title: A proteomics approach to identify the ubiquitinated proteins in mouse heart.
abstract: There is a growing need for the large-scale identification of the ubiquitinated proteins and ubiquitin attachment sites. As part of this effort, we generated a transgenic mouse expressing a tagged ubiquitin in the heart. We found that the majority of ubiquitinated proteins in mouse heart are insoluble in detergent-free buffer and were chemically cleaved after methionine with CNBr. CNBr cleaved the proteins into smaller polypeptides while preserving the ubiquitin chains. Ubiquitin-conjugated polypeptides were then purified under denaturing conditions, digested with Lys-C and trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. We identified 121 proteins that were ubiquitinated in mouse heart, and we detected 33 ubiquitination sites in 21 of the proteins. Components of cardiac muscle and many mitochondrial proteins were identified as substrates for ubiquitination, strongly suggesting that proteins related to major heart functions such as contraction and energy production are under continuous quality control by the ubiquitin system.
authors: Department of Life Science, Gwangju Institute of Science & Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea.
authors: Hong Bae
authors: HB
authors: Jeon
authors: Eun Soo
authors: ES
authors: Choi
authors: Jong Hyuk
authors: JH
authors: Yoon
authors: Jin Ha
authors: JH
authors: Hwang
authors: Jong Wook
authors: JW
authors: Chang
authors: Eun Kyung
authors: EK
authors: Lee
authors: Hyun Woo
authors: HW
authors: Choi
authors: Zee-Yong
authors: ZY
authors: Park
authors: Yung Joon
authors: YJ
authors: Yoo
mesh_terms: D000818:Animals; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008822:Mice, Transgenic; D009206:Myocardium / Q000378:metabolism*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins; D025801:Ubiquitin; D014357:Trypsin; D003488:Cyanogen Bromide