Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging.
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.
Dieterich
Daniela C
DC
Division of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA. dieterd@caltech.edu
Lee
Jennifer J
JJ
Link
A James
AJ
Graumann
Johannes
J
Tirrell
David A
DA
Schuman
Erin M
EM
D000345:Affinity Labels / Q000378:metabolism; D000409:Alanine / Q000031:analogs & derivatives; D000596:Amino Acids / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001386:Azides; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D011506:Proteins; C506098:azidohomoalanine; D000409:Alanine