Universal protein immobilization and digestion reactor facilitates deep quantitative proteomic analysis of single Caenorhabditis elegans individuals

Single-organism proteomics can reveal heterogeneity masked by population-level measurements, but deep profiling of individual Caenorhabditis elegans remains challenging because of low protein input, inefficient extraction through the collagen-rich cuticle, and lengthy sample processing. Herein, we present SPIDR-C (single-organism protein immobilization and digestion reactor for C. elegans), a streamlined workflow enabling deep single-worm proteomics. SPIDR-C seamlessly integrates worm disruption, strong surfactant-assisted extraction, covalent protein immobilization, in-reactor sample cleanup, and rapid enzymatic digestion in a single-tube, transfer-free format. Built from commercially available insert tubes and requiring neither microfluidic devices nor custom instrumentation, the workflow is low-cost, easy to operate, and compatible with standard liquid chromatography (LC) autosamplers, allowing parallel batch processing of multiple samples within 2 h. By coupling aggressive extraction with LC−MS/MS-compatible cleanup, it enabled identification of more than 7100 proteins from a single adult worm, representing one of the deepest single-worm proteomes reported to date. Across eight single-worm-equivalent technical replicates, the median protein-abundance CV was 13.34%, with pairwise Pearson correlation coefficients above 0.977. We applied SPIDR-C to a high-glucose and high-fat (HGHF) model as an exploratory demonstration of its analytical utility, where single-worm proteomics captured HGHF-associated proteome alterations and individual-level heterogeneity under the same dietary challenge. Proteome profiling further pointed to two candidate HGHF-associated response patterns: one marked by higher abundance of ER proteostasis-related proteins and the other characterized by stronger lipid- and membrane-associated remodeling. Thus, SPIDR-C provides a practical strategy for deep single-organism proteomics and facilitates the investigation of molecular heterogeneity in complex low-input biological samples.