Positional Low Mass MS/MS Tracer Analysis of 13C Isotopologues Reveals Carbon Routes During Ammonia Metabolism of Mosquitoes

Shai Dagan, Analytical Chemistry, Israel Institute For Biological Research (iibr), Ness Ziona, Israel
Thomas Horvath, University of Texas , Houston, USA
Philip Lorenzi, University of Texas , Houston, USA
David Hawke, University of Texas , Houston, USA
Patricia Scaraffia, Tulane University, New Orleans, USA

In Aedes aegypti mosquito females, the ammonia released during blood meal digestion is partially metabolized to facilitate the disposal of excess nitrogen. In this study, we incorporated several LC-MS methods to investigate carbon routes during this ammonia detoxification process.

Glu, Gln, Pro, and Ala are the main amino acids (AAs) involved in ammonia detoxification in mosquitoes. Thus, various ESI-based MS² tools were employed to study these unlabeled and isotopically-labeled AAs and better understand their dissociation pathways. A broad range of products, including previously-undescribed low m/z products (m/z 130 down to m/z 27(!)) was revealed and their formulae were confirmed by their accurate masses. The structural origins of carbons in these small MS² products was explored (also by IMS) and some of which were selected as tracers for specific carbon positions in the AAs.

Mosquitoes were fed a blood meal supplemented with [1,2-¹³C₂]glucose, and downstream metabolites were measured for 24 h. Quantification of [¹³C] amino acids in the entire mosquito body was conducted without sample derivatization by LC-QQQ-MS/MS, using MRM with mass transitions that are indicative of the structural position of [¹³C] incorporated atom. Identification of unlabeled and [¹³C] isotopologues of 43 compounds, including amino acids, amino acid derivatives and organic acids, was performed by high-resolution (Orbitrap) LC/MS.

Blood-fed mosquitoes synthesized [¹³C] metabolites in mainly 2 carbon positions from [1,2-¹³C₂]glucose. [¹³C₂]Ala and [¹³C₂]Pro were the most abundant and rapidly labeled  AAs synthesized. Additional [¹³C] amino acids, [¹³C] amino acid derivatives, and [¹³C] organic acids labeled in 1 or 2 carbon positions were also identified. Two kinetic routes were proposed.

Our findings provide evidence that ingested carbon atoms (delivered in glucose) take part in ammonia detoxification and [¹³C] uric acid synthesis through multiple metabolic pathways, uncovering a metabolic link at the carbon atomic level in ammonia metabolism of A. aegypti mosquitoes.