Integrating MACSPI and SILAC for Neuron Type-specific Proteomics in Caenorhabditis elegans.

Understanding neuronal differentiation and function requires precise proteomic characterization of distinct neuron types, yet existing methods face challenges in specificity and sensitivity. Here, we combine Methionine Analog-based Cell-Specific Proteomics and Interactomics (MACSPI) and SILAC to achieve neuron type-specific proteomic profiling in Caenorhabditis elegans. We demonstrate the utility of the methods by profiling and comparing the proteomes of two neuron types, namely the eight dopaminergic neurons (DA) and the six touch receptor neurons (TRNs). By expressing in these neurons an engineered methionyl-tRNA synthetase that can attach a methionine analog with a chemical handle to the synthesizing proteins, we chemically label the proteomes of specific neurons in complex tissues and isolate the labeled proteins from the whole-animal lysates through click chemistry and affinity purification. Thus, our approach does not require physical isolation of the neurons through cell sorting. Quantitative mass spectrometry studies through SILAC enable proteomic profiling and comparison of DA and TRNs and reveal distinct functional signatures between these two neuron types, with DA neurons showing enrichment in synaptic and metabolic pathways, while TRNs were characterized by cytoskeletal and signaling components. Moreover, we observed a weak correlation between protein abundance and mRNA levels, underscoring the importance of proteomic measurements. Our study establishes MACSPI-SILAC as a versatile platform for cell-type-specific proteomics in multicellular organisms, bridging a critical gap between transcriptomic and functional analyses. The proteomic datasets provide a resource for exploring the mechanisms of neuronal fate specification and cellular differentiation.