GG antibody
• mass spectrometry
• pharmacoproteomics
• proteome wide
• proteomics
• ubiquitin
• ubiquitylation
• ubiquitylome

Acknowledgements

We acknowledge all of the scientific contributions made by all the other researchers in the field who were not cited in this review. We apologize for not including your work, however, due to space constraints we were unable to include more of the amazing work being performed in the field of ubiquitylation and mass spectrometry-based proteomics. We thank Raymond J Deshaies for his insight and lively discussions. Lastly, we thank the members of the Sonja Hess Laboratory in the Proteome Exploration Laboratory for their lively discussions and helpful suggestions.

Financial & competing interests disclosure

The Proteome Exploration Laboratory is supported by the Beckman Institute, the Gordon and Betty Moore Foundation through Grant GBMF775 and the NIH through grants SRR029594A and 1S10OD010788. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Ubiquitin modifications are complex and the combinations of enzymes that catalyze ubiquitin modifications are intricate.

• The major challenges facing the field of ubiquitylation include identifying proteome-wide proteins that are ubiquitin modified and the combination of enzymes responsible for catalyzing each modification. Additionally, determining the ubiquitin chain topology on specific substrates is at present difficult to perform by shotgun proteomics.

• It is essential to characterize ubiquitin modifications because the homeostasis of many important cellular processes are regulated by ubiquitylation and can result in disease, such as in the case of many types of cancers and certain neurodegenerative diseases.

• Two main methodologies have developed for the proteome-wide analysis of ubiquitylation: affinity purification in combination with mass spectrometry and peptide level enrichment coupled to mass spectrometry.

• Peptide prefractionation prior to K-ε-GG peptide immunoaffinity enrichment followed by mass spectrometry is, at the moment, the most robust method for identifying lysine ubiquitylated proteins proteome wide.

• Proteasomal inhibitors such as MG132, carfilzomib and bortezomib have been shown to aid in the treatment of specific myelomas, prompting the development of second-generation proteasome inhibitors for the treatment of cancer.

• Pharmacoproteomic studies employing efficient and robust enrichment techniques and mass spectrometry analysis tools will be essential for the rapid evaluation of future drugs on global ubiquitylation.

• The combinatorial effects or cross-talk between post-transitional modifications will also be aided by further advancements in peptide-enrichment strategies, next generation mass spectrometers and more advanced bioinformatics tools.