title: Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach.
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.
authors.affiliation: Institute of Organic Chemistry III/Macromolecular Chemistry and Biological Chemistry, University of Ulm, Albert-Einstein 11, 89069 Ulm, Germany.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Lars
authors.initials: L
authors.lastname: Henschke
authors.forename: Qianzhen
authors.initials: Q
authors.lastname: Wu
authors.forename: Kasturi
authors.initials: K
authors.lastname: Muthoosamy
authors.forename: Boris
authors.initials: B
authors.lastname: Neumann
authors.forename: Tanja
authors.initials: T
authors.lastname: Weil
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism*; D001710:Biotin / Q000138:chemical synthesis / Q000737:chemistry; D002625:Chemistry, Organic / Q000379:methods*; D018360:Crystallography, X-Ray; D005027:Ethylene Oxide / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010720:Phosphines / Q000138:chemical synthesis / Q000737:chemistry; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013388:Succinimides / Q000138:chemical synthesis / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D001386:Azides; D010720:Phosphines; D012235:Rhodamines; D013388:Succinimides; D014867:Water; D001710:Biotin; D012259:Ribonuclease, Pancreatic; C044646:phosphine; D005027:Ethylene Oxide; C029773:rhodamine B