A Highly Efficient Click Linker for Enrichment of Alkyne-Tagged Proteins in Living Cells

Alkyne tags have been widely used for the enrichment of labeled proteins to enable their profiling at a proteome-wide scale by mass spectrometry. The key component in the enrichment process is an azido-terminated cleavable linker for capturing the labeled proteins/peptides via click reaction. Herein, we report a new efficient click linker (APA biotin) featuring an acid-cleavable acetal linkage end-caped with a highly reactive picolyl azido head and a biotin handle for anchoring onto streptavidin-coated supports. Using an amine-reactive probe to profile the proteome structural changes in living S. cerevisiae cells within 5 minutes of heat shock, we demonstrated that the linker allowed identification of >9400 labeled sites, among which 457–1656 with significantly altered reactivity upon heat shock. This study represented the first chemical labeling mass spectrometry (CL–MS)-based profiling of proteome structural changes in living cells in response to external stimuli. Data are available via ProteomeXchange with identifier PXD051279.