Study of the fragmentation of arginine isobutyl ester applied to arginine quantification in Aedes aegypti mosquito excreta

Abstract

It has been demonstrated that argininolysis and uricolysis are involved in the synthesis and excretion of urea in Aedes aegypti female mosquitoes. To further investigate the metabolic regulation of urea in female mosquitoes, it is desirable to have a rapid and efficient method to monitor arginine (Arg) concentration in mosquito excreta. Thus, a procedure currently used for the identification of Arg in urea cycle disorders in newborn babies was adapted to analyze Arg in A. aegypti excreta. The fragmentation patterns of the isobutyl esters of Arg and 15N2-Arg (labeled at the guanidino group) were explored by electrospray ionization (ESI)-tandem mass spectrometry and fragmentation pathways not described before were characterized. In addition, Arg, 18O2-Arg, 15N2-Arg and 15N2-18O2-Arg were also analyzed to elucidate some of the minor fragments in greater detail. Mosquito excreta from individual females were collected before and at different times after feeding a blood meal, mixed with 15N2-Arg, an internal standard, and then derivatized as isobutyl esters. Based on the fragmentation mechanisms of Arg standards, studied by MS2 and MS3, Arg in the mosquito excreta was successfully analyzed by ESI-multiple-reaction monitoring (MRM) in a triple-quadrupole mass spectrometer. Arg excretion was monitored over a 120 h window before and after feeding female mosquitoes with a blood meal, with the maximum level of Arg excretion observed at 36–48 hours post blood feeding. This method provides an efficient and rapid tool to quantify Arg in individual blood-fed mosquitoes, and can be applied to other organisms, whose small size severally limits the use of conventional biochemical analysis.

Factors Affecting the Production of Aromatic Immonium Ions in MALDI 157 nm Photodissociation Studies

“…This increased a 2 ion intensity suggests that its stability is enhanced, possibly due to a fivemembered cyclic structure [34][35][36]. A strong a 2 -NH 3 ion may result from an increased stability of the arginine side chain forming a seven-membered ring with the N-terminus [37].…”

“…The intensities for the aromatic immonium (blue) and a-type (red) ions at each position. The peptides with four aromatic residues (1-4) are in the left charts, and those with one aromatic residue (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) are in the right by losing ammonia [37]. When arginine is at the N-terminus, the average relative abundance of the 112 Da peak is 41%, and at the C-terminus, the abundance is 13%.…”

Effective disposal of nitrogen waste in blood‐fedAedes aegyptimosquitoes requires alanine aminotransferase

“…Mosquito excreta were collected as previously described (9). For 6, 12, 18, and 24 h time points, mosquito excreta were collected by removing the female and storing the vial.…”

“…The term ammonia is defined here as NH 3 or NH 4 + or a combination of both. The application of isotopically labeled compounds and mass spectrometry, as well as RNA interference (RNAi), has demonstrated that A. aegypti females efficiently detoxify ammonia through 3 phases: fixation, assimilation, and excretion (5)(6)(7)(8)(9)(10). During this complex metabolic challenge, female mosquitoes synthesize specific amino acids, such as Gln, Glu, Ala, and Pro, through reactions catalyzed by several enzymes, including glutamine synthetase, glutamate synthase, glutamate dehydrogenase, pyrroline-5-carboxylate synthase, pyrroline-5-carboxylaye reductase, and alanine aminotransferase (ALAT).…”

Low Mass MS/MS Fragments of Protonated Amino Acids Used for Distinction of Their 13C- Isotopomers in Metabolic Studies

“…By feeding A. aegypti females with isotopically-labeled 15 N-compounds (such as 15 NH 4 Cl), we discovered that mosquitoes have an extraordinary biochemical machine to efficiently detoxify ammonia. Indeed, they synthesize specific amino acids and excrete several nitrogen wastes through multiple metabolic pathways [6-9]. By using this strategy, female mosquitoes are able to survive toxic ammonia concentrations released during the digestion of a blood meal.…”