Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition

Abstract

The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via the azide -alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na 2 S 2 O 4 , 2% HOCH 2 CH 2 SH, 10% HCO 2 H, 95% CF 3 CO 2 H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO 2 H for 0.5 h. A model GFP protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.

Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis

“…1 and Supplementary Methods). Several cleavable linkers have been developed for proteomics applications; 29 we selected the silane scaffold 30 because of our observation that glycosidic linkages tolerate its mild acid cleavage conditions (2% formic acid in H 2 O). The natural abundance of the stable isotopes, 79 Br and 81 Br (1:1), provided a ready source for isotope recoding.…”

Identification of secreted bacterial proteins by noncanonical amino acid tagging

“…Proteins were incubated with 400 μL of Streptavidin Plus Ultralink resin (Pierce) for 1.5 h at room temperature. Affinity purification was performed according to a previously published protocol (49). Elution fractions were combined with Amicon Ultra 0.5 centrifuge filters (3 kDa molecular weight cutoff; Millipore).…”

Quantitative, Time-Resolved Proteomic Analysis by Combining Bioorthogonal Noncanonical Amino Acid Tagging and Pulsed Stable Isotope Labeling by Amino Acids in Cell Culture

“…azides or alkynes) are used as metabolic labels to distinguish new proteins from old (1,2). Labeled proteins can be conjugated to fluorescent reporters for visualization or affinity tags for purification and subsequent identification by mass spectrometry (3). Because the ncAA probe can be introduced to cells in a well-defined "pulse," affinity purification removes pre-existing proteins and provides both reduced sample complexity and excellent time resolution.…”