Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)
摘要：
In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We describe here a technology, based on the cotranslational introduction of azide groups into proteins and the chemoselective tagging of azide-labeled proteins with an alkyne affinity tag, to separate and identify, specifically, the newly synthesized proteins in mammalian cells. Incorporation of the azide-bearing amino acid azidohomoalanine is unbiased, not toxic, and does not increase protein degradation. As a first demonstration of the method, we report the selective purification and identification of 195 metabolically labeled proteins with multidimensional liquid chromatography in-line with tandem MS. Furthermore, in combination with leucine-based mass tagging, candidates were immediately validated as newly synthesized proteins. The identified proteins, synthesized in a 2-h window, possess a broad range of biochemical properties and span most functional gene ontology categories. This technology makes it possible to address the temporal and spatial characteristics of newly synthesized proteomes in any cell type.
展开
关键词：
chemical reporter protein synthesis proteomics COPPER(I)-CATALYZED AZIDE-ALKYNE N-LINKED GLYCOPROTEINS CODED AFFINITY TAGS MASS-SPECTROMETRY PROTEOMIC ANALYSIS SHOTGUN PROTEOMICS SACCHAROMYCES-CEREVISIAE QUANTITATIVE-ANALYSIS BIOMARKER DISCOVERY STAUDINGER LIGATION
DOI：
www.pnas.org/cgi/doi/10.1073/pnas.0601637103
被引量：
年份：
2006
国家科技图书文献中心
(权威机构)
Wiley
万方医学
掌桥科研
Semantic Scholar
查看更多
Semantic Scholar
(全网免费下载)
doi.org
Pubmed Central
(全网免费下载)
NCBI
NCBI
JSTOR
Europe PMC
(全网免费下载)
Europe PMC
Cell Press
JBC
adsabs.harvard.edu
dialnet.unirioja.es
ResearchGate
(全网免费下载)
ResearchGate
EBSCO
mcponline.org
jneurosci.org
pubget.com
jlr.org
scienceopen.com
mysciencework.com
molbiolcell.org
brainience.com
(全网免费下载)
msb.embopress.org
authors.library.caltech.edu
(全网免费下载)
brain.mpg.de
(全网免费下载)
pnas.org
(全网免费下载)
plantphysiol.org
The National Academy of Sciences of the United States of America
(全网免费下载)
mcb.asm.org
genesdev.cshlp.org
core.ac.uk
pnas.org
f1000.com
mendeley.com
frontiersin.org
core.ac.uk
(全网免费下载)
rstb.royalsocietypublishing.org
学术范
(全网免费下载)
core.kmi.open.ac.uk
学术范
pubfacts.com
authors.library.caltech.edu
Semantic Scholar
(全网免费下载)
Pubmed Central
(全网免费下载)
Europe PMC
(全网免费下载)
ResearchGate
(全网免费下载)
学术范
(全网免费下载)
brainience.com
(全网免费下载)
The National Academy of Sciences of the United States of America
(全网免费下载)
pnas.org
(全网免费下载)
brain.mpg.de
(全网免费下载)
authors.library.caltech.edu
(全网免费下载)
core.ac.uk
(全网免费下载)
通过文献互助平台发起求助，成功后即可免费获取论文全文。
相似文献
参考文献
引证文献
辅助模式
引用
文献可以批量引用啦~
欢迎点我试用！
