A proteomics approach to identify the ubiquitinated proteins in mouse heart.
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.
Jeon
Hong Bae
HB
Department of Life Science, Gwangju Institute of Science & Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea.
Choi
Eun Soo
ES
Yoon
Jong Hyuk
JH
Hwang
Jin Ha
JH
Chang
Jong Wook
JW
Lee
Eun Kyung
EK
Choi
Hyun Woo
HW
Park
Zee-Yong
ZY
Yoo
Yung Joon
YJ
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*
D011506:Proteins; D025801:Ubiquitin; D014357:Trypsin; D003488:Cyanogen Bromide