Please use this identifier to cite or link to this item:
|Title:||Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach|
|Source:||Chen, X., Henschke, L., Wu, Q., Muthoosamy, K., Neumann, B., Weil, T. (2013-01-14). Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach. Organic and Biomolecular Chemistry 11 (2) : 353-361. ScholarBank@NUS Repository. https://doi.org/10.1039/c2ob26561c|
|Abstract:||Site-selective labeling of endogenous proteins represents a major challenge in chemical biology, mainly due to the absence of unique reactive groups that can be addressed selectively. Recently, we have shown that surface-exposed lysine residues of two endogenous proteins and a peptide exhibit subtle changes in their individual reactivities. This feature allows the modification of a single residue in a highly site-selective fashion if kinetically controlled labeling conditions are applied. In order to broaden the scope of the "kinetically-controlled protein labeling" (KPL) approach and highlight additional applications, the water-soluble bioorthogonal reagent, biotin-TEO-azido-NHS (11), is developed which enables the site-selective introduction of an azido group onto endogenous proteins/peptides. This bioconjugation reagent features a biotin tag for affinity purification, an azido group for bioorthogonal labeling, a TEO (tetraethylene oxide) linker acting as a spacer and to impart water solubility and an N-hydroxysuccinimidyl (NHS) ester group for reacting with the exposed lysine residue. As a proof of concept, the native protein ribonuclease A (RNase A) bearing ten available lysine residues at the surface is furnished with a single azido group at Lys 1 in a highly site-selective fashion yielding azido-(K1)RNase A. The K1 site-selectivity is demonstrated by the combined application and interpretation of high resolution MALDI-ToF mass spectroscopy, tandem mass spectroscopy and extracted ion chromatography (XIC). Finally, the water soluble azide-reactive phosphine probe, rho-TEO-phosphine (21) (rho: rhodamine), has been designed and applied to attach a chromophore to azido-(K1)RNase A via Staudinger ligation at physiological pH indicating that the introduced azido group is accessible and could be addressed by other established azide-reactive bioorthogonal reaction schemes. © 2013 The Royal Society of Chemistry.|
|Source Title:||Organic and Biomolecular Chemistry|
|Appears in Collections:||Staff Publications|
Show full item record
Files in This Item:
There are no files associated with this item.
checked on Feb 14, 2018
WEB OF SCIENCETM
checked on Jan 22, 2018
checked on Feb 19, 2018
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.