Work

Analysis of Ubiquitinated Proteome by Quantitative Mass Spectrometry

Year: 2012

Type: book-chapter

Abstract: Protein modification by ubiquitin (Ub) is one of the most common posttranslational events in eukaryotic cells. Ubiquitinated proteins are destined to various fates such as proteasomal degradation, protein trafficking, DNA repair, and immune response. In the last decade, vast improvements of mass spectrometry make it feasible to analyze the minute amount of ubiquitinated components in vivo. When combined with quantitative strategies, such as stable isotope labeling with amino acids in cell culture (SILAC), it is capable of profiling ubiquitinated proteome under different experimental conditions. Here, we describe a procedure to perform such a study, including differential protein labeling by the SILAC method, enrichment of ubiquitinated species, mass spectrometric analysis, and quality control to reduce false positives. The potential challenges and limitations of the procedure are also discussed.

Source: Methods in molecular biology

DOI: 10.1007/978-1-61779-885-6_26

Authors: Chan Hyun Na, Junmin Peng

Institution: St. Jude Children's Research Hospital

Language: English

Topic: Ubiquitin and proteasome pathways

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: American Cancer Society, National Institutes of Health, National Institute of Neurological Disorders and Stroke, National Center for Research Resources

Awards: G2654164304, G299548475, Analysis of Ubiquitinated Proteome, Emory Neuroscience NINDS Core Facilities (ENNCF)