Introduction: Understanding the spatial distribution of proteins within cells is critical for unraveling cellular function and signaling, particularly in specialized organelles such as cilia. Cilia are dynamic, microtubule-based structures essential for motility, sensory transduction, and signaling pathways. Given their small size and structural complexity, precise mapping of the ciliary proteome has posed significant technical challenges. Our approach overcomes limitations of traditional spatial proteomics by employing the Microscoop® system for hypothesis-free, submicron-resolution proteomic analysis.
Methods: We integrated the Syncell Microscoop® platform with Thermo Scientific™ Orbitrap™ Astral™ mass spectrometry for high-resolution spatial proteomic analysis. The workflow includes: (1) sample preparation using photoactivatable biotin-based probes (Synlight™ kit), (2) AI-guided detection of regions of interest, (3) automated laser-guided photolabeling, (4) protein enrichment via streptavidin-biotin pulldown (Synpull™ kit), (5) LC-MS/MS analysis with Orbitrap Astral mass spectrometer, and (6) label-free identification and data analysis. This approach enables selective labeling of proteins within precisely defined subcellular regions with submicron resolution.
Preliminary Results: From 20 ng of sample input, we quantified over 5,300 proteins and 41,000 peptides across a dynamic range exceeding 5.5 orders of magnitude. Quantitative reproducibility showed a median protein abundance CV of 13% across replicates. Principal Component Analysis confirmed successful enrichment, with photolabeled samples clearly separated from unlabeled controls, demonstrating effective isolation of cilia-specific proteins. Differential analysis identified 1,781 proteins significantly enriched in photolabeled ciliary regions. Ontology analysis confirmed enrichment of proteins associated with ciliary and centrosomal compartments, including intraciliary transport particles, ciliary base, centriole, ciliary tip, and transition zone.
Novel Aspect: Subcellular labeling with submicron resolution enabled comprehensive analysis of cilia-related proteins with unprecedented spatial resolution and sensitivity, revealing previously uncharacterized components of the ciliary proteome. This technology establishes a powerful platform for exploring diverse subcellular structures or specific cell populations within complex tissues, enabling researchers to uncover novel protein interactions and functional relationships previously inaccessible to proteomic approaches.