Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

被引:0
作者
Kaiser S.E. [1 ]
Riley B.E. [1 ]
Shaler T.A. [2 ]
Trevino R.S. [1 ]
Becker C.H. [2 ]
Schulman H. [2 ]
Kopito R.R. [1 ]
机构
[1] Department of Biology, Stanford University, Stanford, CA
[2] Caprion Proteomics U.S., LLC, Menlo Park, CA
关键词
D O I
10.1038/nmeth.1649
中图分类号
学科分类号
摘要
The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule. © 2011 Nature America, Inc. All rights reserved.
引用
收藏
页码:691 / 696
页数:5
相关论文
共 30 条
[1]  
Ravid T., Hochstrasser M., Diversity of degradation signals in the ubiquitin-proteasome system, Nat. Rev. Mol. Cell Biol, 9, pp. 679-690, (2008)
[2]  
Mukhopadhyay D., Riezman H., Proteasome-independent functions of ubiquitin in endocytosis and signaling, Science, 315, 5809, pp. 201-205, (2007)
[3]  
Zeng W., Et al., Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity, Cell, 141, pp. 315-330, (2010)
[4]  
Hoeller D., Dikic I., Targeting the ubiquitin system in cancer therapy, Nature, 458, pp. 438-444, (2009)
[5]  
Lee B.H., Et al., Enhancement of proteasome activity by a small-molecule inhibitor of USP14, Nature, 467, pp. 179-184, (2010)
[6]  
Takada K., Hibi N., Tsukada Y., Shibasaki T., Ohkawa K., Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains, Biochimica et Biophysica Acta - General Subjects, 1290, 3, pp. 282-288, (1996)
[7]  
Haas A.L., Bright P.M., The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates, Journal of Biological Chemistry, 260, 23, pp. 12464-12473, (1985)
[8]  
Bennett E.J., Shaler T.A., Woodman B., Ryu K.-Y., Zaitseva T.S., Becker C.H., Bates G.P., Schulman H., Kopito R.R., Global changes to the ubiquitin system in Huntington's disease, Nature, 448, 7154, pp. 704-708, (2007)
[9]  
Gerber S.A., Rush J., Stemman O., Kirschner M.W., Gygi S.P., Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS, Proceedings of the National Academy of Sciences of the United States of America, 100, 12, pp. 6940-6945, (2003)
[10]  
Kirkpatrick D.S., Et al., Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology, Nat. Cell Biol, 8, pp. 700-710, (2006)