Automated Spatially Targeted Optical Micro Proteomics (autoSTOMP) to Determine Protein Complexity of Subcellular Structures.

Abstract

Spatially Targeted Optical Micro Proteomics (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 photo-driven crosslinking reaction so that they can be affinity purified and identified by mass spectrometry1. However, the use of a custom photo-crosslinker 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 accessiblity, we implemented a commercially available biotin-benzophenone photo-crosslinking and purification protocol. Here we demonstrate that autoSTOMP can efficiently label, purify, and identify proteins belonging to 1-2 \uf06dm 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.