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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.
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Biotin-Conjugated Cellulose Nanofibers Prepared via Copper-Catalyzed Alkyne-Azide Cycloaddition (CuAAC) “Click” Chemistry
As potential high surface area for selective capture in diagnostic or filtration devices, biotin-cellulose nanofiber membranes were fabricated to demonstrate the potential for specific and bio-orthogonal attachment of biomolecules onto nanofiber surfaces. Cellulose acetate was electrospun and substituted with alkyne groups in either a one- or two-step process. The alkyne reaction, confirmed by FTIR and Raman spectroscopy, was dependent on solvent ratio, time, and temperature. The two-step process maximized alkyne substitution in 10/90 volume per volume ratio (v/v) water to isopropanol at 50 °C after 6 h compared to the one-step process in 80/20 (v/v) at 50 °C after 48 h. Azide-biotin conjugate “clicked” with the alkyne-cellulose via copper-catalyzed alkyne-azide cycloaddition (CuAAC). The biotin-cellulose membranes, characterized by FTIR, SEM, Energy Dispersive X-ray spectroscopy (EDX), and XPS, were used in proof-of-concept assays (HABA (4′-hydroxyazobenzene-2-carboxylic acid) colorimetric assay and fluorescently tagged streptavidin assay) where streptavidin selectively bound to the pendant biotin. The click reaction was specific to alkyne-azide coupling and dependent on pH, ratio of ascorbic acid to copper sulfate, and time. Copper (II) reduction to copper (I) was successful without ascorbic acid, increasing the viability of the click conjugation with biomolecules. The surface-available biotin was dependent on storage medium and time: Decreasing with immersion in water and increasing with storage in air.
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- Award ID(s):
- 1719875
- PAR ID:
- 10227974
- Date Published:
- Journal Name:
- Nanomaterials
- Volume:
- 10
- Issue:
- 6
- ISSN:
- 2079-4991
- Page Range / eLocation ID:
- 1172
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/nano10061172
