(172f) Untargeted metabolomic and lipidomic profiling for Golgi molecular content via immunoprecipitation (Golgi-IP)

Organelle-specific metabolic dysfunctions have been implicated in neurodegenerative diseases, but it is challenging to achieve high resolution for compartmentalized metabolism and detect metabolites at low nanomolar concentrations in small organelles such as lysosomes and Golgi. In order to quantitatively assess novel subcellular proteins and compartmentalized metabolism, we developed a high-resolution mass spectrometry (HRMS) method in conjunction with a technique to harvest organelle-specific metabolites and lipids via immunoprecipitation (IP). In addition to uncovering novel Golgi proteins, we applied our HRMS-Golgi IP workflow to study the Golgi-specific metabolome and identified a class of uridine-diphosphate (UDP) sugars and their derivatives that were specifically enriched in the Golgi. Moreover, we validated Golgi-specific reduction of a UDP species after genetically knocking out its transporter on the Golgi. Further isotope tracing analysis using 13C-labeled glucose suggests a potentially new metabolic coupling between UDP species within the Golgi. In addition, we also performed untargeted lipidomics using Golgi IP and identified a group of phospholipids to be selectively enriched in the Golgi. Our work established a robust HRMS-IP method to investigate subcellular metabolism and provided novel insights into the Golgi proteome, metabolome and lipidome. These insights can also lead to discoveries of new metabolic reactions within cellular compartments that still remain elusive by conventional whole-cell approaches.