English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging

Dieterich, D. C., Lee, J. J., Link, A. J., Graumann, J., Tirrell, D. A., & Schuman, E. M. (2007). Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat Protoc, 2(3), 532-40. doi:10.1038/nprot.2007.52.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files

Locators

show
hide
Description:
-

Creators

show
hide
 Creators:
Dieterich, D. C., Author
Lee, J. J., Author
Link, A. J., Author
Graumann, J., Author
Tirrell, D. A., Author
Schuman, Erin M.1, Author              
Affiliations:
1Synaptic Plasticity Department, Max Planck Institute for Brain Research, Max Planck Society, ou_2461710              

Content

show
hide
Free keywords: Affinity Labels/metabolism Alanine/analogs & derivatives Amino Acids/*isolation & purification/*metabolism Animals Azides Gene Expression Profiling/*methods Mice Proteins/*isolation & purification Proteomics/*methods
 Abstract: A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.

Details

show
hide
Language(s):
 Dates: 2007-04-05
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: Other: 17406607
DOI: 10.1038/nprot.2007.52
ISSN: 1750-2799 (Electronic)1750-2799 (Linking)
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Nat Protoc
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: -
Pages: - Volume / Issue: 2 (3) Sequence Number: - Start / End Page: 532 - 40 Identifier: -