| Autor: |
Yin B; Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center , University of Virginia School of Medicine , Charlottesville , Virginia 22908-0395 , United States., Mendez R; Department of Chemistry , University of Virginia , Charlottesville , Virginia 22904-4132 , United States., Zhao XY; Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center , University of Virginia School of Medicine , Charlottesville , Virginia 22908-0395 , United States., Rakhit R; Mitokinin Inc , 953 Indiana Street , San Francisco , California 94107-3007 , United States., Hsu KL; Department of Chemistry , University of Virginia , Charlottesville , Virginia 22904-4132 , United States., Ewald SE; Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center , University of Virginia School of Medicine , Charlottesville , Virginia 22908-0395 , United States. |
| Abstrakt: |
Spatially targeted optical microproteomics (STOMP) is a method to study region-specific protein complexity in primary cells and tissue samples. STOMP uses a confocal microscope to visualize structures of interest and to tag the proteins within those structures by a photodriven cross-linking reaction so that they can be affinity purified and identified by mass spectrometry ( eLife 2015 , 4 , e09579). However, the use of a custom photo-cross-linker and the requirement for extensive user intervention during sample tagging have posed barriers to the utilization of STOMP. To address these limitations, we built automated STOMP (autoSTOMP) which uses a customizable code in SikuliX to coordinate image capture and cross-linking functions in Zeiss Zen Black with image processing in FIJI. To increase protocol accessibility, we implemented a commercially available biotin-benzophenone photo-cross-linking and purification protocol. Here we demonstrate that autoSTOMP can efficiently label, purify, and identify proteins belonging to 1-2 μm structures in primary human foreskin fibroblasts or mouse bone marrow-derived dendritic cells infected with the protozoan parasite Toxoplasma gondii ( Tg ). AutoSTOMP can easily be adapted to address a range of research questions using Zeiss Zen Black microscopy systems and LC-MS protocols that are standard in many research cores. |
