Isolation of signal transduction complexes using biotin and crosslinking methodologies.
We have developed a strategy to preferentially label the N-terminal alpha-amino groups of intact proteins allowing the internal epsilon-amino groups to remain free to react with chemical crosslinking reagents. The convergence of these methodologies allows biotinylated ligands to bind to their receptors within the cell membrane followed by removal of the crosslinked complex from cell lysate. This technique allows for the isolation of protein complexes in an MS-compatible system, thus providing a tool for furthering our understanding of signal transduction.
Freed
Julie K
JK
Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Smith
Julia R
JR
Li
Peigang
P
Greene
Andrew S
AS
D000818:Animals; D001710:Biotin / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D011485:Protein Binding; D051381:Rats; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D003432:Cross-Linking Reagents; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D001710:Biotin