Analysis of sulfur containing metabolites by mass spec

Our laboratory utilizes a combination of gas chromatography mass spectrometry (GC-MS), targeted liquid chromatography mass spectrometry (LC-MS/MS, with triple quadrupole) and non-targeted LC-MS (with orbitrap) to profile and quantify metabolite changes in cells, fluids and tissues. With these technologies we perform both metabolite quantitation and flux analysis using stable isotope labeled substrates. However, thiol-containing metabolites including cysteine and glutathione are very labile and require special derivatization during sample preparation to prevent their oxidation. We have optimized the derivatization and quantification of sulfur containing metabolites in various tissues, cell types and fluids for the study of sulfur metabolism (Kang et al. eLife 2019).

 

 

Adapted from Ward and DeNicola. Int Rev Cell Mol Biol. 2019;347:39-103. doi: 10.1016/bs.ircmb.2019.05.001.

Tracing the source and fate of sulfur in vivo

By delivering stable isotope labeled serine, methionine and cystine via the jugular catheter to mice, we are investigating how various tissues and tumors that arise within these tissues obtain cysteine and use it to synthesize downstream metabolites.

 

 

The essentiality of sulfur-dependent processes in tumor initiation and progression

Cysteine plays an essential role in many NRF2-regulated processes. However, little is known about how different cellular compartments respond to cystine starvation and how cysteine is prioritized for cysteine-dependent processes. One such process is iron-sulfur (Fe-S) cluster synthesis, which requires cysteine-derived sulfur. Fe-S clusters are highly sensitive to ROS, which are detoxified by NADPH-dependent enzymes in the mitochondria. We found that the mitochondrial NADPH-generating enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly enhances tumor formation and aggressiveness in mouse models of lung tumor initiation and progression by preventing mitochondrial Fe-S cluster oxidation to maintain mitochondrial function (Ward et al., J Exp Med 2020). Interestingly, loss of NNT does not influence global mitochondrial antioxidant capacity, suggesting that NNT serves a specific role in mitigating the oxidation of critical Fe-S cofactors in lung cancer and represents an attractive target for therapy.

 

 

Open questions:

  • How do different subcellular compartments respond to cystine starvation?
  • How is cysteine prioritized for cysteine-dependent processes?
  • Which tissues are capable of de novo cysteine synthesis (transsulfuration)?
  • How is cystine uptake/use influenced by tumor formation?