Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry.
Article Details
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Denis NJ, Vasilescu J, Lambert JP, Smith JC, Figeys D
Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry.
Proteomics. 2007 Mar;7(6):868-74.
- PubMed ID
- 17370265 [ View in PubMed]
- Abstract
Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.
DrugBank Data that Cites this Article
- Polypeptides
Name UniProt ID 2-oxoglutarate dehydrogenase, mitochondrial Q02218 Details Gamma-aminobutyric acid receptor subunit alpha-5 P31644 Details von Willebrand factor P04275 Details Gamma-aminobutyric acid receptor subunit beta-2 P47870 Details Serine/threonine-protein kinase Chk1 O14757 Details Centromere-associated protein E Q02224 Details Proteasome subunit alpha type-6 P60900 Details Phosphoglycerate mutase 2 P15259 Details Protein RIC-3 Q7Z5B4 Details