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

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 azide−alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na_2S_2O_4, 2% HOCH_2CH_2SH, 10% HCO_2H, 95% CF_3CO_2H, 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_2H for 0.5 h. A model green fluorescent 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

The vein-banding disease syndrome: A synergistic reaction between grapevine viroids and fanleaf virus

Viroid-free Vitis vinifera cultivars Cabernet Sauvignon and Sauvignon blanc were established in controlled field trials in California to evaluate the relationship between grapevine viroids and fanleaf virus for induction of the vein-banding disease. Vein-banding symptoms were observed only on vines which contained the three principal grapevine viroids, grapevine yellow speckle viroids (GYSVd-1, GYSVd-2), and hop stunt viroid (HSVd-g), as well as grapevine fanleaf virus (GFLV). Sauvignon blanc vines which contained the single viroid, HSVd-g, and GFLV were non-symptomatic indicating an absence of a correlation between HSVd-g and the vein-banding disease. The intensity of vein-banding symptoms was directly correlated with an enhanced titer of GYSVd-1 and GYSVd-2. Vein-banding and yellow speckle symptomatic as well as non-symptomatic vines in Italy contained two viroids, GYSVd-1 and HSVd-g. However, symptomatic vines displayed a higher titer of GYSVd-1 than non-symptomatic materials and vein-banding symptomatic vines were GFLV infected. These data experimentally demonstrate that expression of the vein-banding disease is induced by an unique synergistic reaction between a viroid, GYSVd-1 and a virus, GFLV

Relationship and patterns of distribution among grapevine viroids from California and Europe

Analyses of California and European grapevine sources indicated a ubiquitous occurrence of viroids in these materials. Hybridization results indicated sequence homology to both GV-1 and GV-3 for viroids of varieties grown in California as well as from European sources. Wine and rootstock varieties contained a greater proportion of the more common GV-1 plus GV-3 viroid profile, whereas the table varieties contained a larger proportion of the relatively unusual viroid profile of GV-1, -2, and-3. An unexpected divergence of four viroid profiles emerged in the rootstock species. These profiles were 1) Gv-1, -2, and -3, 2) GV-1 plus GV-3, 3) GV-3, and 4) viroid-free. V. californica was the only grapevine analyzed which was found to be viroid-free

Live-Cell Imaging of Cellular Proteins by a Strain-Promoted Azide–Alkyne Cycloaddition

Live and let dye: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts. All three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold. These conjugates are powerful tools for visualizing biomolecule dynamics in living cells

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Microbial pathogens use complex secretion systems to deliver virulence factors into host cells, where they disrupt host cell function. Understanding these systems is essential to the development of new treatments for infectious disease. A challenge in such studies arises from the abundance of host cell proteins, which interfere with detection of microbial effectors. Here we describe a metabolic labeling strategy that allows selective enrichment of microbial proteins from the host cell cytoplasm. The method enables efficient identification of microbial proteins that have been delivered to the host, identifies distinct secretion profiles for intracellular and extracellular bacteria, and allows for determination of the order of injection of microbial proteins into host cells