Institute for Molecules and Materials, Radboud University Nijmegen, Heijendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Bioorg Med Chem. 2012 Jan 15;20(2):655-61. doi: 10.1016/j.bmc.2011.07.049. Epub 2011 Aug 3.
An enrichment strategy was devised for azide derivatized macromolecules, based on strain-promoted alkyne-azide cycloaddition (SPAAC) and a cleavable linker. A ring-strained alkyne, bicyclo[6.1.0]non-4-yne (BCN), was covalently attached to agarose beads via a hydrazine-sensitive linker. Benchmark studies of the resulting 'azido-trap' beads were performed with a fluorogenic coumarin derivative, leading to efficient capture of the azidocoumarin with concomitant fluorescence staining of the beads via SPAAC. The versatility of the beads for specific protein enrichment was shown by an effective and highly specific capture-release strategy for enrichment of azido-containing Candida antarctica lipase B (CalB) from a mixture of proteins. This approach is suited for selective enrichment of (glyco)proteins after metabolic incorporation of azides for subsequent (glyco)proteomics studies.
设计了一种基于应变促进炔叠氮环加成（SPAAC）和可裂解连接子的叠氮化物衍生的大分子的富集策略。通过肼敏感连接子将环张力炔，双环[6.1.0]壬-4-炔（BCN）共价连接到琼脂糖珠上。用荧光香豆素衍生物对所得的“叠氮陷阱”珠进行了基准研究，导致叠氮香豆素的有效捕获，并通过 SPAAC 同时对珠进行荧光染色。通过从蛋白质混合物中有效且高度特异性地捕获和释放含有叠氮化物的南极假丝酵母脂肪酶 B（CalB）的策略，证明了珠子在特定蛋白质富集方面的多功能性。该方法适用于在代谢掺入叠氮化物后用于随后的（糖）蛋白质组学研究的（糖）蛋白质的选择性富集。
