Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry.

Article Details

Citation

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
NameUniProt ID
2-oxoglutarate dehydrogenase, mitochondrialQ02218Details
Gamma-aminobutyric acid receptor subunit alpha-5P31644Details
von Willebrand factorP04275Details
Gamma-aminobutyric acid receptor subunit beta-2P47870Details
Serine/threonine-protein kinase Chk1O14757Details
Centromere-associated protein EQ02224Details
Proteasome subunit alpha type-6P60900Details
Phosphoglycerate mutase 2P15259Details
Protein RIC-3Q7Z5B4Details