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Abstract 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 (APAbiotin) 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 livingS. cerevisiaecells 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.